tetrodotoxin and Hypoxia

Carotid bodies (CBs) are secondary sensory receptors in which the sensing elements, chemoreceptor cells, are activated by decreases in arterial PO(2) (hypoxic hypoxia). Upon activation, chemoreceptor cells (also known as Type I and glomus cells) increase their rate of release of neurotransmitters that drive the sensory activity in the carotid sinus nerve (CSN) which ends in the brain stem where reflex responses are coordinated. When challenged with hypoxic hypoxia, the physiopathologically most relevant stimulus to the CBs, they are activated and initiate ventilatory and cardiocirculatory reflexes. Reflex increase in minute volume ventilation promotes CO(2) removal from alveoli and a decrease in alveolar PCO(2) ensues. Reduced alveolar PCO(2) makes possible alveolar and arterial PO(2) to increase minimizing the intensity of hypoxia. The ventilatory effect, in conjunction the cardiocirculatory components of the CB chemoreflex, tend to maintain an adequate supply of oxygen to the tissues. The CB has been the focus of attention since the discovery of its nature as a sensory organ by de Castro (1928) and the discovery of its function as the origin of ventilatory reflexes by Heymans' group (1930). A great deal of effort has been focused on the study of the mechanisms involved in O(2) detection. This review is devoted to this topic, mechanisms of oxygen sensing. Starting from a summary of the main theories evolving through the years, we will emphasize the nature and significance of the findings obtained with veratridine and tetrodotoxin (TTX) in the genesis of current models of O(2)-sensing.

Topics: Animals; Arteries; Calcium; Calcium Channels; Carotid Body; Chemoreceptor Cells; Humans; Hypoxia; Neurotransmitter Agents; Oxygen; Potassium; Rats; Reflex; Sodium Channels; Tetrodotoxin; Veratridine

Anti-invasive effects of riluzole and ranolazine, a neuro-protectant and an anti-anginal drug, respectively, on Mat-LyLu rat prostate cancer (PCa) cells were tested in vitro (a) at non-toxic doses and (b) under both normoxic and hypoxic conditions, the latter common to growing tumours. Tetrodotoxin (TTX) was used as a positive control. Hypoxia had no effect on cell viability but reduced growth at 48 hours. Riluzole (5 μmol/L) or ranolazine (20 μmol/L) had no effect on cell viability or growth under normoxia or hypoxia over 24 hours. Matrigel invasion was not affected by hypoxia but inhibited by TTX, ranolazine and riluzole under a range of conditions. The expression of Nav1.7 mRNA, the prevailing, pro-invasive voltage-gated sodium channel α-subunit (VGSCα), was up-regulated by hypoxia. Riluzole had no effect on Nav1.7 mRNA expression in normoxia but significantly reduced it in hypoxia. VGSCα protein expression in plasma membrane was reduced in hypoxia; riluzole increased it but only under hypoxia. It was concluded (a) that riluzole and ranolazine have anti-invasive effects on rat PCa cells and (b) that Nav1.7 mRNA and protein expression can be modulated by riluzole under hypoxia. Overall, therefore, riluzole and ranolazine may ultimately be "repurposed" as anti-metastatic drugs against PCa.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Humans; Hypoxia; Male; NAV1.7 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Prostatic Neoplasms; Rats; Riluzole; Tetrodotoxin

Functional expression of voltage-gated Na

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; RNA, Small Interfering; Tetrodotoxin

Recently, mitochondria have been localized to astrocytic processes where they shape Ca(2+) signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca(2+) channel blocker), two inhibitors of reversed Na(+)/Ca(2+) exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA1. Using the genetic Ca(2+) indicator Lck-GCaMP-6S, we observed two types of Ca(2+) signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ∼2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca(2+) through reversed Na(+)/Ca(2+) exchange triggers mitochondrial loss and dramatic increases in Ca(2+) signaling in astrocytic processes.. Astrocytes, the most abundant cell type in the brain, are vital integrators of signaling and metabolism. Each astrocyte consists of many long, thin branches, called processes, which ensheathe vasculature and thousands of synapses. Mitochondria occupy the majority of each process. This occupancy is decreased by ∼50% 24 h after an in vitro model of ischemia/reperfusion injury, due to delayed fragmentation and mitophagy. The mechanism appears to be independent of neuropathology, instead involving an extended period of high glutamate uptake into astrocytes. Our data suggest that mitochondria serve as spatial buffers, and possibly even as a source of calcium signals in astrocytic processes. Loss of mitochondria resulted in drastically altered calcium signaling that could disrupt neurovascular coupling and gliotransmission.

Topics: Action Potentials; Animals; Astrocytes; Calcium Channel Blockers; Calcium Signaling; Enzyme Inhibitors; GAP-43 Protein; Glial Fibrillary Acidic Protein; Glucose; Hippocampus; Hypoxia; In Vitro Techniques; Microtubule-Associated Proteins; Mitochondria; Organ Culture Techniques; Rats; Rats, Transgenic; Sodium Channel Blockers; Tacrolimus; Tetrodotoxin; Time Factors

Harmful algal blooms are increasing in frequency and extent worldwide and occur nearly annually off the west coast of Florida where they affect both humans and wildlife. The dinoflagellate Karenia brevis is a key organism in Florida red tides that produces a suite of potent neurotoxins collectively referred to as the brevetoxins (PbTx). Brevetoxins bind to and open voltage gated sodium channels (VGSC), increasing cell permeability in excitable cells and depolarizing nerve and muscle tissue. Exposed animals may thus show muscular and neurological symptoms including head bobbing, muscle twitching, paralysis, and coma; large HABs can result in significant morbidity and mortality of marine life, including fish, birds, marine mammals, and sea turtles. Brevetoxicosis however is difficult to treat in endangered sea turtles as the physiological impacts have not been investigated and the magnitude and duration of brevetoxin exposure are generally unknown. In this study we used the freshwater turtle Trachemys scripta as a model organism to investigate the effects of the specific brevetoxin PbTx-3 in the turtle brain. Primary turtle neuronal cell cultures were exposed to a range of PbTx-3 concentrations to determine excitotoxicity. Agonists and antagonists of voltage-gated sodium channels and downstream targets were utilized to confirm the toxin's mode of action. We found that turtle neurons are highly resistant to PbTx-3; while cell viability decreased in a dose dependent manner across PbTx-3 concentrations of 100-2000nM, the EC

Topics: Animals; Calcium; Cell Survival; Cells, Cultured; Dizocilpine Maleate; Exocytosis; Female; Florida; Harmful Algal Bloom; Humans; Hypoxia; Marine Toxins; Neurons; Oxocins; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Tetrodotoxin; Turtles; Voltage-Gated Sodium Channels; Water Pollutants

Ranolazine (RAN), a novel antianginal agent, inhibits the increased late sodium current (INa.L) under many pathological conditions. In this study, the whole-cell patch-clamp technique was used to explore the effects of RAN on INa.L and reverse Na(+)/Ca(2+) exchange current (INCX) in rabbit ventricular myocytes during hypoxia.Tetrodotoxin (TTX) at 2 μM or RAN at 9 μM decreased significantly INa.L and reverse INCX under normoxia and RAN had no further effects on both currents in the presence of TTX. RAN (3, 6, and 9 μM) attenuated hypoxia-increased INa.L and reverse INCX in a concentration-dependent manner. Hypoxia-increased INa.L and reverse INCX were inhibited by 2 μM TTX, whereas 9 μM RAN applied sequentially did not further decrease both currents. In another group, after both currents were decreased by 9 μM RAN, 2 μM TTX had no further effects in the presence of Ran. In monophasic action potential (MAP) recording, early after-depolarizations (EADs) were suppressed by RAN (9 μM) during hypoxia. In conclusion, RAN decreased reverse INCX by inhibiting INa.L in normoxia, concentration-dependently attenuated the increase of INa.L, which thereby decreased the reverse INCX, and obviously relieved EADs during hypoxia.

Topics: Acetanilides; Action Potentials; Animals; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Heart Ventricles; Hypoxia; Myocytes, Cardiac; Patch-Clamp Techniques; Piperazines; Rabbits; Ranolazine; Sodium; Sodium-Calcium Exchanger; Tetrodotoxin

In response to low ambient oxygen levels the western painted turtle brain undergoes a large depression in metabolic rate which includes a decrease in neuronal action potential frequency. This involves the arrest of N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) currents and paradoxically an increase in γ-aminobutyric acid receptor (GABAR) currents in turtle cortical neurons. In a search for other oxygen-sensitive channels we discovered a Ca(2+)-activated K(+) channel (K(Ca)) that exhibited a decrease in open time in response to anoxia. Single-channel recordings of K(Ca) activity were obtained in cell-attached and excised inside-out patch configurations from neurons in cortical brain sheets bathed in either normoxic or anoxic artificial cerebrospinal fluid (aCSF). The channel has a slope conductance of 223pS, is activated in response to membrane depolarization, and is controlled in a reversible manner by free [Ca(2+)] at the intracellular membrane surface. In the excised patch configuration anoxia had no effect on K(Ca) channel open probability (P(open)); however, in cell-attached mode, there was a reversible fivefold reduction in P(open) (from 0.5 ± 0.05 to 0.1 ± 0.03) in response to 30-min anoxia. The inclusion of the potent protein kinase C (PKC) inhibitor chelerythrine prevented the anoxia-mediated decrease in P(open) while drip application of a phorbol ester PKC activator decreased P(open) during normoxia (from normoxic 0.4 ± 0.05 to phorbol-12-myristate-13-acetate (PMA) 0.1 ± 0.02). Anoxia results in a slight depolarization of turtle pyramidal neurons (∼8 mV) and an increase in cytosolic [Ca(2+)]; therefore, K(Ca) arrest is likely important to prevent Ca(2+) activation during anoxia and to reduce the energetic cost of maintaining ion gradients. We conclude that turtle pyramidal cell Ca(2+)-activated K(+) channels are oxygen-sensitive channels regulated by cytosolic factors and are likely the reptilian analog of the mammalian large conductance Ca(2+)-activated K(+) channels (BK channels).

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Biophysics; Calcium; Cerebral Cortex; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Female; Hypoxia; In Vitro Techniques; Ion Channel Gating; Male; Membrane Potentials; Oxygen; Patch-Clamp Techniques; Phorbol Esters; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Probability; Pyramidal Cells; Sodium Channel Blockers; Tetraethylammonium; Tetrodotoxin; Turtles; Valine

We attempted to measure the regional metabolic rate of glucose (MRglc) in sliced spinal cords in vitro. The thoracic spinal cord of a mature Wister rat was cut into 400-mum slices in oxygenated and cooled (1-4 degrees C) Krebs-Ringer solution. After at least 60 min of preincubation, the spinal cord slices were transferred into double polystyrene chambers and incubated in Krebs-Ringer solution at 36 degrees C, bubbled with 5% O(2)/5% CO(2) gas. To measure MRglc, we used the dynamic positron autoradiography technique (dPAT) with F-18-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG) and the net influx constant of [(18)F]FDG as an index. Uptake curves of [(18)F]FDG were well fitted by straight lines for more than 7 h after the slicing of the spinal cord (linear regression coefficient, r=0.99), indicating a constant uptake of glucose by the spinal cord tissue. The slope (K), which denotes MRglc, is affected by tetrodotoxin, and high K(+) (50 mM) or Ca(2+)-free, high Mg(2+) solution. After 10 min of hypoxia, the K value following reoxygenation was similar to the unloaded control value, but after 45 min of hypoxia, the K value was markedly lower than the unloaded control value, and after >90 min of reoxygenation it was nearly 0. Our results indicate that the living spinal cord slices used retained an activity-dependent metabolism to some extent. This technique may provide a new approach for measuring MRglc in sliced living spinal cord tissue in vitro and for quantifying the dynamic changes in MRglc in response to various interventions such as hypoxia.

Topics: Animals; Autoradiography; Brain; Fluorodeoxyglucose F18; Glucose; Hypoxia; Male; Radiopharmaceuticals; Rats; Rats, Wistar; Spinal Cord; Tetrodotoxin

The aim of the present study was to explore whether endogenous activation of different purine receptors by ATP and adenosine contributes to or inhibits excess glutamate release evoked by ischemic-like conditions in rat hippocampal slices. Combined oxygen-glucose deprivation (OGD) elicited a substantial, [Ca(2+)](o)-independent release of [(3)H]glutamate, which was tetrodotoxin (1 microM)-sensitive and temperature-dependent. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 0.1-10 microM), and the selective P2X(7) receptor antagonist Brilliant Blue G (1-100 nM), decreased OGD-evoked [(3)H]glutamate efflux indicating that endogenous ATP facilitates ischemia-evoked glutamate release. The selective A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1-250 nM) and the selective A(2A) receptor antagonists 4-(2-[7-amino-2-)2-furyl(triazolo-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 0.1-20 nM) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, 2-100 nM) decreased OGD-evoked [(3)H]glutamate efflux, indicating that endogenous adenosine also facilitates glutamate release under these conditions. The effect of DPCPX and ZM241385 was reversed, whereas the action of P2 receptor antagonists was potentiated by the selective ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL67156, 50 microM). The binding characteristic of the A(2A) ligand [(3)H]CGS21680 to hippocampal membranes did not change significantly in response to OGD. Taken together these data suggest that while A(1) receptors might became desensitized, A(2A) and P2X receptor-mediated facilitation of glutamate release by endogenous ATP and its breakdown product adenosine remains operational under long-term OGD. Therefore the inhibition of P2X/A(2A) receptors rather than the stimulation of A(1) adenosine receptors could be an effective approach to attenuate glutamatergic excitotoxicity and thereby counteract ischemia-induced neurodegeneration.

Topics: Adenosine; Adenosine Triphosphate; Analgesics; Animals; Dose-Response Relationship, Drug; Glucose; Glutamic Acid; Hippocampus; Hypoxia; In Vitro Techniques; Ischemia; Male; Phenethylamines; Purinergic Agonists; Purinergic Antagonists; Pyrimidines; Rats; Rats, Wistar; Receptors, Purinergic; Sodium Channel Blockers; Tetrodotoxin; Triazines; Triazoles; Xanthines

Physiological activity-dependent long-term changes in synaptic transmission, as long-term potentiation (LTP) are thought to be the substrate of learning and memory. However, a form of postsynaptic pathological LTP at the CA3-CA1 synapses has been demonstrated following few minutes of anoxia and aglycemia in vitro. The ischemia LTP shared many molecular mechanisms with the physiological LTP, and was believed to be involved in the delayed neuronal death following ischemia. However, the role of the presynaptic component in this regard is not known. Here we show that a short period of oxygen-glucose deprivation can induce a form of LTP (lasting for hours) of the presynaptic response at the CA3-CA1 synapses. This form of LTP is independent of postsynaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, but Ca(2+) dependent. This presynaptic LTP may represent a presynaptic hyperexcitability of the afferent fibers following ischemia, and responsible for the excitotoxicity to the CA1 neurons (ischemia-induced increases of glutamate release that kills neurons) and the postsynaptic pathological ischemic LTP.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Glucose; Hippocampus; Hypoxia; In Vitro Techniques; Long-Term Potentiation; Phosphorus Compounds; Rats; Rats, Sprague-Dawley; Synapses; Tetrodotoxin; Time Factors

Na(+)-K(+)-ATPase pump failure during either anoxia or ouabain perfusion induces rapid axonal depolarization by dissipating ionic gradients. In this study, we examined the interplay between cation and anion transporting pathways mediating axonal depolarization during anoxia or selective Na(+)-K(+)-ATPase inhibition. Compound resting membrane (V(m)) potential of rat optic nerve was measured in a grease gap at 37 degrees C. Chemical anoxia (2 mM NaCN or NaN(3)) or ouabain (1 mM) caused a loss of resting potential to 42 +/- 11% and 47 +/- 2% of control after 30 min, respectively. Voltage-gated Na(+)-channel blockade was partially effective in abolishing this depolarization. TTX (1 microM) reduced depolarization to 73 +/- 10% (chemical anoxia) and 68 +/- 4% (ouabain) of control. Quaternary amine Na(+) channel blockers QX-314 (1 mM) or prajmaline (100 microM) produced similar results. Residual ionic rundown largely representing co-efflux of K(+) and Cl(-) during chemical anoxia in the presence of Na(+)-channel blockade was further spared with DIDS (500 microM), a broad-spectrum anion transport inhibitor (95 +/- 8% of control after 30 min in anoxia + TTX vs. 73 +/- 10% in TTX alone). Addition of DIDS was slightly more effective than TTX alone in ouabain (74 +/- 5% DIDS + TTX vs. 68 +/- 4% in TTX alone, P < 0.05). Additional Na(+)-entry pathways such as the Na-K-Cl cotransporter were examined using bumetanide, which produced a modest albeit significant sparing of V(m) during ouabain-induced depolarization. Although cation-transporting pathways play the more important role in mediating pathological depolarization of central axons, anion-coupled transporters also contribute to a significant, albeit more minor, degree.

Topics: Animals; Bumetanide; Chlorides; Diuretics; Enzyme Inhibitors; Glycolysis; Hypoxia; Iodoacetates; Membrane Potentials; Optic Nerve; Ouabain; Rats; Rats, Long-Evans; Sodium; Sodium Channel Blockers; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin

In this study we use nystatin perforated-patch and conventional whole-cell recording to characterize the biophysical properties of neuronal nitric oxide synthase (nNOS)-expressing paraganglion neurons from the rat glossopharyngeal nerve (GPN), that are thought to provide NO-mediated efferent inhibition of carotid body chemoreceptors. These GPN neurons occur in two populations, a proximal one near the bifurcation of the GPN and the carotid sinus nerve, and a more distal one located further along the GPN. Both populations were visualized in whole mounts by vital staining with the styryl pyridinium dye, 4-Di-2-ASP (D289). Following isolation in vitro, proximal and distal neurons had similar input resistances (mean: 1.5 and 1.6 GOmega, respectively), input capacitances (mean: 25.0 and 27.4 pF, respectively), and resting potentials (mean: -53.9 and -53.3 mV, respectively). All neurons had similar voltage-dependent currents composed of: tetrodotoxin (TTX)-sensitive Na+ currents (IC50 approximately 0.2 microM), prolonged and transient Ca2+ currents, and delayed rectifier-type K+ currents. Threshold activation for the Na+ currents was approximately -30 mV and they were inactivated within 10 ms. Inward Ca2+ currents consisted of nifedipine-sensitive L-type, omega-agatoxin IVA-sensitive P/Q-type, omega-conotoxin GVIA-sensitive N-type, SNX-482-sensitive R-type, and Ni2+-sensitive, but SNX-482-insensitive, T-type channels. The voltage-dependent outward K+ currents were sensitive to tetraethylammonium (TEA; 10 mM) and 4-aminopyridine (4-AP; 2 mM). Exposure to a chemosensory stimulus, hypoxia (PO2 range: 80-5 Torr), caused a dose-dependent decrease in K+ current which persisted in the presence of TEA and 4-AP, consistent with the involvement of background K+ channels. Under current clamp, GPN neurons generated TTX-sensitive action potentials, and in spontaneously active neurons, hypoxia caused membrane depolarization and an increase in firing frequency. These properties endow GPN neurons with an exquisite ability to regulate carotid body chemoreceptor function during hypoxia, via voltage-gated Ca2+-entry, activation of nNOS, and release of NO.

Topics: 4-Aminopyridine; Action Potentials; Animals; Animals, Newborn; Cadmium; Calcium Channels; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Glossopharyngeal Nerve; Hypoxia; Immunohistochemistry; Ion Channels; Membrane Potentials; Neurons; Nickel; Oxygen; Patch-Clamp Techniques; Potassium Channel Blockers; Pyridinium Compounds; Rats; Rats, Wistar; Sodium Channel Blockers; Tetraethylammonium; Tetrodotoxin

Dopamine (DA), released from the lateral olivocochlear (LOC) efferent terminals, the efferent arm of the short-loop feedback in the cochlea, is considered as a protective factor in the inner ear since it inhibits auditory nerve dendrite firing in ischemia- or noise-induced excitotoxicity leading to sensorineural hearing loss (SNHL). In the present study we investigated the effect of oxygen-glucose deprivation (OGD), an in vitro ischemia model, on guinea-pig cochlear [(3)H]DA release in a microvolume superfusion system. We found that OGD alone failed to induce a detectable elevation of [(3)H]DA level, but in the presence of specific D(2) receptor antagonists, sulpiride and L-741,626, it evoked a significant increase in the extracellular concentration of [(3)H]DA. D(2) negative feedback receptors are involved not exclusively in the regulation of synthesis and vesicular release of DA, but also in the activation of its reuptake. Thus, D(2) receptor antagonism interferes with the powerful reuptake of DA from the extracellular space. To explore the underlying mechanism of this DA-releasing effect we applied nomifensine and found that the effect of OGD on cochlear DA release in the presence of D(2) antagonists could be inhibited by this selective DA uptake inhibitor. This finding indicates that the OGD-evoked DA release was mainly mediated through the reverse operation of the DA transporter. The two structurally different D(2) antagonists also augmented the electrical field stimulation-evoked release of DA proving the presence of D(2) autoreceptors on dopaminergic LOC terminals. Our results confirm the presence and role of D(2) DA autoreceptors in the regulation of DA release from LOC efferents, and suggest a protective local mechanism during ischemia which involves the direct transporter-mediated release of DA. Increasing the release of the protective transmitter DA locally in the inner ear may form the basis of future new therapeutic strategies in patients suffering from SNHL.

Topics: Animals; Cochlea; Dopamine; Dopamine Antagonists; Dopamine Uptake Inhibitors; Drug Interactions; Electric Stimulation; Glucose; Guinea Pigs; Hypoxia; In Vitro Techniques; Indoles; Male; Neurons; Nomifensine; Piperidines; Receptors, Dopamine D2; Sulpiride; Tetrodotoxin; Time Factors; Tritium

Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.

Topics: Amino Acids; Animals; Aspartic Acid; Brain; Calcium; Citrulline; Corpus Striatum; Female; gamma-Aminobutyric Acid; Glutamic Acid; Hypoxia; Ions; Ischemia; L-Lactate Dehydrogenase; Male; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; Taurine; Tetrodotoxin; Time Factors

The role of alpha-synuclein (alpha-Syn) has recently received considerable attention because it seems to play a role in Parkinson's disease (PD). Missense mutations in the alpha-Syn gene were found in autosomal dominant PD and alpha-Syn was shown to be a major constituent of protein aggregates in sporadic PD and other synucleinopathies. Under normal conditions, alpha-Syn protein is found exclusively in synaptic terminals. However, the potential participation of alpha-synuclein in maintaining and regulating synaptic efficacy is unknown. We have investigated the excitatory synaptic modulation of alpha-synuclein in CA1 pyramidal neurons, using the in vitro hippocampal slice technique. The 4-aminopyridine-induced increase of both spontaneous excitatory postsynaptic current (EPSC) frequency and amplitude was significantly higher in alpha-Syn wild-type than knockout mice, whereas basal spontaneous EPSC frequency and amplitude was similar in both animals. As the spontaneous synaptic activity was abolished by tetrodotoxin, which indicates that it was a result of action potential-mediated transmitter release from presynaptic terminals, spontaneous EPSC changes observed in alpha-Syn knockout mice suggest that these animals present a modification of synaptic transmission with a presynaptic origin. Presynaptic depression of evoked EPSCs by hypoxia or adenosine was significantly larger in alpha-Syn knockout than in wild-type mice, further supporting the hypothesis of regulation of synaptic transmission by alpha-Syn. Together, these observations indicate that the loss of alpha-Syn reduces synaptic efficacy when the probability of transmitter release is modified. We conclude that alpha-Syn might have important actions on the maintenance of the functional integrity of synaptic transmission and its regulation in hippocampus.

Topics: 4-Aminopyridine; Adenosine; alpha-Synuclein; Analgesics; Anesthetics, Local; Animals; Blotting, Western; Drug Interactions; Excitatory Postsynaptic Potentials; Hippocampus; Hypoxia; In Vitro Techniques; Mice; Mice, Knockout; Nerve Tissue Proteins; Neurons; Potassium Channel Blockers; Synaptic Transmission; Synucleins; Tetrodotoxin; Theophylline; Time Factors

Rundown of ionic gradients is a central feature of white matter anoxic injury; however, little is known about the contribution of anions such as Cl-. We used the in vitro rat optic nerve to study the role of aberrant Cl- transport in anoxia/ischemia. After 30 min of anoxia (NaN3, 2 mm), axonal membrane potential (V(m)) decreased to 42 +/- 11% of control and to 73 +/- 11% in the presence of tetrodotoxin (TTX) (1 microm). TTX + 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid disodium salt (500 microm), a broad spectrum anion transport blocker, abolished anoxic depolarization (95 +/- 8%). Inhibition of the K-Cl cotransporter (KCC) (furosemide 100 microm) together with TTX was also more effective than TTX alone (84 +/- 14%). The compound action potential (CAP) area recovered to 26 +/- 6% of control after 1 hr anoxia. KCC blockade (10 microm furosemide) improved outcome (40 +/- 4%), and TTX (100 nm) was even more effective (74 +/- 12%). In contrast, the Cl- channel blocker niflumic acid (50 microm) worsened injury (6 +/- 1%). Coapplication of TTX (100 nm) + furosemide (10 microm) was more effective than either agent alone (91 +/- 9%). Furosemide was also very effective at normalizing the shape of the CAPs. The KCC3a isoform was localized to astrocytes. KCC3 and weaker KCC3a was detected in myelin of larger axons. KCC2 was seen in oligodendrocytes and within axon cylinders. Cl- gradients contribute to resting optic nerve membrane potential, and transporter and channel-mediated Cl- fluxes during anoxia contribute to injury, possibly because of cellular volume changes and disruption of axo-glial integrity, leading to propagation failure and distortion of fiber conduction velocities.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Action Potentials; Animals; Astrocytes; Axons; Chloride Channels; Chlorides; Hypoxia; In Vitro Techniques; Ion Transport; K Cl- Cotransporters; Membrane Potentials; Myelin Sheath; Neural Conduction; Niflumic Acid; Oligodendroglia; Optic Neuropathy, Ischemic; Protein Isoforms; Rats; Rats, Long-Evans; Sodium Nitrite; Symporters; Tetrodotoxin

Substitution of extracellular Na+ with N-methyl D-glucamine caused marked hyperpolarisation in rat isolated carotid body type I cells, suggesting the presence of a standing Na+ conductance. Choline substitution produced smaller hyperpolarisations, whilst Li+ was virtually without effect. This Na+ conductance was not blocked by amiloride, tetrodotoxin, Zn2+ or Gd3+ and did not arise from electrogenic Na-glucose co-transport, since substitution of glucose with sucrose could not mimic the effects of Na+ substitution. Hypoxia and acidosis did not modify the tonic Na+ influx. Our results suggest that Na+ influx provides a constant depolarising influence on type I cells which acts to shift membrane potential beyond that required for initiation of neurosecretion, an essential step in carotid body chemotransduction.

Topics: Acid-Base Equilibrium; Acidosis, Respiratory; Amiloride; Animals; Carotid Body; Cells, Cultured; Choline; Gadolinium; Homeostasis; Hydrogen-Ion Concentration; Hypoxia; Lithium; Meglumine; Membrane Potentials; Oxygen; Patch-Clamp Techniques; Potassium Channels; Rats; Signal Transduction; Sodium; Sodium Channels; Tetrodotoxin; Zinc

Synaptic inhibition by GABA(A) and glycine receptors, which are ligand-gated anion channels, depends on the electrochemical potential for chloride. Several potassium-chloride cotransporters can lower the intracellular chloride concentration [Cl(-)](i), including the neuronal isoform KCC2. We show that KCC2 knockout mice died immediately after birth due to severe motor deficits that also abolished respiration. Sciatic nerve recordings revealed abnormal spontaneous electrical activity and altered spinal cord responses to peripheral electrical stimuli. In the spinal cord of wild-type animals, the KCC2 protein was found at inhibitory synapses. Patch-clamp measurements of embryonic day 18.5 spinal cord motoneurons demonstrated an excitatory GABA and glycine action in the absence, but not in the presence, of KCC2, revealing a crucial role of KCC2 for synaptic inhibition.

Topics: Action Potentials; Animals; Animals, Newborn; Carrier Proteins; Embryo, Mammalian; Embryonic and Fetal Development; gamma-Aminobutyric Acid; Gene Expression Regulation, Developmental; Glycine; Hypoxia; K Cl- Cotransporters; Mice; Mice, Knockout; Motor Neurons; Patch-Clamp Techniques; Potassium; Protein Isoforms; Sodium; Symporters; Synaptic Transmission; Tetrodotoxin

In the rhythmic brain stem slice preparation, spontaneous respiratory activity is generated endogenously and can be recorded as output activity from hypoglossal XII rootlets. Here we combine these recordings with measurements of the intrinsic optical signal (IOS) of cells in the regions of the periambigual region and nucleus hypoglossus of the rhythmic slice preparation. The IOS, which reflects changes of infrared light transmittance and scattering, has been previously employed as an indirect sensor for activity-related changes in cell metabolism. The IOS is believed to be primarily caused by cell volume changes, but it has also been associated with other morphological changes such as dendritic beading during prolonged neuronal excitation or mitochondrial swelling. An increase of the extracellular K(+) concentration from 3 to 9 mM, as well as superfusion with hypotonic solution induced a marked increase of the IOS, whereas a decrease in extracellular K(+) or superfusion with hypertonic solution had the opposite effect. During tissue anoxia, elicited by superfusion of N(2)-gassed solution, the biphasic response of the respiratory activity was accompanied by a continuous rise in the IOS. On reoxygenation, the IOS returned to control levels. Cells located at the surface of the slice were observed to swell during periods of anoxia. The region of the nucleus hypoglossus exhibited faster and larger IOS changes than the periambigual region, which presumably reflects differences in sensitivities of these neurons to metabolic stress. To analyze the components of the hypoxic IOS response, we investigated the IOS after application of neurotransmitters known to be released in increasing amounts during hypoxia. Indeed, glutamate application induced an IOS increase, whereas adenosine slightly reduced the IOS. The IOS response to hypoxia was diminished after application of glutamate uptake blockers, indicating that glutamate contributes to the hypoxic IOS. Blockade of the Na(+)/K(+)-ATPase by ouabain did not provoke a hypoxia-like IOS change. The influences of K(ATP) channels were analyzed, because they contribute significantly to the modulation of neuronal excitability during hypoxia. IOS responses obtained during manipulation of K(ATP) channel activity could be explained only by implicating mitochondrial volume changes mediated by mitochondrial K(ATP) channels. In conclusion, the hypoxic IOS response can be interpreted as a result of cell and mitochondrial swelling. C

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine; Adenosine Triphosphate; Animals; Animals, Newborn; Anti-Bacterial Agents; Antihypertensive Agents; Diazoxide; Energy Metabolism; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glyburide; Hypoglossal Nerve; Hypoglycemic Agents; Hypoxia; Kainic Acid; Macrolides; Mice; Mitochondrial Swelling; N-Methylaspartate; Optics and Photonics; Organ Culture Techniques; Ouabain; Potassium Channels; Respiratory Center; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin

We studied the effects of lidocaine and tetrodotoxin (TTX) on hypoxic changes in CA1 pyramidal neurons to examine the ionic basis of neuronal damage. Lidocaine (10 and 100 microM) and TTX (6 and 63 nM) delayed and attenuated the hypoxic depolarization and improved recovery of the resting and action potentials after 10 min of hypoxia. Lidocaine (10 and 100 microM) and TTX (63 nM) reduced the number of morphologically damaged CA1 cells and improved protein synthesis measured after 10 min hypoxia. Lidocaine (10 microM) attenuated the increase in intracellular sodium (181 vs. 218%) and the depolarization (-21 vs. -1 mV) during hypoxia but did not significantly attenuate the changes in ATP, potassium, or calcium measured at 10 min of hypoxia. Lidocaine (100 microM) attenuated the changes in membrane potential, sodium, potassium, ATP, and calcium during hypoxia. TTX (63 nM) attenuated the changes in membrane potential (-36 vs. -1 mV), sodium (179 vs. 226%), potassium (78 vs. 50%), and ATP (24 vs. 11%) but did not significantly attenuate the increase in calcium during hypoxia. These data indicate that the primary blockade of sodium channels can secondarily alter other cellular parameters. The hypoxic depolarization and the increase in intracellular sodium appear to be important triggers of hypoxic damage independent of their effect on cytosolic calcium; a treatment that selectively blocked sodium influx (lidocaine 10 microM) improved recovery. Our data indicate that selective blockade of sodium channels with a low concentration of lidocaine or TTX improves recovery after hypoxia by attenuating the rise in cellular sodium and the hypoxic depolarization. This blockade improves the resting and action potentials, histologic state, and protein synthesis of CA1 pyramidal neurons after 10 min of hypoxia to rat hippocampal slices. A higher concentration of lidocaine, which also improved ATP, potassium, and calcium concentrations during hypoxia was more potent. In conclusion, the depolarization and increased sodium concentration during hypoxia account for a portion of the neuronal damage after hypoxia independent of changes in calcium.

Topics: Adenosine Triphosphate; Anesthetics, Local; Animals; Calcium; Cytosol; Electrophysiology; Hippocampus; Hypoxia; Lidocaine; Male; Membrane Potentials; Nerve Tissue Proteins; Patch-Clamp Techniques; Potassium; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Tetrodotoxin

In order to better understand the post-natal increase in peripheral chemoreceptor responsiveness to hypoxia, chemoreceptors of newborn (1-2 days) and older (10-12 days, 30 days, adult) rabbits were isolated and superfused, in vitro. The free tissue catecholamine concentration was measured using carbon-fiber voltammetry and pauci-fiber nerve activity was recorded from the sinus nerve during stimulation (4 min) with graded hypoxia or increased potassium. Both the peak catecholamine and peak nerve responses to stimulation with 10% and 0% oxygen increased with age, particularly between 10 and 30 days of age. In contrast, peak nerve and peak catecholamine responses to increased potassium did not significantly change with age. For a better understanding of how responsiveness increases with age, the fast Na+ and the Ca2+ currents were measured from isolated glomus cells of newborn and older rabbits, but the magnitude of the currents when normalized to membrane area was not significantly different between ages. We conclude that: (1) rabbit chemoreceptors mature in the newborn period (10-30 days) and part of this maturation is an increase in catecholamine secretion, (2) maturation of hypoxia transduction primarily occurs in steps prior to depolarization since potassium-evoked responses were not affected, and (3) an increase in the magnitude of glomus cell fast Na+ or Ca2+ currents is not a likely mechanism for the maturational change, but changes in the oxygen sensitivity of these currents cannot be excluded.

Topics: Age Factors; Animals; Calcium; Calcium Channels; Carotid Body; Chemoreceptor Cells; Dopamine; Electrophysiology; Hypoxia; Membrane Potentials; Oxygen; Peripheral Nerves; Potassium; Rabbits; Signal Transduction; Sodium; Sodium Channels; Tetrodotoxin

We have previously observed that prolonged O(2) deprivation alters membrane protein expression and membrane properties in the central nervous system. In this work, we studied the effect of prolonged O(2) deprivation on the electrical activity of rat cortical and hippocampal neurons during postnatal development and its relationship to Na(+) channels. Rats were raised in low O(2) environment (inspired O(2) concentration = 9.5+/-0.5%) for 3-4 weeks, starting at an early age (2-3 days old). Using electrophysiologic recordings in brain slices, RNA analysis (northern and slot blots) and saxitoxin (a specific ligand for Na(+) channels) binding autoradiography, we addressed two questions: (1) does long-term O(2) deprivation alter neuronal excitability in the neocortical and hippocampal neurons during postnatal development? and (2) if so, what are the main mechanisms responsible for the change in excitability in the exposed brain? Our results show that (i) baseline membrane properties of cortical and hippocampal CA1 neurons from rats chronically exposed to hypoxia were not substantially different from those of naive neurons; (ii) acute stress (e.g., hypoxia) elicited a markedly exaggerated response in the exposed neurons as compared to naive ones; (iii) chronic hypoxia tended to increase Na(+) channel mRNA and saxitoxin binding density in the cortex and hippocampus as compared to control ones; and (iv) the enhanced neuronal response to acute hypoxia in the exposed cortical and CA1 neurons was considerably attenuated by applying tetrodotoxin, a voltage-sensitive Na(+) channel blocker, in a dose-dependent manner. We conclude that prolonged O(2) deprivation can lead to major electrophysiological disturbances, especially when exposed neurons are stressed acutely, which renders the chronically exposed neurons more vulnerable to subsequent micro-environmental stress. We suggest that this Na(+) channel-related over-excitability is likely to constitute a molecular mechanism for some neurological sequelae, such as epilepsy, resulting from perinatal hypoxic encephalopathy.

Topics: Animals; Animals, Newborn; Brain; Cell Membrane; Cerebral Cortex; Hippocampus; Hypoxia; In Vitro Techniques; Neocortex; Neurons; Rats; Rats, Sprague-Dawley; Saxitoxin; Sodium Channels; Tetrodotoxin; Transcription, Genetic

Calpains are ubiquitous Ca(2+)-activated neutral proteases that have been implicated in ischemic and traumatic CNS injury. Ischemia and trauma of central white matter are dependent on Ca2+ accumulation, and calpain overactivation likely plays a significant role in the pathogenesis. Adult rat optic nerves, representative central white matter tracts, were studied in an in vitro anoxic model. Functional recovery following 60 min of anoxia and reoxygenation was measured electrophysiologically. Calpain activation was assessed using western blots with antibodies against calpain-cleaved spectrin breakdown products. Sixty minutes of in vitro anoxia increased the amount of spectrin breakdown approximately 20-fold over control, with a further increase after reoxygenation to >70 times control, almost as much as 2 h of continuous anoxia. Blocking voltage-gated Na+ channels with tetrodotoxin or removing bath Ca2+ was highly neuroprotective electrophysiologically and resulted in a marked reduction of spectrin degradation. The membrane-permeable calpain inhibitors MDL 28,170 and calpain inhibitor-I (10-100 microM) were effective at reducing spectrin breakdown in anoxic and reoxygenated optic nerves, but no electrophysiological improvement was observed. We conclude that calpain activation is an important step in anoxic white matter injury, but inhibition of this Ca(2+)-dependent process in isolation does not improve functional outcome, probably because other deleterious Ca(2+)-activated pathways proceed unchecked.

Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Electrophysiology; Glycoproteins; Hypoxia; Ion Channel Gating; Male; Optic Nerve; Oxygen; Rats; Rats, Long-Evans; Sodium Channel Blockers; Sodium Channels; Spectrin; Tetrodotoxin; Time Factors

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na(+) and Ca(2+), which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca(2+) concentration [Ca(2+)](i), whereas the elevation of intracellular Na(+) has received less attention. In the present study we investigated the effects of inhibitors of different Na(+) channels and of the Na(+)/Ca(2+) exchanger, which couples the Na(+) to the Ca(2+) gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na(+) channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca(2+) accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca(2+) extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca(2+)](i) increase caused by the exchanger.

Topics: Animals; Brain Ischemia; Cell Death; Culture Techniques; Dizocilpine Maleate; Electrophysiology; Hippocampus; Homeostasis; Hypoglycemia; Hypoxia; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium; Sodium Channel Blockers; Sodium Channels; Sodium-Calcium Exchanger; Tetrodotoxin; Thiourea

Hypoxia initiates the neurosecretory response of the carotid body (CB) by inhibiting one or more potassium channels in the chemoreceptor cells. Oxygen-sensitive K(+) channels were first described in rabbit CB chemoreceptor cells, in which a transient outward K(+) current was reported to be reversibly inhibited by hypoxia. Although progress has been made to characterize this current with electrophysiological and pharmacological tools, no attempts have been made to identify which Kv channel proteins are expressed in rabbit CB chemoreceptor cells and to determine their contribution to the native O(2)-sensitive K(+) current. To probe the molecular identity of this current, we have used dominant-negative constructs to block the expression of functional Kv channels of the Shaker (Kv1.xDN) or the Shal (Kv4.xDN) subfamilies, because members of these two subfamilies contribute to the transient outward K(+) currents in other preparations. Delivery of the constructs into chemoreceptor cells has been achieved with adenoviruses that enabled ecdysone-inducible expression of the dominant-negative constructs and reporter genes in polycistronic vectors. In voltage-clamp experiments, we found that, whereas adenoviral infections of chemoreceptor cells with Kv1.xDN did not modify the O(2)-sensitive K(+) current, infections with Kv4.xDN suppressed the transient outward current in a time-dependent manner, significantly depolarized the cells, and abolished the depolarization induced by hypoxia. Our work demonstrate that genes of the Shal K(+) channels underlie the transient outward, O(2)-sensitive, K(+) current of rabbit CB chemoreceptor cells and that this current contributes to the cell depolarization in response to low pO(2).

Topics: Adenoviridae; Animals; Carotid Body; Chemoreceptor Cells; CHO Cells; Cricetinae; Electrophysiology; Gene Expression; Gene Transfer Techniques; Genes, Dominant; Humans; Hypoxia; Kidney; Membrane Potentials; Mutagenesis; Oxygen; Potassium; Potassium Channels; Potassium Channels, Voltage-Gated; Rabbits; Shaker Superfamily of Potassium Channels; Shal Potassium Channels; Tetrodotoxin; Transfection

There is ample evidence that ischaemia is associated with partial denervation of the detrusor muscle and that this is responsible for much of its abnormal contractile behaviour, resulting in bladder dysfunction (instability). In guinea-pig nerves are very susceptible to the ischaemic damage as compared to the muscle cells. The purpose of this study was to assess the neuroprotection afforded by taurine on guinea-pig detrusor under ischaemic-like conditions. Guinea-pig detrusor strips were subjected for 60 min to ischaemic-like conditions, followed by 150 min reperfusion. Intrinsic nerves underwent every 30 min electrical field stimulation (EFS) by 5-s trains of square voltage pulses of 0.05 ms duration (15 Hz, 50 V). Detrusor strips were perfused with 0.1, 1, 3 or 10 mM taurine during the ischaemia-like exposure and the first 30 min of reperfusion. Taurine (1 and 3 mM) significantly improved the response of the strips to EFS both at the end of ischaemia and reperfusion. On the contrary, neither 0.1 nor 10 mM taurine had significant effects. It is concluded that taurine can partially counteract the ischaemia-reperfusion injury in the guinea-pig urinary bladder.

Topics: Aminoethylphosphonic Acid; Animals; Atropine; Electric Stimulation; Evoked Potentials; Glucose; Guinea Pigs; Hypoxia; Ischemia; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neuroprotective Agents; Oxygen; Purinergic P2 Receptor Antagonists; Reperfusion Injury; Suramin; Taurine; Tetrodotoxin; Urinary Bladder

Hypoxia can dramatically disrupt neural processing because energy-dependent homeostatic mechanisms are necessary to support normal neuronal function. In a human context, the long-term effects of such disruption may become all too apparent after a "stroke," in which blood-flow to part of the brain is compromised. We used an insect preparation to investigate the effects of hypoxia on neuron membrane properties. The preparation is particularly suitable for such studies because insects respond rapidly to hypoxia, but can recover when they are restored to normoxic conditions, whereas many of their neurons are large, identifiable, and robust. Experiments were performed on the "fast" coxal depressor motoneuron (Df) of cockroach (Periplaneta americana). Five-minute periods of hypoxia caused reversible multiphasic depolarizations (10-25 mV; n = 88), consisting of an initial transient depolarization followed by a partial repolarization and then a slower phase of further depolarization. During the initial depolarizing phase, spontaneous plateau potentials normally occurred, and inhibitory postsynaptic potential frequency increased considerably; 2-3 min after the onset of hypoxia all electrical activity ceased and membrane resistance was depressed. On reoxygenation, the membrane potential began to repolarize almost immediately, becoming briefly more negative than the normal resting potential. All phases of the hypoxia response declined with repeated periods of hypoxia. Blockade of ATP-dependent Na/K pump by 30 microM ouabain suppressed only the initial transient depolarization and the reoxygenation-induced hyperpolarization. Reduction of aerobic metabolism between hypoxic periods (produced by bubbling air through the chamber instead of oxygen) had a similar effect to that of ouabain. Although the depolarization seen during hypoxia was not reduced by tetrodotoxin (TTX; 2 microM), lowering extracellular Na+ concentration or addition of 500 microM Cd2+ greatly reduced all phases of the hypoxia-induced response, suggesting that Na influx occurs through a TTX-insensitive Cd2+-sensitive channel. Exposure to 20 mM tetraethylammonium and 1 mM 3,4-diaminopyridine increased the amplitude of the hypoxia-induced depolarization, suggesting that activation of K channels may normally limit the amplitude of the hypoxia response. In conclusion we suggest that the slow hypoxia-induced depolarization on motoneuron Df is mainly carried by a TTX-resistant, Cd2+-sensitive sodium influx. C

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channel Blockers; Electrophysiology; Enzyme Inhibitors; Ganglia, Invertebrate; Hypoxia; In Vitro Techniques; Ion Channels; Male; Membrane Potentials; Motor Neurons; Ouabain; Patch-Clamp Techniques; Periplaneta; Potassium Channel Blockers; Potassium Channels; Sodium; Tetrodotoxin

In an in vitro preparation of the intact carotid body (CB) of the rabbit, adenosine (100 microM) inhibited hypoxia-induced catecholamine release by 25%. The specific A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 microM) prevented the inhibition and increased the response to hypoxia further. In isolated chemoreceptor cells from the same species, adenosine inhibited voltage-dependent Ca2+ currents by 29% at 1 microM (concentration producing half-maximal inhibition, IC50 = 50 nM). This inhibition was mimicked by R(-)N6-(2-phenylisopropyl)-adenosine and 2-chloroadenosine (1 microM), two purinergic agonists poorly active at the intracellular ('P') site, and persisted in the presence of dipyridamole (a blocker of adenosine uptake; 1 microM) and was fully inhibited by 8-phenyltheophylline (10 microM). The A1 antagonists DPCPX (10 microM) and 8-cyclopentyl-1,3-dimethylxantine (0.1 microM) inhibited the effect of adenosine by 93% (IC50 = 0.14 microM) and 59%, respectively. The inhibition of the Ca2+ current (I(Ca)) was reduced by nisoldipine (an L-type Ca2+ channel antagonist) by nearly 50%, and was unaltered by omega-conotoxin GVIA, a blocker of N-type Ca2+ channels. Adenosine did not affect the voltage-dependent Na+ current (I(Na)) or K+ current (I(K)). We conclude that adenosine A1 receptors are located in chemoreceptor cells and mediate the inhibition of L-type Ca2+ channels and thereby the release of catecholamines produced by hypoxia. The data also indicate that endogenous adenosine acts as a physiological negative modulator of the chemoreceptor cell function. The previously reported excitatory action of adenosine on the activity of the sensory nerve of the CB is discussed in terms of a balance between the inhibition mediated by A1 receptors and the excitation mediated by A2 receptors.

Topics: Adenosine; Adrenergic alpha-Antagonists; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Carotid Body; Catecholamines; Chemoreceptor Cells; Dose-Response Relationship, Drug; Electrophysiology; Hypoxia; Ion Channel Gating; Membrane Potentials; Neuroprotective Agents; omega-Conotoxin GVIA; Peptides; Potassium; Rabbits; Sodium; Tetrodotoxin; Theophylline; Tritium; Xanthines

Previous reports from this laboratory have shown that a high percentage of neurons in the caudal hypothalamus are stimulated by hypoxia both in vivo and in vitro. This stimulation is in the form of an increase in firing frequency and significant membrane depolarization. The goal of the present study was to determine if this hypoxia-induced excitation is influenced by development. In addition, we sought to determine the mechanism by which hypoxia stimulates caudal hypothalamic neurons. Caudal hypothalamic neurons from neonatal (4-16 days) or juvenile (20-40 days) rats were patch-clamped, and the whole cell voltage and current responses to moderate (10% O2) or severe (0% O2) hypoxia were recorded in the brain slice preparation. Analysis of tissue oxygen levels demonstrated no significant difference in the levels of tissue oxygen in brain slices between the different age groups. A significantly larger input resistance, time constant and half-time to spike height was observed for neonatal neurons compared with juvenile neurons. Both moderate and severe hypoxia elicited a net inward current in a significantly larger percentage of caudal hypothalamic neurons from rats aged 20-40 days (juvenile) as compared with rats aged 4-16 days (neonatal). In contrast, there was no difference in the magnitude of the inward current response to moderate or severe hypoxia between the two age groups. Those cells that were stimulated by hypoxia demonstrated a significant decrease in input resistance during hypoxic stimulation that was not observed in those cells unaffected by hypoxia. A subset of neurons were tested independent of age for the ability to maintain the inward current response to hypoxia during synaptic blockade (11.4 mM Mg2+/0. 2 mM Ca2+). Most of the neurons tested (88.9%) maintained a hypoxic excitation during synaptic blockade, and this inward current response was unaffected by addition of 2 mM cobalt chloride to the bathing medium. In contrast, perfusion with the Na+ channel blocker, tetrodotoxin (1-2 microM) or Na+ replacement with N-methyl-D-glucamine (NMDG) significantly reduced the inward current response to hypoxia. Furthermore, the input resistance decrease observed during hypoxia was attenuated significantly during perfusion with NMDG. These results indicate the excitation elicited by hypoxia in hypothalamic neurons is age dependent. In addition, the inward current response of caudal hypothalamic neurons is not dependent on synaptic input but results from

Topics: Age Factors; Animals; Calcium; Cell Size; Cobalt; Fluorescent Dyes; Hypothalamus; Hypoxia; Hypoxia, Brain; Isoquinolines; Membrane Potentials; Neurons; Oxygen; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Tetrodotoxin

Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation. The effects of combined O2/glucose deprivation were investigated on large aspiny (LA) interneurons recorded from a striatal slice preparation by means of simultaneous electrophysiological and optical recordings. LA interneurons were visually identified and impaled with sharp microelectrodes loaded with the calcium (Ca2+)-sensitive dye bis-fura-2. These cells showed the morphological, electrophysiological, and pharmacological features of large striatal cholinergic interneurons. O2/glucose deprivation induced a membrane hyperpolarization coupled to a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, this [Ca2+]i elevation was more pronounced in dendritic branches rather than in the somatic region. The O2/glucose-deprivation-induced membrane hyperpolarization reversed its polarity at the potassium (K+) equilibrium potential. Both membrane hyperpolarization and [Ca2+]i rise were unaffected by TTX or by a combination of ionotropic glutamate receptors antagonists, D-2-amino-5-phosphonovaleric acid and 6cyano-7-nitroquinoxaline-2, 3-dione. Sulfonylurea glibenclamide, a blocker of ATP-sensitive K+ channels, markedly reduced the O2/glucose-deprivation-induced membrane hyperpolarization but failed to prevent the rise in [Ca2+]i. Likewise, charybdotoxin, a large K+-channel (BK) inhibitor, abolished the membrane hyperpolarization but did not produce detectable changes of [Ca2+]i elevation. A combination of high-voltage-activated Ca2+ channel blockers significantly reduced both the membrane hyperpolarization and the rise in [Ca2+]i. In a set of experiments performed without dye in the recording electrode, either intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid or external barium abolished the membrane hyperpolarization induced by O2/glucose deprivation. The hyperpolarizing effect on membrane potential was mimicked by oxotremorine, an M2-like muscarinic receptor agonist, and by baclofen, a GABAB receptor agonist. However, this membrane hyperpolarization was not coupled to an increase but rather to a decrease of the basal [Ca2+]i. Furthermore glibenclamide did not reduce the oxotremorine- and baclofen-induced membrane hyperpolarization. In conclusion, the present results suggest that in striatal LA cells, O2/glucose deprivation activates a membrane hyperpolarization that does not invo

Topics: Animals; Calcium; Calcium Channels; Chelating Agents; Corpus Striatum; Egtazic Acid; Electrophysiology; Excitatory Amino Acid Antagonists; Glucose; Hypoxia; Interneurons; Male; Potassium Channel Blockers; Rats; Rats, Wistar; Tetrodotoxin

Studies in slices suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by D(-)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-D-aspartate (NMDA) and GABAA (gamma-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Aging; Animals; Animals, Newborn; Benzodiazepines; Bicuculline; Cations, Divalent; Electric Stimulation; Embryonic and Fetal Development; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Hippocampus; Hypoxia; Lysine; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Synapses; Tetrodotoxin

Fresh rat brain slices were incubated with 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, and serial two-dimensional time-resolved images of [18F]FDG uptake were obtained from these specimens on imaging plates. The fractional rate constant (= k3*) of [18F]FDG proportional to the cerebral glucose metabolic rate (CMRglc) was evaluated by applying the Gjedde-Patlak graphical method to the image data. With hypoxia loading (oxygen deprivation) or glucose metabolism inhibitors acting on oxidative phosphorylation, the k3* value increased dramatically suggesting enhanced glycolysis. After relieving hypoxia < or = 10-min, the k3* value returned to the pre-loading level. In contrast, with > or = 20-min hypoxia only partial or no recovery was observed, indicating that irreversible neuronal damage had been induced. However, after loading with tetrodotoxin (TTX), the k3* value also decreased but returned to the pre-loading level even after 70-min TTX-loading, reflecting a transient inhibition of neuronal activity. This technique provides a new means of quantifying dynamic changes in the regional CMRglc in living brain slices in response to various interventions such as hypoxia and neurotoxic chemical-loading as well as determining the viability and prognosis of brain tissues.

Topics: Animals; Autoradiography; Brain; Culture Media; Electrons; Fluorodeoxyglucose F18; Glucose; Hypoxia; In Vitro Techniques; Lactates; Male; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Tetrodotoxin

A 10 min exposure of rat hippocampal slices to hypoxic/hypoglycemic medium decreased tissue adenosine 5'-triphosphate (ATP) levels. Hypoxia/hypoglycemia also caused an anoxic depolarization and essentially no recovery of the synaptically evoked population spike from CA1 region recorded 30 min after re-introduction of normoxic/normoglycemic medium. Removal of Ca2+ or the addition of either the non-competitive N-methyl-D-aspartate antagonist dizocilpine maleate, the inorganic Ca2+ channel antagonist Co2+; or the Na+ channel blocker tetrodotoxin to hypoxic/hypoglycemic medium improved recovery of the evoked population spike upon re-oxygenation. Dizocilpine maleate, Co2+, and tetrodotoxin spared ATP during exposure to hypoxia/hypoglycemia. In contrast, Ca(2+)-free medium facilitated recovery of the population spike but did not preserve ATP during hypoxia/hypoglycemia. Dizocilpine maleate, Co2+ or dantrolene, when added to Ca(2+)-free medium, did not preserve ATP. Tetrodotoxin, when added to Ca(2+)-free medium, was effective in sparing ATP in hypoxic/hypoglycemic medium. To determine the effect of anoxic depolarization on ATP levels, hippocampal slices were collected just before and after the depolarization. There appeared to be an abrupt drop in ATP associated with the anoxic depolarization. We conclude that Na+ influx plays a relatively larger role in ATP consumption during hypoxia/hypoglycemia than Ca2+ influx. In addition, the anoxic depolarization imposes a large and rapid drop in ATP levels.

Topics: Adenosine Triphosphate; Animals; Electrophysiology; Hippocampus; Hypoglycemia; Hypoxia; In Vitro Techniques; Male; Rats; Rats, Sprague-Dawley; Sodium; Tetrodotoxin

The principal finding of the present study with rat spinal cord slices was the novel demonstration of the [Ca2+]o-independent effect of ischemia on norepinephrine release and its antagonism by tetrodotoxin and low temperature (10 degrees C). Our finding that tetrodotoxin antagonized the effects of glucose deprivation on norepinephrine release in a [Ca2+]o-independent way suggests that Na+ channel block alone, i.e., the prevention of Na+ accumulation, may account for the protective action. Low temperature completely prevented the effect of ischemia on norepinephrine release but did not change the release associated with axonal activity. This finding is in good agreement with the observation that small changes in brain temperature critically determine the extent of neuronal injury from ischemia and suggests that both [Ca2+]o-independent release and cell injury are associated with the norepinephrine membrane carrier. It is suggested, therefore, that drugs able to attenuate the increase in [Na+]i during ischemia may be useful agents to protect against ischemic damage if given before the insult.

Topics: 4-Aminopyridine; Anesthetics, Local; Animals; Calcium; Calcium Channel Blockers; Cold Temperature; Hypoglycemia; Hypoxia; Ischemia; Lidocaine; Male; Norepinephrine; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spinal Cord; Tetrodotoxin

The intracellular sodium concentration ([Na+]i) and resting potential (Em) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na+-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO2 122 Torr), [Na+]i was 12-13 mM corresponding to a Na+ equilibrium potential (ENa) of about 58 mV. Em was about -42 mV. Hypoxia, induced by Na2S2O4 1 mM (PO2 10 Torr), depolarized the cells by about 20 mV, [Na+]i increased by 21 mM and ENa dropped to about 35 mV. One millimolar of CoCl2 depressed, or blocked, the effects of Na2S2O4 on [Na+]i but did not affect hypoxic depolarization. Voltage-clamping at -70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba2+. All currents were blocked by 1 mM CoCl2 suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na+]i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na+ increases (inflow) probably occurred by disturbing a Na+/K+ exchange mechanism and not by activation of voltage-gated channels.

Topics: Animals; Antimutagenic Agents; Antioxidants; Aortic Bodies; Barium; Calcium Channel Blockers; Carotid Body; Cobalt; Electric Stimulation; Hypoxia; Ion Channel Gating; Membrane Potentials; Mice; Microelectrodes; Patch-Clamp Techniques; Sodium; Tetrodotoxin; Thiosulfates

1. The sensitivity of arterial chemoreceptor spike generation to reductions in excitability was examined using rat chemoreceptors in vitro. Axonal excitability was reduced by reducing extracellular sodium concentration ([Na+]o) by 10-40% or by applying low doses of tetrodotoxin (TTX). 2. In normoxia and in hypoxia, an isosmotic reduction in [Na+]o caused a proportional decrease in single-fibre, spiking nerve activity. For a 20% reduction in [Na+]o, nerve activity decreased to 54 +/- 7% of control in normoxia and 41 +/- 5% in hypoxia. 3. Low doses of TTX (25-50 nM) caused a similar decrease in spiking frequency, but this response was variable amongst fibres, with some fibres unaffected by TTX. 4. A reduction in [Na+]o by 20% caused a slowing of conduction velocity, measured using an electrical stimulus delivered to an electrode placed in the carotid body. Threshold current for spike generation was increased by about 2.7 +/- 1.4%. Threshold current increased by 6.5 +/- 3.7% following a 40% reduction in [Na+]o. 5. The spike generation process was modelled as a Poisson process in which depolarizing events summate and give rise to an action potential. The experimental data were best fitted to a high order process characterized by a large number of events and high event threshold. 6. This result is not consistent with depolarization events caused by episodic transmitter release, but suggests that afferent spike generation is an endogenous process in the afferent nerve fibres, perhaps linked to random channel activity or to thermal noise fluctuations.

Topics: Animals; Axons; Carotid Body; Chemoreceptor Cells; Cobalt; Electric Stimulation; Evoked Potentials; Hypoxia; In Vitro Techniques; Nerve Fibers; Neural Conduction; Poisson Distribution; Rats; Saline Solution, Hypertonic; Sodium; Tetrodotoxin

The urinary bladder stores urine at low intravesical pressure and empties the urine efficiently and completely. Bladder compliance is the property that allows the bladder to fill to near capacity without a large increase in intravesical pressure. The current study utilized an in vitro whole bladder model to determine the effects of hypoxia, alterations in extracellular calcium concentration, carbachol and atropine on bladder capacity and compliance.. Mature male New Zealand White rabbits were used in this study. The urinary bladder was excised from the rabbit together with a short segment of proximal urethra and mounted in a 400 ml. isolated bath containing Tyrode's buffer. Bladder filling was started by opening the bladder to a saline reservoir placed 80 cm. above the bladder. Intravesical pressure, rate of pressure increase, rate of volume increase, and maximal volume were digitally recorded. The bladder filling was repeated while the whole bladder was subjected to hypoxia, high calcium concentration, the presence of EGTA, carbachol, atropine and tetrodotoxin respectively.. Results are summarized as follows: 1) Bladder filling was biphasic. There was an initial rapid rise in intravesical pressure followed by a slower rise. The final bladder volume averaged 46 ml. 2) Hypoxia significantly decreased the initial rate of the rise in intravesical pressure, increased the rate of bladder filling, and increased bladder volume by 43%. 3) Incubation of the bladder in the presence of EGTA also significantly decreased the initial rate of intravesical pressure rise, increased the rate of filling and increased bladder volume by 39%. 4) High concentrations of calcium increased the initial rate of rise in intravesical pressure. 5) Carbachol significantly increased the rate of intravesical pressure rise, decreased the rate of bladder filling, and decreased bladder volume. 6) Atropine and tetrodotoxin (TTX) had no effects on bladder filling.. In summary, alterations in muscle tone had significant effects on bladder capacity and compliance.

Topics: Animals; Atropine; Calcium; Carbachol; Egtazic Acid; Hypoxia; In Vitro Techniques; Male; Rabbits; Tetrodotoxin; Urinary Bladder

The substance 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7) has been developed recently as a cerebroprotective compound with Na+ and Ca2+ channel blocking action. In the present study, the effect of NS-7 in an in vitro model of hypoxic injury was examined and the possible involvement of Na+ and Ca2+ channels in the hypoxic injury subsequently determined. When slices of rat cerebral cortex were exposed to hypoxia/glucose deprivation followed by reoxygenation and restoration of the glucose supply, marked leakage of lactate dehydrogenase (LDH) occurred 3-6 h after reoxygenation. This hypoxia/reoxygenation-induced injury was blocked almost completely by the removal of extracellular Ca2+ or by chelating intracellular Ca2+ with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM). In addition, combined treatment with the N-type Ca2+ channel blocker omega-conotoxin GVIA and the P/Q-type Ca2+ channel blocker omega-agatoxin IVA significantly reduced LDH leakage, although neither of these Ca2+ channel blockers alone, nor nimodipine, an L-type Ca2+ channel blocker, was effective. On the other hand, several Na+ channel blockers, including tetrodotoxin, local anaesthetics and antiepileptics, significantly reduced the hypoxic injury. NS-7 (3-30 microM) concentration-dependently inhibited LDH leakage caused by hypoxia/reoxygenation, but had no influence on the reduction of tissue ATP content and energy charge during hypoxia and glucose deprivation. It is suggested that blockade of Na+ and Ca2+ channels is implicated in the cerebroprotective action of NS-7.

Topics: Adenosine Triphosphate; Anesthetics; Animals; Anticonvulsants; Calcium; Calcium Channel Blockers; Cerebral Cortex; Dose-Response Relationship, Drug; Glucose; Hypoxia; In Vitro Techniques; Male; Neuroprotective Agents; Oxygen; Oxygen Consumption; Pyrimidines; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetrodotoxin; Time Factors

1. Using perforated-patch, whole cell recording, we investigated the membrane mechanisms underlying O2 chemosensitivity in neonatal rat adrenomedullary chromaffin cells (AMC) bathed in extracellular solution containing tetrodotoxin (TTX; 0.5-1 microM), with or without blockers of calcium entry. 2. Under voltage clamp, low PO2 (0-15 mmHg) caused a graded and reversible suppression in macroscopic outward K+ current. The suppression during anoxia (PO2 = 0 mmHg) was approximately 35% (voltage step from -60 to +30 mV) and was due to a combination of several factors: (i) suppression of a cadmium-sensitive, Ca2+-dependent K+ current, IK(CaO2); (ii) suppression of a Ca2+-insensitive, delayed rectifier type K+ current, IK(VO2); (iii) activation of a glibenclamide- (and Ca2+)-sensitive current, IK(ATP). 3. During normoxia (PO2 = 150 mmHg), application of pinacidil (100 microM), an ATP-sensitive potassium channel (KATP) activator, increased outward current density by 45.0 +/- 7.0 pA pF-1 (step from -60 to + 30 mV), whereas the KATP blocker glibenclamide (50 microM) caused only a small suppression by 6.3 +/- 4.0 pA pF-1. In contrast, during anoxia the presence of glibenclamide resulted in a substantial reduction in outward current density by 24.9 +/- 7.9 pA pF-1, which far exceeded that seen in its absence. Thus, activation of IK(ATP) by anoxia appears to reduce the overall K+ current suppression attributable to the combined effects of IK(CaO2) and IK(VO2). 4. Pharmacological tests revealed that IK(CaO2) was carried predominantly by maxi-K+ or BK potassium channels, sensitive to 50-100 nM iberiotoxin; this current also accounted for the major portion (approximately 60%) of the anoxic suppression of outward current. Tetraethylammonium (TEA; 10-20 mM) blocked all of the anoxia-sensitive K+ currents recorded under voltage clamp, i.e. IK(CaO2), IK(VO2) and IK(ATP). 5. Under current clamp, anoxia depolarized neonatal AMC by 10-15 mV from a resting potential of approximately -55 mV. At least part of this depolarization persisted in the presence of either TEA, Cd2+, 4-aminopyridine or charybdotoxin, suggesting the presence of anoxia-sensitive mechanisms additionalto those revealed under voltage clamp. In Na+/Ca2+-free solutions, the membrane hyperpolarized, though at least a portion of the anoxia-induced depolarization persisted. 6. In the presence of glibenclamide, the anoxia-induced depolarization increased significantly to approximately 25 mV, suggesting that activation

Topics: Animals; Animals, Newborn; Chromaffin Cells; Electric Stimulation; Electrophysiology; Glyburide; Hypoglycemic Agents; Hypoxia; In Vitro Techniques; Membrane Potentials; Patch-Clamp Techniques; Pinacidil; Potassium Channels; Rats; Rats, Wistar; Tetrodotoxin

Immediate and delayed effects of glucose deprivation, oxygen deprivation (hypoxia) and both oxygen and glucose deprivation (in vitro ischemia) on glutamate efflux from guinea pig cerebral cortex slices were studied. Immediate effects were evaluated by measuring changes of glutamate efflux during the metabolic insults. Delayed effects were evaluated by measuring the response of the tissue to a 50 mM KCI pulse applied 60 min after the metabolic insults. Deprivation of glucose in the medium did not induce either immediate or delayed effects, while hypoxic condition produced an immediate slight stimulation of glutamate efflux without any delayed effect. Conversely, in vitro ischemia produced both immediate and delayed effects on glutamate efflux. During in vitro ischemia glutamate efflux dramatically increased in a calcium-independent and tetrodotoxin-sensitive manner; this effect was potentiated by a low sodium containing medium. The blockade of the sodium/potassium ATPase exchanger by ouabain caused a glutamate outflow similar to that induced by in vitro ischemia. On the whole, these data demonstrate the central role played by the sodium electrochemical gradient and by the membrane glutamate uptake system in the glutamate overflow induced by in vitro ischemia. Moreover, in slices previously exposed to both oxygen and glucose deprivation the effect of KCI on glutamate efflux was potentiated. This in vitro ischemia-induced delayed potentiation of neurotransmitter efflux, until now unreported in the literature, was found to be selectively restricted to glutamatergic structures and to be mainly due to an enhancement of the exocytotic component of glutamate release.

Topics: Animals; Brain Ischemia; Calcium; Cerebral Cortex; Female; Glutamic Acid; Guinea Pigs; Hypoxia; In Vitro Techniques; Male; Ouabain; Potassium Chloride; Sodium; Tetrodotoxin; Time Factors

To examine anoxic injury in spinal cord white matter, we studied axonal conduction in the dorsal columns during and following a standard 60 min anoxic insult at 36 degrees C. Perfusion of the spinal cord in 0-Ca2+ Ringer solution resulted in significantly improved recovery of the compound action potential. Similarly, removal of Na+ from the perfusate resulted in significantly improved recovery of conduction in dorsal column axons. Exposure of the anoxic spinal cord to the Na+ channel blocker tetrodotoxin (TTX), the Na-Ca exchange blockers benzamil and bepridil, Na(+)-H+ exchange blockers amiloride and harmaline, and perfusion in Ringer solution with pH adjusted to 6.4, all resulted in improved recovery. The tertiary anesthetics procaine and lidocaine, as well as phenytoin and carbamazepine, also resulted in improved recovery of compound action potential amplitude after 60 min of anoxia. These results demonstrate that a significant component of irreversible loss of conduction, following anoxic injury of the dorsal columns, is Ca(2+)-dependent. Moreover, these results demonstrate that TTX-inhibitable Na+ channels participate in the pathophysiology of anoxic injury in spinal cord white matter, and indicate that reverse Na-Ca exchange provides a route for at least part of the damaging influx of Ca2+ into an intracellular compartment in anoxic spinal cord white matter. Our results also suggest that extracellular acidosis may have a protective effect on anoxic spinal cord white matter, and support the hypothesis that anoxic injury of spinal cord white matter may involve the Na(+)-H+ exchanger.

Topics: Action Potentials; Animals; Calcium; Female; Ganglia, Spinal; Hypoxia; Rats; Rats, Wistar; Spinal Cord Injuries; Tetrodotoxin

An important cause of anoxia-induced nerve injury involves the disruption of the ionic balance that exists across the neuronal membrane. This loss of ionic homeostasis results in an increase in intracellular calcium, sodium, and hydrogen and is correlated with cell injury and death. Using time-lapse confocal microscopy, we have previously reported that nerve cell injury is mediated largely by sodium and that removing extracellular sodium prevents the anoxia-induced morphological changes. In this study, we hypothesized that sodium enters neurons via specific mechanisms and that the pharmacologic blockade of sodium entry would prevent nerve damage. In cultured neocortical neurons we demonstrate that replacing extracellular sodium with NMDG+ prevents anoxia-induced morphological changes. With sodium in the extracellular fluid, various routes of sodium entry were examined, including voltage-sensitive sodium channels, glutamate receptor channels, and sodium-dependent chloride-bicarbonate exchange. Blockade of these routes had no effect. Amiloride, however, prevented the morphological changes induced by anoxia lasting 10, 15, or 20 min. At doses of 10 microM-1 mM, amiloride protected neurons in a dose-dependent fashion. We argue that amiloride acts on a Na+-dependent exchanger (e.g., Na+-Ca2+) and present a model to explain these findings in the context of the neuronal response to anoxia.

Topics: Amiloride; Animals; Bicarbonates; Biological Transport; Cell Nucleus; Cells, Cultured; Cerebral Cortex; Coloring Agents; Excitatory Amino Acid Antagonists; Hypoxia; Neurons; Propidium; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Tetrodotoxin

Short-circuit current (Isc) and transepithelial potential difference (PD) of rat distal colon decrease during acute hypoxia and overshoot on reoxygenation. It is not known whether tonic intrinsic nervous activity may influence these responses.. Preparations lacking the submucosal plexus (islet mucosa) and preparations retaining it (mucosa-submucosa) were mounted in Ussing chambers at 37 degrees C and gassed with 95% O2-5% CO2; Isc and PD were monitored. A 5-min hypoxia with 95% N2-5% CO2 was followed by reoxygenation. The procedure was repeated in the presence of the nervous blocking agent, tetrodotoxin (10(-6)M) in the serosal side of the chamber.. In the isolated mucosa (n = 10) hypoxia reduced Isc by -55 +/- 5% and PD by -54 +/- 6% below baseline; reoxygenatory overshoots were, respectively, +60 +/- 17% and +/- 16%. Tetrodotoxin slightly and transiently reduced baseline Isc (-16 +/- 2%) and PD (-14 +/- 3%), with a small resistivity increase. It did not significatively modify the responses to responses to either hypoxia or reoxygenation. In mucosa-submucosa preparations (n = 9) hypoxia reduced Isc (-54 +/- 8%) and PD (-61 +/- 4%). On reoxygenation Isc and PD were increased, respectively, +30 +/- 5% and +19 +/- 6% over baseline. Tetrodotoxin reduced baseline Isc (-59.6 +/- 5%) and PD (61.3 +/- 6%). It enhanced hypoxic Isc and PD decreases (-80 +/- 5%), but not the reoxygenatory overshoots.. 1) Tetrodotoxin affects baseline Isc and PD more intensely in submucosal plexus innervated preparations than in the isolated mucosa. 2) The epithelial electrical response to acute hypoxia appears to be modulated by tonic neural activity.

Topics: Acute Disease; Animals; Colon; Electrophysiology; Hypoxia; Intestinal Mucosa; Male; Rats; Rats, Wistar; Submucous Plexus; Tetrodotoxin

Nitric oxide (NO) contributes to hypoxia-induced pial artery dilation, at least in part, via the formation of guanosine 3',5'-cyclic monophosphate (cGMP) and subsequent release of Met-enkephalin and Leu-enkephalin in the newborn pig. In separate studies, these opioids were also observed to elicit NO-dependent pial dilation. The present study was designed to investigate the role of the neuronal isoform of NO synthase (NOS) in hypoxic pial dilation, associated opioid release, and opioid dilation in piglets equipped with a closed cranial window. Tetrodotoxin (10(-6) M) attenuated the dilation resulting from hypoxia (PO2 approximately 35 mmHg; 25 +/- 1 vs. 14 +/- 1%). Similarly, 7-nitroindazole, sodium salt (7-NINA, 10(-6) M), a purported neuronal NOS inhibitor, attenuated hypoxic pial dilation (26 +/- 1 vs. 14 +/- 2%). Hypoxic dilation was accompanied by elevated cerebrospinal (CSF) cGMP, which was blocked by 7-NINA (433 +/- 19 and 983 +/- 36 vs. 432 +/- 19 and 441 +/- 19 fmol/ml for control and hypoxia in absence and presence of 7-NINA, respectively). Additionally, hypoxic dilation was also accompanied by elevated CSF Met-enkephalin, which was attenuated by 7-NINA (1,027 +/- 47 and 2,871 +/- 134 vs. 779 +/- 78 and 1,551 +/- 42 pg/ml for control and hypoxia in absence and presence of 7-NINA, respectively). In contrast, Met-enkephalin (10(-10), 10(-8), and 10(-6) M) induced dilation that was unchanged by 7-NINA (7 +/- 1, 12 +/- 1, and 18 +/- 1 vs. 6 +/- 1, 10 +/- 1, and 17 +/- 1%, respectively). N-methyl-D-aspartate (NMDA, 10(-8) and 10(-6) M), an activator of neuronal NOS, induced pial dilation that was blocked by 7-NINA (10 +/- 1 and 20 +/- 2 vs. 1 +/- 1 and 2 +/- 1%, respectively). However, sodium nitroprusside-induced dilation was unchanged by 7-NINA. These data indicate that neuronal NOS contributes to hypoxic pial artery dilation but not to opioid-induced dilation. Furthermore, these data suggest that neuronally derived NO contributes to hypoxic dilation, at least in part, via formation of cGMP and the subsequent release of opioids.

Topics: Animals; Cerebral Arteries; Cyclic GMP; Endorphins; Enkephalins; Enzyme Inhibitors; Female; Hypoxia; Indazoles; Male; Neurons; Nitric Oxide; Nitric Oxide Synthase; Pia Mater; Swine; Tetrodotoxin; Vasodilation

1. Presynaptic modulation of noradrenaline release in human atrial tissue specimens was investigated under normoxic and anoxic conditions. 2. Noradrenaline release was induced by electrical stimulation and release during experimental intervention (S2) was compared with release during a preceding control stimulation (S1). The results were expressed as the geometric means and 95% confidence intervals of the S2/S1 ratio. 3. The alpha 2-adrenoceptor agonist, UK 14304 (0.1 mumol-1) significantly inhibited noradrenaline release, resulting in a S2/S1 ratio of 0.49 (0.40-0.59), and the a 2-adrenoceptor antagonist, yohimbine (1 mumol l-1) increased noradrenaline release (S2/S1 1.83 [1.43-2.35]) during normoxia. Both compounds were ineffective during anoxia. 4. Adenosine (30 mumol-1) inhibited noradrenaline release with a S2/S1 ratio of 0.54 (0.42-0.66). The adenosine antagonist, 8-phenyltheophylline, alone had no effect during normoxia. During anoxia, neither adenosine nor 8-phenyltheophylline altered noradrenaline release. 5. The beta 2-adrenoceptor agonist, terbutaline (1 mumol l-1) increased (1.53 [1.14-2.01]) and the beta-adrenoceptor antagonist, pindolol (1 mumol l-1) suppressed noradrenaline release (0.62 [0.49-0.79]) under normoxic conditions. During anoxia, pindolol significantly inhibited noradrenaline release with a S2/S1 ratio of 0.66 (0.51-0.85), whereas terbutaline did not influence noradrenaline release. 6. Angiotensin II (0.1 mumol l-1 enhanced noradrenaline release resulting in a S2/S1 ratio of 1.44 (1.34-1.54), while the angiotensin II antagonist, losartan (1 mumol l-1) had no effect on noradrenaline release during normoxia. Conversely, angiotensin II did not increase noradrenaline release and losartan significantly inhibited noradrenaline release to a S2/S1 ratio of 0.60 (0.46-0.77) during anoxia. 7. In conclusion, human cardiac tissue possesses presynaptic inhibitory alpha 2-adrenoceptors and adenosine receptors, as well as facilitatory beta 2-adrenoceptors and angiotensin II receptors regulating noradrenaline release under normoxic conditions. During anoxia the responses to alpha 2-adrenoceptors and adenosine receptor stimulation are lost, whereas facilitatory responses to beta 2-adrenoceptors and adenosine II receptor stimulation are maintained and these receptors appear to be maximally stimulated. This differential presynaptic modulation in anoxia may contribute to enhanced sympathetic activity in ischaemia.

Topics: Adenosine; Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-2 Receptor Antagonists; Adrenergic beta-2 Receptor Antagonists; Adrenergic beta-Antagonists; Angiotensin II; Angiotensin Receptor Antagonists; Electric Stimulation; Heart Atria; Humans; Hypoxia; In Vitro Techniques; Myocardium; Norepinephrine; Receptors, Angiotensin; Receptors, Presynaptic; Tetrodotoxin; Vasoconstrictor Agents

1. A persistent inward current activated by depolarization was recorded using the whole-cell, tight seal technique in rat isolated cardiac myocytes. The amplitude of the inward current increased when cells were exposed to a solution with low oxygen tension. 2. The persistent inward current had the characteristics of the persistent Na+ current described previously in rat ventricular myocytes: it was activated at negative potentials (-70 mV), reversed close to the equilibrium potential for Na+ (ENa), was blocked by TTX and was resistant to inactivation. 3. Persistent single Na+ channel currents activated by long (200-400 ms) depolarizations were recorded in cell-attached patches on isolated ventricular myocytes. Hypoxia increased the frequency of opening of the persistent Na+ channels. 4. Persistent Na+ channels recorded during hypoxia had characteristics similar to those of persistent Na+ channels recorded at normal oxygen tensions. They had a null potential at ENa, their amplitude varied with [Na+], they were resistant to inactivation and their mean open time increased with increasing depolarization. 5. The persistent Na+ channels in cell-attached patches were blocked by TTX (50 microM) in the patch pipette and by lidocaine (100 microM). 6. It was concluded that hypoxia increases the open probability of TTX-sensitive, inactivation-resistant Na+ channels. The voltage dependence of these channels, and their greatly increased activity during hypoxia, suggest that they may play an important role in the generation of arrhythmias during hypoxia.

Topics: Animals; Anti-Arrhythmia Agents; Electrophysiology; Heart Ventricles; Hypoxia; Ion Channel Gating; Lidocaine; Membrane Potentials; Myocardium; Rats; Rats, Wistar; Sodium; Sodium Channels; Tetrodotoxin; Time Factors

Membrane potentials were recorded from neuronal somata in the substantia nigra pars reticulata of the rat midbrain slice using the whole-cell patch-clamp technique. Hypoxia induced a consistent decrease in input resistance often accompanied by membrane hyperpolarization and cessation of firing. The membrane hyperpolarization was mediated by K+ as indicated by its reversal potential at -88 +/- 9 mV, which is close to the equilibrium potential of K+. The hypoxic response was not sensitive to 1 microM tetrodotoxin or superfusion with Ca2(+)-free medium. While glibenclamide at 30 microM and tolbutamide at 300 microM had no effect on the resting membrane properties of the neurones, these sulphonylureas reversed the hypoxia-induced membrane hyperpolarization and restored firing. Inclusion of 2 mM of ATP in the recording pipette also prevented the hyperpolarization. These observations suggest that post-synaptic ATP-sensitive potassium channels exist on the GABA neurones of SNR and that these channels are activated in energy-depleting conditions exemplified by hypoxia.

Topics: Animals; Glyburide; Hypoxia; Membrane Potentials; Potassium Channels; Rats; Substantia Nigra; Tetrodotoxin

Functionally intact coronary artery segments studied in vitro responded to 15 min of hypoxia with relaxations of preexisting contractions. The hypoxic relaxations were obtained in preparations routinely denuded of endothelium and were unaffected by tetrodotoxin, by indomethacin or by the blockers of calcium-dependent potassium channels, apamin and charybdotoxin. Relaxations from contractions to the calcium channel opener Bay K 8644 and to spontaneous tone were attenuated most by hypoxia, and those to carbamylcholine and 5-hydroxytryptamine were inhibited to an intermediate extent. Contractions to the thromboxane A2 analog U 46619, dependent largely on intracellular calcium, were the least reduced during 15 min of hypoxia. Pretreatment of contracted preparations with glibenclamide, the potent antagonist of ATP-dependent potassium channels, before exposure to 95% N2/5% CO2 significantly attenuated, but did not eliminate, hypoxic relaxations. Hypoxic relaxations from contractions to the calcium channel opener Bay K 8644 and to spontaneous tone were antagonized most by glibenclamide, and those to U 46619 were reduced the least. In the presence of the calcium channel antagonist nifedipine, tissues contracted with carbamylcholine or 5-hydroxytryptamine relaxed during hypoxia, but these relaxations were insensitive to glibenclamide. Contractions of cattle radial artery and rabbit aorta were variably reduced during hypoxia but were insensitive to glibenclamide. We conclude that K+ ATP channels participate in hypoxia-induced coronary artery smooth muscle relaxation and may do so particularly with contractions that utilize principally extracellular calcium.

Topics: Adenosine; Adenosine Triphosphate; Animals; Arteries; Calcium; Calcium Channel Blockers; Cattle; Coronary Vessels; Glyburide; Guanidines; Hypoxia; In Vitro Techniques; Indomethacin; Muscle Relaxation; Pinacidil; Potassium Channel Blockers; Potassium Channels; Rabbits; Tetrodotoxin; Vasodilator Agents

Chronic hypoxic acclimatization modifies ventilatory reflexes arising from carotid body stimulation. To explore this, the effects of in vivo chronic hypoxia on membrane currents were quantified in chemoreceptive carotid body glomus cells. Pregnant rats were maintained in either normoxia (NORM: inspired oxygen tension 141 mmHg), or hypoxia (CHX: inspired oxygen tension 80 mmHg) from day 3 of gestation, to day 5-10 postpartum. Whole cell patch clamp recordings were then made from the mechanically and enzymatically dissociated carotid body glomus cells of the rat pups (NORM: 41 cells, CHX: 36 cells) and comparisons of means +/- S.E.M. were made with unpaired t-tests. Glomus cells were bright under phase contrast illumination, formed clusters, were histochemically positive for catecholamines and possessed voltage-gated potassium currents that were depressed by acute hypoxia. Acclimatization to chronic hypoxia did not affect rat pup whole body mass (CHX: 12.0 +/- 0.7 g vs. NORM: 11.0 +/- 0.2 g), but it significantly increased blood hematocrit (CHX: 48.7 +/- 0.9% vs. NORM: 37.8 +/- 0.5%, P < 0.05). Sodium current was not uniformly present in glomus cells from either group, but sodium current was observed in a greater proportion of glomus cells isolated from the chronically hypoxic pups (CHX: 72% vs. NORM: 46%, P < 0.05). The mean peak tetrodotoxin-sensitive sodium current evoked by -70 mV to +10 mV depolarizations was greater after hypoxic acclimatization (CHX: -100 +/- 25 pA vs. NORM: -38 +/- 15 pA, P < 0.05), but the sodium current density (pA/pF) was unchanged. In contrast, the mean peak voltage-gated potassium current (pA) evoked by -70 mV to 0 mV depolarizations was unchanged by acclimatization, but the potassium current density (pA/pF) was reduced (P < 0.05). Unchanged sodium current density coupled with decreased potassium current density may make glomus cells more excitable during exposure to chronic in vivo hypoxia.

Topics: Animals; Animals, Newborn; Carotid Body; Female; Hypoxia; Membrane Potentials; Patch-Clamp Techniques; Potassium Channels; Pregnancy; Rats; Sodium Channels; Tetrodotoxin

Topics: Animals; Calcium; Carotid Body; Catecholamines; Chemoreceptor Cells; Dose-Response Relationship, Drug; Hypoxia; In Vitro Techniques; Kinetics; Rabbits; Sodium; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Veratridine

Ionic membrane currents are hypothesized to play a major role in determining secretion from carotid body glomus cells, and increased secretion likely mediates the increase in nerve activity in response to hypoxia. The hypothesis that Na+ and K+ channels play an important role in determining secretion and nerve activity was tested by measuring single-fiber afferent nerve activity along with an estimate of free tissue catecholamine using Nafion-covered carbon-fiber micro-electrodes placed in rat carotid bodies in vitro. Baseline and anoxia-stimulated (1 min duration; PO2 of approximately 0 Torr at nadir) levels were quantified. Sham treatment had no significant effect. Tetrodotoxin (2 microns) ablated the nerve activity and reduced peak catecholamine (19.5 +/- 3.1 to 14.5 +/- 3.4 microM; P < 0.05). Cesium (10 microns) had no effect on catecholamine but reduced the nerve response (19.8 +/- 2.7 to 7.8 +/- 2.0 Hz; P < 0.05). 4-Aminopyridine (4 mM) significantly reduced the nerve response (17.2 +/- 3.7 to 4.9 +/- 1.9 Hz; P < 0.05) and increased the baseline (0.9 +/- 0.2 to 3.1 +/- 0.8 microM; P < 0.05) and reduced the peak catecholamine (10.0 to 4.3 +/- 0.8 microM; P < 0.05) levels. These results demonstrate that Na+ and K+ channels play an important role in modulating the secretory and nerve responses. However, channel blockers do not emulate severe hypoxia, suggesting that hypoxia transduction procedes, at least in part, through an alternate pathway.

Topics: 4-Aminopyridine; Animals; Carotid Body; Catecholamines; Cesium; Electrophysiology; Hypoxia; Potassium Channel Blockers; Rats; Reference Values; Sodium Channel Blockers; Tetrodotoxin

The role played by Na+ channels of carotid body (CB) chemoreceptor cells was investigated by studying the effects of tetrodotoxin (TTX) on the release of 3H-labeled catecholamines ([3H]CA) by adult rabbit CBs previously incubated with the precursor [3H]tyrosine. TTX inhibited partially the release of [3H]CA elicited by mild hypoxia (10 or 7% O2) or by depolarizing incubation medium containing 20 or 30 mM KCl, but the response to more intense hypoxia (5 or 2% O2) or to higher KCl concentration (40 or 50 mM) was not significantly affected. The release of [3H]CA elicited by acidic stimuli, either 20% CO2 (pH 6.6) or the protonophore dinitrophenol (100 microM), although comparable in magnitude to that elicited by mild hypoxia, was not modified by TTX. These results provide evidence for the first time that Na+ channels of chemoreceptor cells participate in the transduction of hypoxic stimuli into the neurotransmitter release response of these cells and suggest that Na+ current operates as an amplifying device that enhances the initial cell depolarization mediated by the closure of the O2-sensitive K+ channels. Sympathetic denervation of CBs was followed by a marked reduction in the release of [3H]CA elicited by veratridine or by 20 mM KCl, suggesting that the number of Na+ channels in chemoreceptor cells decreases after denervation.

Topics: Acids; Animals; Carotid Body; Catecholamines; Chemoreceptor Cells; Hypoxia; Potassium; Potassium Chloride; Rabbits; Sodium Channels; Sympathectomy; Tetrodotoxin; Veratridine

1. The effects of NG-monomethyl-L-arginine, tetrodotoxin and glibenclamide on hypoxia-induced coronary artery relaxation, induced by bubbling Krebs solution with 95% N2 and 5% CO2 instead of 95% O2 and 5% CO2, were assessed by measuring the changes in isometric tension in isolated epicardial coronary artery rings of the rabbit. In addition, the effects of glibenclamide on the relaxation induced by adenosine were investigated. 2. Hypoxia caused a transient relaxation of 38 +/- 3% (P < 0.01) and 17 +/- 2% (P < 0.01) in endothelium-intact or -denuded arteries respectively. NG-monomethyl-L-arginine (30 and 100 microM) inhibited the relaxation in endothelium-intact rings to 31 +/- 2% (P < 0.05) and 16 +/- 2% (P < 0.01) respectively and slightly but significantly attenuated the relaxation in endothelium-denuded rings to 15 +/- 1% and 13 +/- 1% (P < 0.05) respectively. 3. Glibenclamide, a potassium channel inhibitor, did not significantly after the hypoxia-induced relaxation. 4. Incubation with tetrodotoxin (3 and 10 microM) for 30 min reduced the relaxation to 31 +/- 3% (P < 0.05) and 14 +/- 2% (P < 0.01), and 14 +/- 2% (P < 0.05) and 11 +/- 1% (P < 0.05) in endothelium-intact and -denuded rings respectively. However, indomethacin (10 microM), atropine (1 microM), propranolol (10 microM) and phentolamine (10 microM) did not significantly affect the relaxation. 5. Adenosine (1, 10 and 100 MicroM) caused relaxation of 6 +/- 1%, 52 +/-3% and 97 +/-2% respectively in endothelium-denuded rings precontracted with prostaglandin F2alpha (PGF2 alpha, 3 MicroM) and the relaxation was markedly inhibited by 8-phenyltheophylline. Furthermore, glibenclamide (1 and 10 MicroM) reduced the relaxation induced by adenosine (1, 10 and 100 MicroM) to 2 +/-1% (P<0.05), 38 =/-3% (P<0.05) and 85 +/-2%(P<0.05), and 0.6 +/- 0.4% (P<0.05), 27 +/- 4% (P<0.05) and 72 +/- 4% (P<0.01) respectively, in these endothelium-denuded preparations.6. These data suggest that hypoxia-induced relaxation is mediated by the release of nitric oxide rather than by the activation of glibenclamide-sensitive potassium channels in rabbit isolated coronary arteries. A neurogenic mechanism partially modulates the relaxation, possibly by activating non-adrenergic and noncholinergic nerve endings. The inhibition by glibenclamide on adenosine-induced relaxation in isolated coronary arteries may help to explain the fact that glibenclamide inhibits hypoxic coronary relaxation in perfused hearts but not in isolated co

Topics: Adenosine; Adenosine Triphosphate; Animals; Arginine; Capsaicin; Coronary Vessels; Endothelium, Vascular; Glyburide; Hypoxia; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; omega-N-Methylarginine; Potassium Channels; Rabbits; Tetrodotoxin; Theophylline

Studies have shown that the rise in intracellular ionized calcium, [Ca2+]i, in hypoxic myocardium is driven by an increase in sodium, [Na+]i, but the source of Na+ is not known.. Inhibitors of the voltage-gated Na+ channel were used to investigate the effect of Na+ channel blockade on hypoxic Na+ loading, Na(+)-dependent Ca2+ loading, and reoxygenation hypercontracture in isolated adult rat cardiac myocytes. Single electrically stimulated (0.2 Hz) cells were loaded with either SBFI (to index [Na+]i) or indo-1 (to index [Ca2+]i) and exposed to glucose-free hypoxia (PO2 < 0.02 mm Hg). Both [Na+]i and [Ca2+]i increased during hypoxia when cells became inexcitable following ATP-depletion contracture. The hypoxic rise in [Na+]i and [Ca2+]i was significantly attenuated by 1 mumol/L R 56865. Tetrodotoxin (60 mumol/L), a selective Na(+)-channel blocker, also markedly reduced the rise in [Ca2+]i during hypoxia and reoxygenation. Reoxygenation-induced cellular hypercontracture was reduced from 83% (45 of 54 cells) under control conditions to 12% (4 of 32) in the presence of R 56865 (P < .05). Lidocaine reduced hypercontracture dose dependently with 13% of cells hypercontracting in 100 mumol/L lidocaine, 42% in 50 mumol/L lidocaine, and 93% in 25 mumol/L lidocaine. The Na(+)-H+ exchange blocker, ethylisopropylamiloride (10 mumol/L) was also effective, limiting hypercontracture to 12%. R 56865, lidocaine, and ethylisopropylamiloride were also effective in preventing hypercontracture in normoxic myocytes induced by 75 mumol/L veratridine, an agent that impairs Na+ channel inactivation. Ethylisopropylamiloride prevented the veratridine-induced rise in [Ca2+]i without affecting Na(+)-Ca2+ exchange, suggesting that amiloride derivatives can reduce Ca2+ loading by blocking Na+ entry through Na+ channels, an action that may in part underlie their ability to prevent hypoxic Na+ and Ca2+ loading.. Na+ influx through the voltage-gated Na+ channel is an important route of hypoxic Na+ loading, Na(+)-dependent Ca2+ loading, and reoxygenation hypercontracture in isolated rat cardiac myocytes. Importantly, the Na+ channel appears to serve as a route for hypoxic Na+ influx after myocytes become inexcitable.

Topics: Amiloride; Animals; Benzofurans; Benzothiazoles; Calcium; Calcium Channel Blockers; Cell Separation; Ethers, Cyclic; Fluorescent Dyes; Hypoxia; Lidocaine; Myocardial Contraction; Myocardium; Piperidines; Rats; Sodium; Sodium Channel Blockers; Sodium-Hydrogen Exchangers; Tetrodotoxin; Thiazoles

We examined the effects of brief periods of hypoxia or application of cyanide on the discharge and membrane properties of medullary pacemaker neurones in slices of the rostral ventrolateral reticular nucleus (RVL) of the medulla oblongata of rats. Stable intracellular recordings were obtained from seventy-nine neurones within the RVL which exhibited spontaneous rhythmic discharge in the absence of excitatory postsynaptic potentials (EPSPs). The membrane potential cycles of these neurones could be reset with an evoked spike without eliciting EPSPs or inhibitory postsynaptic potentials and hence met criteria of RVL pacemaker neurones. Hypoxia, produced by reducing O2 from 95 to 20% for 40 s or exposure to cyanide (30-300 microM for 40 s), reversibly increased neuronal discharge 1.6-fold (20% O2) or 2.6-fold (300 microM cyanide), respectively, in association with membrane depolarization and a significant fall in membrane resistance. The membrane responses to hypoxia and cyanide were observed in the presence of tetrodotoxin (TTX) at a concentration (10 microM) which eliminated spontaneous spikes or spikes evoked by intracellular depolarization. When recorded at a holding potential of -70 mV by single-electrode voltage clamp, hypoxia or cyanide (300 microM) elicited inward currents of 0.44 +/- 0.06 and 0.58 +/- 0.08 nA, respectively, which are attenuated by reducing the concentration of extracellular Ca2+ ions, and abolished by 2 mM CoCl2 and 100 microM NiCl2, but not affected by 50 microM CdCl2, replacement of 83% extracellular Na+, or adenosine deaminase (2U ml-1). We conclude that hypoxia and cyanide directly excite RVL pacemaker neurones in vitro by a common mechanism: activation of Ca2+ channel conductance.

Topics: Action Potentials; Adenosine; Animals; Biological Clocks; Calcium Channel Blockers; Calcium Channels; Cations, Divalent; Cyanides; Electrophysiology; Hypoxia; In Vitro Techniques; Medulla Oblongata; Membrane Potentials; Microelectrodes; Neurons; Rats; Rats, Sprague-Dawley; Tetrodotoxin

We tested the hypothesis that the intracellular Ca2+ overload of ventricular myocardium during the period of posthypoxic reoxygenation is mediated by transsarcolemmal Ca2+ influx via Na+/Ca2+ exchange. In aequorin-loaded, ferret right ventricular papillary muscles, blockers of the sarcolemmal and the sarcoplasmic reticulum Ca2+ channels, slowed the Cai2+ transient, producing a convex ascent during membrane depolarization, followed by a concave descent during repolarization. The magnitude of the Cai2+ transient was affected by changes in the membrane potential, Nai+, Nao+, and Cao2+, and was blocked by Ni2+, or dichlorbenzamil. The calculated Na+/Ca2+ exchange current was in the reverse mode (Ca2+ influx) during the ascending phase of the Cai2+ transient, and was abruptly switched to the forward mode (Ca2+ efflux) at repolarization, matching the time course of the Cai2+ transient. During hypoxic superfusion, the Cai2+ transient was abbreviated, which was associated with a shorter action potential duration. In contrast, immediately after reoxygenation, the Cai2+ transient increased to a level greater than that of the control, even though the action potential remained abbreviated. This is the first demonstration on a beat-to-beat basis that, during reoxygenation, Ca2+ influx via Na+/Ca2+ exchange is augmented and transports a significant amount of Ca2+ into the ventricular myocardial cell. The activation of the exchanger at the time of reoxygenation appears to be mediated by Nai+ accumulation, which occurs during hypoxia.

Topics: Action Potentials; Aequorin; Animals; Calcium; Ferrets; Hypoxia; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Sodium; Tetrodotoxin

Synaptosomes of rat cerebral cortex were used to study the effect of veratridine-induced Na+ load on postanoxic recovery of respiration and on aerobic and anaerobic ATP turnover, calculated from rates of oxygen consumption and lactate production. Non-stimulated synaptosomes: after onset of anoxia lactate synthesis of synaptosomes rose immediately from 0.8 to 17.7 nmol lactate/min/mg protein indicating an anaerobic ATP turnover of 17.7 nmol ATP/min/mg protein. This value accounts for 80% of ATP synthesized during oxygenated conditions and seems to cover the energetic demand of anoxic synaptosomes. This assumption was supported by linearity of lactate production throughout anoxia (90 min), by unaffected synaptosomal integrity and by complete recovery of postanoxic respiration after 90 min of anoxia. Stimulated synaptosomes: stimulation of oxygenated synaptosomes with 10(-5) mol/l veratridine enhanced ATP turnover 5-fold, due to activation of Na+/K+ ATPase, as a result of veratridine-induced Na+ influx. Consequently, if not limited in capacity, anaerobic ATP synthesis should be enhanced after addition of veratridine during anoxia. However, the opposite effect was observed. Veratridine reduced anaerobic glycolysis in a concentration-dependent manner. This inhibitory effect could be prevented by tetrodotoxin applied 5 min prior to veratridine. Inhibition of anaerobic glycolysis was independent of extrasynaptosomal glucose (1-30 mmol/l) and Ca2+ concentration (Ca(2+)-free and 1.2 mmol/l Ca2+). Veratridine stimulation of anoxic synaptosomes reduced also the recovery of postanoxic respiration. The data indicate that Na+ load inhibits anaerobic ATP synthesis, the only energy source during anaerobic conditions. To our knowledge, inhibition of anaerobic glycolysis due to increased Na+ influx has not been shown so far.

Topics: Anaerobiosis; Animals; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cerebral Cortex; Dose-Response Relationship, Drug; Glucose; Glycolysis; Hypoxia; Kinetics; L-Lactate Dehydrogenase; Lactates; Male; Oxygen Consumption; Rats; Rats, Wistar; Sodium; Synaptosomes; Tetrodotoxin; Veratridine

The ionic current underlying the upstroke of axonal action potentials is carried by rapidly activating, voltage-dependent Na+ channels. Termination of the action potential is mediated in part by the rapid inactivation of these Na+ channels. We previously demonstrated that an influx of Na+ plays a critical role in the cascade leading to irreversible anoxic injury in central nervous system white matter. We speculated that a noninactivating Na+ conductance mediates this pathological Na+ influx and persists at depolarized membrane potentials as seen in anoxic axons. In the present study we measured the resting compound membrane potential of rat optic nerves using a modified "grease-gap" technique. Application of tetrodotoxin (2 microM) to resting nerves ([K+]o = 3 mM) or to nerves depolarized by 15 or 40 mM K+ resulted in hyperpolarizing shifts of membrane potential. We interpret these shifts as evidence for a persistent, noninactivating Na+ conductance. This conductance is present at rest and persists in nerves depolarized sufficiently to abolish classical transient Na+ currents. PK/PNa ratios were estimated at 35.5, 23.2, and 88 in 3 mM, 15 mM, and 40 mM K+, respectively. We suggest that this noninactivating Na+ conductance may provide an inward pathway for Na+ ions, necessary for the operation of Na+, K(+)-ATPase. Under pathological conditions, such as anoxia, this conductance is the likely route of Na+ influx, which causes damaging Ca2+ entry through reverse operation of the Na(+)-Ca2+ exchanger. The presence of this conductance in white matter axons may provide a therapeutic opportunity for diseases such as stroke and spinal cord injury.

Topics: Animals; Electric Conductivity; Hypoxia; In Vitro Techniques; Membrane Potentials; Optic Nerve; Rats; Sodium; Sodium Channels; Tetrodotoxin

White matter of the mammalian CNS suffers irreversible injury when subjected to anoxia/ischemia. However, the mechanisms of anoxic injury in central myelinated tracts are not well understood. Although white matter injury depends on the presence of extracellular Ca2+, the mode of entry of Ca2+ into cells has not been fully characterized. We studied the mechanisms of anoxic injury using the in vitro rat optic nerve, a representative central white matter tract. Functional integrity of the nerves was monitored electrophysiologically by quantitatively measuring the area under the compound action potential, which recovered to 33.5 +/- 9.3% of control after a standard 60 min anoxic insult. Reducing Na+ influx through voltage-gated Na+ channels during anoxia by applying Na+ channel blockers (TTX, saxitoxin) substantially improved recovery; TTX was protective even at concentrations that had little effect on the control compound action potential. Conversely, increasing Na+ channel permeability during anoxia with veratridine resulted in greater injury. Manipulating the transmembrane Na+ gradient at various times before or during anoxia greatly affected the degree of resulting injury; applying zero-Na+ solution (choline or Li+ substituted) before anoxia significantly improved recovery; paradoxically, the same solution applied after the start of anoxia resulted in more injury than control. Thus, ionic conditions that favored reversal of the normal transmembrane Na+ gradient during anoxia promoted injury, suggesting that Ca2+ loading might occur via reverse operation of the Na+)-Ca2+ exchanger. Na(+)-Ca2+ exchanger blockers (bepridil, benzamil, dichlorobenzamil) significantly protected the optic nerve from anoxic injury. Together, these results suggest the following sequence of events leading to anoxic injury in the rat optic nerve: anoxia causes rapid depletion of ATP and membrane depolarization leading to Na+ influx through incompletely inactivated Na+ channels. The resulting rise in the intracellular [Na+], coupled with membrane depolarization, causes damaging levels of Ca2+ to be admitted into the intracellular compartment through reverse operation of the Na(+)-Ca2+ exchanger. These observations emphasize that differences in the pathophysiology of gray and white matter anoxic injury are likely to necessitate multiple strategies for optimal CNS protection.

Topics: Amiloride; Animals; Calcium; Carrier Proteins; Central Nervous System; Electric Stimulation; Hypoxia; In Vitro Techniques; Kinetics; Models, Neurological; Optic Nerve; Rats; Saxitoxin; Sodium; Sodium Channels; Sodium-Calcium Exchanger; Tetrodotoxin

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Chickens; Dizocilpine Maleate; Edema; Electron Transport; Glycolysis; Hypoglycemia; Hypoxia; Iodoacetates; Iodoacetic Acid; Ischemia; N-Methylaspartate; Potassium Cyanide; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Retina; Tetrodotoxin

1. The effects of anoxia on membrane properties of 119 dorsal vagal motoneurones (DVMs) were investigated in an in vitro slice preparation of the rat medulla. 2. Membrane potential was unaffected by anoxia in 11% of DVMs. An hyperpolarization accompanied by a decrease in input resistance occurred in 44% of DVMs; the remaining 45% depolarized with either an increase (60%) or decrease in input resistance (40%). TTX at a concentration of 0.3-1 microM did not significantly affect these responses. 3. Anoxic artificial cerebrospinal fluid (ACSF) containing 20 mM-TEA reversed the response of DVMs that hyperpolarized in standard ACSF to reveal a depolarization of 7.4 +/- 2.1 mV, and increased the anoxic depolarization from 5.0 +/- 0.7 to 8.7 +/- 1.4 mV. 4. Anoxic depolarization was converted to an hyperpolarization of 7.3 +/- 2.1 mV in ACSF containing 5 mM-4-aminopyridine (4-AP) and 1 microM-TTX. A residual depolarization of 4.5 +/- 3.5 mV was then observed in ACSF containing 5 mM-4-AP, 1 microM-TTX and 20 mM-TEA. Anoxic hyperpolarization was increased from 7.8 +/- 1.8 to 10.0 +/- 3.9 mV in 5 mM-4-AP and 1 microM-TTX and converted to a depolarization of 5.3 +/- 4.5 mV in 5 mM-4-AP, 1 microM-TTX and 20 mM-TEA. 5. In anoxic ACSF containing TEA, the action potential width was increased from 0.92 +/- 0.04 to 8.1 +/- 1.1 ms in hyperpolarizing DVMs, and from 0.85 +/- 0.01 to 2.4 +/- 1.0 ms in depolarizing DVMs. The increase in width was prevented by 2-3 mM-Mn2+. 6. The long after-hyperpolarization (AHP) of DVMs, which is contributed to by both an apamin-sensitive IK(Ca) and an apamin, charybdotoxin and TEA insensitive IK(Ca) was decreased in duration from 2.59 +/- 0.14 to 1.94 +/- 0.12 s during anoxia. 7. It is concluded that anoxia enhances the delayed rectifier current (IK(DR)) and an inward current, probably ICa, but suppresses the A currents (IA). In DVMs that hyperpolarize during anoxia, the increase in IK(DR) outweighs the increase in ICa and the decrease in IA. In depolarizing DVMs the decrease in IA and increase in ICa outweight the increase in IK(DR). The change in input resistance is determined by the relative sizes of current enhancement or suppression.

Topics: 4-Aminopyridine; Action Potentials; Animals; Apamin; Hypoxia; Ions; Male; Manganese; Membrane Potentials; Motor Neurons; Oxygen; Rats; Rats, Wistar; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Vagus Nerve

Rat hippocampal tissue slices were made hypoxic in control medium and in medium containing the ion channel blockers tetraethylammonium (TEA), 4-aminopyridine (4-AP), or tetrodotoxin (TTX). Postsynaptic evoked potentials, extracellular DC potential Vec, and in some experiments extracellular potassium concentration [K+]o were monitored in stratum pyramidale of the CA1 region. TEA (10 mM) decreased the latency of hypoxia-induced spreading depression (SD), and reduced the amplitudes of the changes in Vec and [K+]o. 4-AP (50 microM) also decreased the latency of SD but had no effect on the Vec shift. In most slices, TTX (1 microM) increased SD latency but had no effect on the Vec shift. In some slices, TTX blocked the occurrence of SD.

Topics: 4-Aminopyridine; Animals; Cortical Spreading Depression; Evoked Potentials; Female; Hippocampus; Hypoxia; In Vitro Techniques; Ion Channels; Rats; Rats, Inbred Strains; Reaction Time; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihydroxyphenylethylamine (dopamine) were examined in the rat striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O2 in N2) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 microM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.

Topics: Animals; Corpus Striatum; Dialysis; Dopamine; Hypoxia; Male; Microchemistry; Neurons; Nomifensine; Rats; Rats, Inbred Strains; Tetrodotoxin

Under in vitro conditions the rat cecum transported HCO3- from the serosal to an unbuffered solution in contact with the mucosal side [Js----m = 7.12 +/- 0.18 mumol.cm-2.h-1 (n = 149)]. With reversed tissues, a significantly lower flux was obtained [Jm----s = 2.47 +/- 0.11 mumol.cm-2.h-1 (n = 42)]. Both fluxes were stable for several hours. Increasing the H+ gradient across the tissue for 60 min did not change either flux. Anoxia for 45 min reversibly reduced Js----m by 65 +/- 3% (n = 20) but had no effect on Jm----s. Both fluxes were linearly related to HCO3- concentration on the buffered side, but the slope for Js----m was 3.5 times that for Jm----s. When tissues were initially set up in HEPES buffer rather than HCO3-, Js----m was 0.12 +/- 0.05 mumol.cm-2.h-1 (n = 6), which is not significantly different from zero. Replacement of Na+ by choline reduced Js----m by 40 +/- 3% (n = 11) and ouabain (1 mM) by 24 +/- 3% (n = 5). Replacement of Cl- with isethionate or K+ with Na+ for 60 min did not alter Js----m. Serosal application of DIDS (0.5 mM) reduced Js----m by 24 +/- 6% (n = 6), but SITS (0.5 mM), furosemide (1 mM), acetazolamide (0.1 mM), amiloride (1 mM), and a proton pump inhibitor (Sch 28080, 50 microM) had no effect. Mucosal application of DIDS, furosemide, and amiloride had no effect on Js----m. Serosal tetrodotoxin (1 microM) and indomethacin (28 microM) were also without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bicarbonates; Cecum; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Ions; Kinetics; Male; Mannitol; Muscle, Smooth; Rats; Rats, Inbred Strains; Tetrodotoxin

Microscopic fluorometry was used to examine the effects of anoxia and cyanide (CN-) on cytosolic calcium [Ca2+]i of cultured carotid body (CB) glomus cells from newborn rabbits. Applications of high K+ and veratridine (VRT), a sodium channel activator, induced rapid and marked increases in [Ca2+]i. These effects were inhibited by D600 a calcium channel blocker. [Ca2+]i changes induced by VRT were also blocked by tetrodotoxin (TTX). Glomus cells exhibited a slow increase in [Ca2+]i in response to anoxia and CN-, and a slight decrease during hyperoxia. The effects of anoxia and CN- were blocked by D600 but not by TTX. We conclude that these stimuli induce calcium entry into glomus cells via voltage-dependent Ca2+ channels. Voltage-dependent Na+ channels were not involved.

Topics: Animals; Animals, Newborn; Calcium; Carotid Body; Cells, Cultured; Cyanides; Cytosol; Gallopamil; Hypoxia; Rabbits; Tetrodotoxin; Veratridine

To clarify the effects of hypoxia on adrenergic transmission, we examined the contractile responses of isolated rabbit aortic strips to electrical stimulation, the concentration-response relationships for noradrenaline and KCl, and the electrical stimulation-evoked overflows of total [3H] and [3H]noradrenaline from strips preloaded with [3H]noradrenaline in media equilibrated with gas mixtures containing various concentrations of O2. Contractile responses to electrical stimulation were completely inhibited by tetrodotoxin and alpha-adrenoceptor antagonists such as phentolamine and phenoxybenzamine, but were not affected by indomethacin. When the concentration of O2 in the gas mixture was decreased from 95% to 20%, the contractile responses to electrical stimulation remained unchanged, but as the concentration of O2 was further decreased, the responses were inhibited concentration-dependently. At 0% O2, the response was inhibited by about 80% when compared with control values obtained at 95% O2, and the electrical stimulation-evoked overflows of total [3H] and [3H]noradrenaline into the superfusates were decreased by about 55%. At 0% O2, the concentration-response curve for exogenous noradrenaline was shifted to the right about 50-fold and the maximum response was decreased by 25%. The maximum contractile responses of aortic strips from animals pretreated with reserpine or 6-hydroxydopamine to high KCl were decreased slightly (about 15%). These results suggest that inhibition of adrenergic transmission under hypoxic conditions is mainly the result of a decrease in the stimulus-evoked release of noradrenaline and of a decrease in the affinity of alpha-adrenoceptor for noradrenaline and/or inhibition of signal transduction mechanisms, although hypoxia also causes a slight decrease in the contractility of vascular smooth muscle.

Topics: Animals; Aorta, Thoracic; Electric Stimulation; Hypoxia; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Potassium Chloride; Rabbits; Tetrodotoxin; Time Factors

Effects of hypoxia and pH alterations on the spontaneous contractions and responses to cholinergic stimuli or KCl were investigated in isolated human intestines. The longitudinal strip of human intestine showed spontaneous contractions. The spontaneous contraction was abolished by hypoxia with nitrogen gas substitution (95% N2 and 5% CO2) but not by substitution with acidic (pH 6.53) or alkaline (pH 7.75) solution. Contractile responses to acetylcholine (ACh) were not altered by treatment with hypoxia or pH alterations. KCl (10 mM)-induced contraction was inhibited by hypoxia but not by pH alterations. Transmural electrical stimulation elicited a transient contraction that was blocked by tetrodotoxin or atropine. Contractions induced by electrical stimulation at a low frequency (5 Hz) was not altered by hypoxia or pH alterations. The metabolic pathway related to energy generation and utilization for the spontaneous contraction and KCl-induced contraction seems to be more dependent on oxygen supply than that for ACh-induced contraction. The contractile mechanism in human intestine seems to be resistant to pH alterations.

Topics: Acetylcholine; Adult; Atropine; Electric Stimulation; Gastrointestinal Motility; Humans; Hydrogen-Ion Concentration; Hypoxia; Ileum; In Vitro Techniques; Jejunum; Middle Aged; Muscle Contraction; Muscle, Smooth; Potassium Chloride; Receptors, Cholinergic; Tetrodotoxin

The effects of carotid body chemoreceptor stimulation by NaCN and sustained lung inflation on TTX-induced apneustic respiration were studied before and after picrotoxin in the rabbit. Intravenous and intracarotid injections of TTX (30-100 micrograms) induced the apneusis. The characteristic phrenic responses induced by NaCN and lung inflation were not observed during the TTX-induced apneusis. The results indicate that apneustic respiration induced by TTX may cause a reduction of spontaneous cell activity in the medulla and pons respiratory neurons. In TXX-treated animals, picrotoxin partially restored the excitatory phrenic responses by NaCN, whereas the lung inflation effects were not restored. Therefore, it is most likely that the TTX-resistant brain stem respiratory neurons may be stimulated by picrotoxin and this effect also acts to elevate the sensitivity of inspiratory neurons closely related to the PC neurons.

Topics: Animals; Carotid Body; Chemoreceptor Cells; Cyanides; Electric Stimulation; Hypoxia; Lung; Oxygen Consumption; Phrenic Nerve; Picrotoxin; Rabbits; Respiration; Tetrodotoxin; Vagotomy

The effect of anoxia and ischemia on the release of amino acid transmitters from cerebellar slices induced by veratridine or high [K+] was studied. Synaptic specificity was tested by examining the tetradotoxin (TTX)-sensitive and the Ca2+-dependent components of stimulated release. Evoked release of endogenous amino acids was investigated in addition to more detailed studies on the stimulated efflux of preloaded [14C]GABA and D-[3H]aspartate (a metabolically more stable anologue of acidic amino acids). [14C]GABA release evoked by either method of stimulation was unaffected by periods of up to 35 min of anoxia and declined moderately by 45 min. In contrast, induced release of D-[3H]Asp increased markedly during anoxia to a peak at about 25 min, followed by a decline when anoxia was prolonged to 45 min. Evidence was obtained that the increased evoked efflux of D'[3H]Asp from anoxic slices was not due to impaired reuptake of the released amino acid and that it was completely reversible by reoxygenation of the slices. Results of experiments examining the evoked release of endogenous amino acids in anoxia were consistent with those obtained with the exogenous amino acids. Only 4 of the 10 endogenous amino acids studied exhibited TTX-sensitive veratridine-induced release under aerobic conditions (glutamate, aspartate, GABA, and glycine). Anoxia for 25 min did not affect the stimulated efflux of these amino acids with the exception of glutamate, which showed a significant increase. Compared with anoxia, effects of ischemia on synaptic function appeared to be more severe. Veratridine-evoked release of [14C]GABA was already depressed by 10 min and that of D-[3H[Asp showed a modest elevation only a 5 min. Stimulated release of D-Asp and labelled GABA declined progressively after 5 min. These findings were compared with changes in tissue ATP concentrations and histology. The latter studies indicated that in anoxia the earliest alterations are detectable in glia and that nerve terminals were the structures by far the most resistant to anoxic damage. The results thus indicated that evoked release of amino acid transmitters in the cerebellum is compromised only by prolonged anoxia in vitro. In addition, it would appear that the stimulated release of glutamate is selectively accentuated during anoxia. This effect may have a bearing on some hypoxic behavioral changes and, perhaps, also on the well-known selective vulnerability of certain neurons during hypoxia.

Topics: Amino Acids; Animals; Aspartic Acid; Brain Ischemia; Cerebellum; Female; gamma-Aminobutyric Acid; Glutamates; Glutamic Acid; Glycine; Hypoxia; In Vitro Techniques; Kinetics; Male; Neurotransmitter Agents; Potassium; Rats; Rats, Inbred Strains; Tetrodotoxin

The early effects of glucose and oxygen deprivation on the spontaneous acetylcholine output from the myenteric plexus - longitudinal muscle preparation of the guinea pig ileum were studied using an incubation chamber that permitted rapid sample collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine output. However, glucose deprivation plus hypoxia caused an acceleration in acetylcholine output within 10-15 min, which attained a rate seven times greater than observed under normal conditions. Recovery of low resting rates was obtained by reintroduction of oxygen and glucose into the bath medium. Neither morphine (2.7 x 10(-5) M) nor tetrodotoxin (1.6 x 10(-6) M) prevented the increase in acetylcholine output induced by energy deprivation. The substitution of Ca2+ by Mg2+, in the presence of EGTA, greatly reduced the acetylcholine output induced by energy deprivation. However, a small transitory output of acetylcholine was observed under these conditions which was resistant to tetrodotoxin and ot affected by depolarizing amounts of K+. The transitory output was repeatable by reintroduction of glucose and oxygen to the Ca2+-free medium with subsequent return to conditions of hypoxia and glucose deprivation. These results suggest that energy deprivation initially stimulates normal acetylcholine secretion by (a) increasing Ca2+ influx across the plasma membrane and (b) mobilizing an intracellular Ca2+ poll. This implies that processes involved in maintenance of normal low transmitter release are more sensitive to energy lack than the neurosecretion process itself.

Topics: Acetylcholine; Animals; Calcium; Dose-Response Relationship, Drug; Egtazic Acid; Glucose; Guinea Pigs; Hypoxia; In Vitro Techniques; Magnesium; Male; Morphine; Myenteric Plexus; Potassium; Tetrodotoxin; Time Factors

Topics: Acidosis; Action Potentials; Animals; Dogs; Electrophysiology; Endocardium; Hyperkalemia; Hypoxia; Membrane Potentials; Papillary Muscles; Purkinje Fibers; Solutions; Tetrodotoxin; Verapamil

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Calcium; Coronary Disease; Guinea Pigs; Heart; Hydrogen-Ion Concentration; Hypoxia; Isoproterenol; Potassium; Sodium; Tetrodotoxin

Topics: Adenine Nucleotides; Aerobiosis; Animals; Coronary Disease; Guanosine Triphosphate; Heart; Hypoxia; Insulin; Male; Muscle Proteins; Myocardium; Perfusion; Phenylalanine; Rats; Tetrodotoxin

Topics: Action Potentials; Animals; Coronary Disease; Dogs; Electric Stimulation; Endocardium; Heart Conduction System; Hypoxia; In Vitro Techniques; Lidocaine; Membrane Potentials; Purkinje Fibers; Tetrodotoxin

Excitation conduction in Meissner's plexus of the rabit small intestine was investigated by analyzing the records of potentials evoked by a single electrical stimulus applied to this plexus. Experiments were performed on the Meissner's plexus that remained attached to the circular muscle after the longitudinal muscle and mucous membrane were removed from intestinal segment. Conduction velocities of nerve impulses were 0.3-0.7 m/s, chronaxie of the nerve bundle was 0.06-0.12 ms. While the distance between the stimulating and recording electrodes was increased, the latency of evoked potentials was prolonged, the number increased, and the amplitude decreased; no potentials could be recorded when the distance was more than 4 mm. Evoked potentials recorded at relatively long conduction distance were reduced in amplitude or abolished after a repeated stimulation with high frequencies above 50/s, after hexamethonium application, and in a state of lack of oxygen. It was concluded that, in Meissner's plexus, nerve impulses spread through multiple pathways and make synaptic transmission at a relatively short conduction distance.

Topics: Animals; Electric Stimulation; Electrophysiology; Evoked Potentials; Hexamethonium Compounds; Hypoxia; Ileum; Intestine, Small; Jejunum; Rabbits; Submucous Plexus; Synapses; Synaptic Transmission; Tetrodotoxin

Excitation conduction in Auerbach's plexus of the rabbit small intestine was investigated by analyzing its evoked potentials as the response to a single electrical stimulus given to this plexus. When the conduction distance was 1 mm, two spike waves were recorded. Conduction velocities of nerve impulses were 0.3-0.5 m/s, and chronaxie was 0.06-0.11 ms. When the distance between stimulating and recording electrodes was further increased, evoked potential waves became multiple and small, until beyond 15 mm in the longitudinal direction and beyond 3 mm in the circular direction they could no longer be recorded. Evoked potentials recorded at a distance greater than 2 mm were reduced in their amplitude, and some potential waves were abolished after repeated stimulation with high frequency above 50/s, and also after hexamethonium application and lack of oxygen. It was concluded that in Auerbach's plexus nerve impulses spread through multiple pathways, conducting mainly on the longitudinal axis of the small intestine, and that some impulses make synaptaic transmission at ganglia.

Topics: Animals; Electric Stimulation; Electrophysiology; Evoked Potentials; Hexamethonium Compounds; Hypoxia; Intestine, Small; Myenteric Plexus; Neural Conduction; Rabbits; Tetrodotoxin

Canine vein strips were mounted for isometric tension recording. Anoxia did not affect basal tension of saphenous and pulmonary strips mounted in standard Krebs-Ringer solution or after 30 min of incubation in glucose-free solution. Anoxia depressed the strength of spontaneous contractions of mesenteric veins; in glucose-free solution (30 min), anoxia relaxed the strips. Veins placed in glucose-free solution for more than 60 min contracted with anoxia; this contraction was not inhibited by iproveratril. When the vein strips were contracted by norepinephrine or KCl, anoxia depressed the contractions, most in mesenteric and least in saphenous preparations; this depression was greater in the absence of glucose. When oxygen was present, the absence of glucose had little effect on the response to vasoactive agents. Contractions with acetylcholine were depressed by anoxia in mesenteric and pulmonary strips but were augmented in saphenous veins; the latter potentiation was inhibited by iproveratril and by incubation in glucose-free solution. Thus, especially in the saphenous vein, anaerobic glycolysis can provide most of the energy requirements, and intracellular substrates are available for oxidative metabolism.

Topics: Acetylcholine; Animals; Dogs; Glucose; Hypoxia; In Vitro Techniques; Mesenteric Veins; Muscle Contraction; Muscle, Smooth; Norepinephrine; Phentolamine; Potassium Chloride; Pulmonary Veins; Saphenous Vein; Tetrodotoxin; Verapamil

Topics: Action Potentials; Animals; Biological Transport, Active; Choline; Deoxyglucose; Dogs; Glucose; Heart Conduction System; Hypoxia; In Vitro Techniques; Magnesium; Norepinephrine; Potassium; Purkinje Fibers; Strophanthidin; Temperature; Tetrodotoxin

Topics: Acetylcholine; Animals; Cocaine; Cold Temperature; Drug Resistance; Electric Stimulation; Guinea Pigs; Histamine H1 Antagonists; Hypoxia; Ileum; In Vitro Techniques; Lidocaine; Methysergide; Morphine; Muscle Contraction; Muscle, Smooth; Phloretin; Potassium Chloride; Prostaglandins; Scopolamine; Serotonin Antagonists; Tetrodotoxin

1. Tetrodotoxin, at concentrations at which it abolishes generation of action potentials in the nervous system, enhances by about 300% the rate of anaerobic glycolysis of brain-cortex slices from adult rats, or from adult and infant guinea pigs. This occurs to a greater extent in Ca(2+)-deficient incubation media than in Ca(2+)-rich media. Tetrodotoxin has no accelerative effect on cerebral aerobic glycolysis. 2. Tetrodotoxin does not affect the rate of anaerobic glycolysis of 2-day-old rat brain-cortex slices, nor that of adult rat kidney medulla, nor that of an extract of an acetone-dried powder of brain. 3. Tetrodotoxin does not affect the rate of penetration of glucose into brain slices. 4. Its effect is not apparent if it is added 10min or later after the onset of anoxia. 5. Its effect diminishes as the concentration of K(+) in the incubation medium is increased while that of Na(+) is decreased. 6. Its salient effect, at the onset of anoxia, is to diminish influx of Na(+) into, and efflux of K(+) from, the brain slices. 7. Substances that promote cerebral influx of Na(+), e.g. protoveratrine, sodium l-glutamate, diminish the accelerative action of tetrodotoxin. 8. It is concluded that tetrodotoxin exerts its effect on anaerobic glycolysis by suppressing, at the onset of anoxia, the generation of action potentials and thereby the accompanying influx of Na(+) and efflux of K(+). It is suggested that glycolytic stimulation occurs because a rate-limiting step, e.g. operation of pyruvate kinase, is stimulated by K(+) and depressed by Na(+). 9. Local anaesthetics behave in a manner similar to that of tetrodotoxin in enhancing cerebral anaerobic glycolysis. 10. Sodium Amytal has a marked effect at relatively high concentration. 11. Tetrodotoxin diminishes efflux of amino acids, particularly glutamate and aspartate, at the onset of anoxia.

Topics: Action Potentials; Amobarbital; Anesthetics; Anesthetics, Local; Animals; Aspartic Acid; Calcium; Carbon Isotopes; Cerebral Cortex; Glutamates; Glycine; Glycolysis; Hypoxia; In Vitro Techniques; Potassium; Protoveratrines; Pyruvate Kinase; Rats; Sodium; Tetrodotoxin

1. A period of stimulation of intrinsic or extrinsic nerves to intestinal muscle is often followed by a secondary contraction. In the present work, the basis for such secondary contractions in the longitudinal muscle of the large bowel of guinea-pigs and rabbits was examined.2. In general, the action of cholinergic nerves did not contribute significantly to the secondary contractions. Concentrations of atropine or hyoscine, which completely blocked primary cholinergic contractions, potentiated or did not significantly reduce secondary contractions.3. Atropine resistant, nerve mediated primary contractions of the guinea-pig ileum were inhibited by anticholinesterases, although these drugs potentiated secondary contractions in other segments of the gut.4. The occurrence of a secondary contraction following inhibition of smooth muscle activity did not depend on the nature of the initial inhibition. Thus, secondary contractions were observed following the responses to both nonadrenergic and adrenergic inhibitory nerves, adenosine triphosphate, noradrenaline and brief periods of anoxia. The secondary contractions following hyperpolarizing drugs or anoxia were not prevented by tetrodotoxin in a concentration sufficient to paralyse all nerves.5. It is concluded that cholinergic nerves do not contribute significantly to secondary contractions except at frequencies of stimulation higher than about 50 Hz or after the inhibition of cholinesterases. In the segments of gut examined, the secondary contractions were principally myogenic.

Topics: Animals; Atropine; Bretylium Compounds; Cecum; Cholinesterase Inhibitors; Colon; Electric Stimulation; Female; Guinea Pigs; Hypoxia; Ileum; In Vitro Techniques; Intestines; Male; Muscle Contraction; Muscle, Smooth; Parasympathomimetics; Rabbits; Scopolamine; Tetrodotoxin

Topics: Adenosine Triphosphatases; Animals; Biological Transport; Crustacea; Cyanides; Hypoxia; Neurons; Ouabain; Peripheral Nerves; Phenobarbital; Phenytoin; Potassium; Schwann Cells; Sodium; Tetrodotoxin; Trimethadione

Topics: Acetylcholine; Action Potentials; Animals; Carotid Body; Cats; Chlorides; Cyanides; Electrophysiology; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Injections, Intra-Arterial; Injections, Intravenous; Isotonic Solutions; Potassium Chloride; Procaine; Sodium Chloride; Tetrodotoxin

Topics: Animals; Chick Embryo; Evoked Potentials; Hypoxia; Optic Lobe, Nonmammalian; Ouabain; Oxygen; Tectum Mesencephali; Tetrodotoxin

The effect of various inhibitors on insulin release from pieces of rabbit pancreas incubated in vitro was studied. Insulin release was stimulated by glucose (3mg./ml.), leucine (5mm), tolbutamide (200mug./ml.), ouabain (10mum), a raised extracellular K(+) concentration (60mm) and substitution of the Ca(2+) content of the incubation medium by Ba(2+) (2.5mm). (a) Mannoheptulose (6mg./ml.) inhibited glucose-stimulated insulin release only. (b) Anoxia abolished or inhibited insulin release stimulated by glucose, leucine, tolbutamide and K(+), but had little or no effect on release stimulated by ouabain or Ba(2+). (c) 2,4-Dinitrophenol (0.25mm) abolished or inhibited insulin release stimulated by glucose, ouabain or Ba(2+). (d) Diazoxide (250mug./ml.) abolished or inhibited insulin release stimulated by glucose, leucine, tolbutamide, ouabain or Ba(2+) (0.25 or 1mm). Diazoxide had no effect on insulin release stimulated by Ba(2+) (2.5mm) and potentiated release stimulated by K(+). (e) Adrenaline (1mum) abolished insulin release stimulated by glucose, leucine, tolbutamide, ouabain or Ba(2+). K(+)-stimulated release was inhibited by adrenaline. (f) Tetrodotoxin (1mum) had no effect on insulin release stimulated by glucose, leucine, tolbutamide, ouabain, K(+) or Ba(2+). (g) Nupercaine (1mm) abolished insulin release stimulated by glucose or Ba(2+).

Topics: Animals; Barium; Diazoxide; Dibucaine; Dinitrophenols; Epinephrine; Glucose; Heptoses; Hypoxia; In Vitro Techniques; Insulin; Insulin Secretion; Leucine; Ouabain; Pancreas; Potassium; Rabbits; Tetrodotoxin; Tolbutamide

1. The electrical events evoked in smooth muscle cells of the chick and pigeon gizzards by vagal, perivascular sympathetic and transmural stimulation were recorded with intracellular micro-electrodes.2. Single stimulating pulses applied to the extrinsic nerves, or to intrinsic fibres, produced excitatory junction potentials (E.J.P.s) which were blocked by hyoscine. Repetitive stimulation caused facilitation of E.J.P.s and, at higher frequencies, summation. When the tissue was well oxygenated, maximal stimulation evoked action potentials and the tissue contracted. If the tissue was allowed to become anoxic, action potentials were blocked and thus junctional transmission could be observed uncomplicated by tissue contraction.3. Cholinergic E.J.P.s appeared to be due to stimulation of both post-ganglionic neurones and the pre-ganglionic input to post-ganglionic neurones, since ganglion-blocking drugs abolished the late phase of complex E.J.P.s while leaving unaffected their initial components.4. Repetitive perivascular sympathetic stimulation, after cholinergic E.J.P.s had been blocked by hyoscine, evoked a slow, long-lasting depolarization of the muscle cells. This depolarization was blocked by guanethidine (5 x 10(-6) g/ml.) and was mimicked by noradrenaline (10(-7) g/ml.).5. Under non-anoxic conditions, the tissue underwent rhythmical contractions which were associated with action potential firing. When the tissue was anoxic action potentials were not seen, but spontaneous membrane depolarizations resembling E.J.P.s were observed. Spontaneous miniature excitatory junction potentials (M.E.J.P.s) were observed only infrequently whether the tissue was well oxygenated or not.6. Single stimulating pulses applied to extrinsic or intrinsic nerves frequently evoked inhibitory junction potentials (I.J.P.s) in smooth muscle cells of the gizzards of both birds. I.J.P.s were blocked by tetrodotoxin (10(-7) g/ml.). Repetitive stimulation of inhibitory nerves gave rise to a membrane hyperpolarization; when stimulation was stopped the membrane potential showed rebound depolarization.7. These results are discussed in terms of the autonomic innervation of the avian gizzard, and possible explanations of the various types of activity observed are considered.

Topics: Action Potentials; Animals; Chickens; Columbidae; Electric Stimulation; Electrophysiology; Evoked Potentials; Gizzard, Avian; Guanethidine; Hypoxia; Membrane Potentials; Muscle, Smooth; Neurons; Norepinephrine; Scopolamine; Tetrodotoxin; Vagus Nerve

Topics: Animals; Brain; Chick Embryo; Culture Techniques; Evoked Potentials; Hypoxia; Membrane Potentials; Ouabain; Tetrodotoxin; Visual Cortex