omega-agatoxin-iva and 1-4-dihydropyridine

To determine if the properties of Ca2+ channels in cerebellar Purkinje cells change during postnatal development, we recorded Ca2+ channel currents from Purkinje cells in cerebellar slices of mature (postnatal days (P) 40-50) and immature (P13-20) rats. We found that at P40-50, the somatic Ca2+ channel current was inhibited by omega-agatoxin IVA at concentrations selective for P-type Ca2+ channels (approximately 85%; IC50, <1 nM) and by the dihydropyridine (-)-(S)-Bay K8644 (approximately 70%; IC50, approximately 40 nM). (-)-(S)-Bay K8644 is known to activate L-type Ca2+ channels, but the decrease in current was not secondary to the activation of L-type channels because inhibition by (-)-(S)-Bay K8644 persisted in the presence of the L-type channel blocker (R,S)-nimodipine. By contrast, at P13-20, the current was inhibited by omega-agatoxin IVA (approximately 86%; IC50, approximately 1 nM) and a minor component was inhibited by (R,S)-nimodipine (approximately 8%). The dihydropyridine (-)-(S)-Bay K8644 had no clear effect when applied alone, but in the presence of (R,S)-nimodipine it reduced the current (approximately 40%), suggesting that activation of L-type channels by (-)-(S)-Bay K8644 masks its inhibition of non-L-type channels. Our findings indicate that Purkinje neurons express a previously unrecognized type of Ca2+ channel that is inhibited by omega-agatoxin IVA, like prototypical P-type channels, and by (-)-(S)-Bay K8644, unlike classical P-type or L-type channels. During maturation, there is a decrease in the size of the L-type current and an increase in the size of the atypical Ca2+ channel current. These changes may contribute to the maturation of the electrical properties of Purkinje cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Cerebellar Cortex; Dihydropyridines; Male; Membrane Potentials; omega-Agatoxin IVA; Patch-Clamp Techniques; Purkinje Cells; Rats; Rats, Wistar

Cerebral aneurysm rupture and subarachnoid hemorrhage (SAH) inflict disability and death on thousands of individuals each year. In addition to vasospasm in large diameter arteries, enhanced constriction of resistance arteries within the cerebral vasculature may contribute to decreased cerebral blood flow and the development of delayed neurological deficits after SAH. In this study, we provide novel evidence that SAH leads to enhanced Ca2+ entry in myocytes of small diameter cerebral arteries through the emergence of R-type voltage-dependent Ca2+ channels (VDCCs) encoded by the gene CaV 2.3. Using in vitro diameter measurements and patch clamp electrophysiology, we have found that L-type VDCC antagonists abolish cerebral artery constriction and block VDCC currents in cerebral artery myocytes from healthy animals. However, 5 days after the intracisternal injection of blood into rabbits to mimic SAH, cerebral artery constriction and VDCC currents were enhanced and partially resistant to L-type VDCC blockers. Further, SNX-482, a blocker of R-type Ca2+ channels, reduced constriction and membrane currents in cerebral arteries from SAH animals, but was without effect on cerebral arteries of healthy animals. Consistent with our biophysical and functional data, cerebral arteries from healthy animals were found to express only L-type VDCCs (CaV 1.2), whereas after SAH, cerebral arteries were found to express both CaV 1.2 and CaV 2.3. We propose that R-type VDCCs may contribute to enhanced cerebral artery constriction after SAH and may represent a novel therapeutic target in the treatment of neurological deficits after SAH.

Topics: Animals; Blood; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Channels, R-Type; Cerebral Arteries; Cisterna Magna; Dihydropyridines; Diltiazem; Disease Models, Animal; Drug Resistance; Injections; Ion Transport; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nifedipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Rabbits; Spider Venoms; Subarachnoid Hemorrhage; Vasoconstriction; Vasospasm, Intracranial

The relative contribution of Ca2+ and Na+ channels to the mechanism underlying the action of the dihydropiridines (DHPs), nimodipine, nitrendipine and nifedipine was investigated in rat striatum synaptosomes. The rise in internal Ca2+ (Ca(i), as determined with fura-2) induced by high K+ was unchanged by the DHPs, which like tetrodotoxin (TTX) inhibited both the rise in internal Na+ (Na(i), as determined with the Na+ selective indicator dye, SBFI) and the rise in Ca(i) induced by veratridine. Nimodipine and nitrendipine were much more potent than nifedipine. Oppositely to TTX and to the DHPs, the P/Q type Ca2+ channel blocker, omega-agatoxin IVA did not inhibit the rise in Ca(i) induced by veratridine, but inhibited the rise in Ca(i) induced by high K+. Veratridine-evoked release of dopamine, GABA, Glu, and Asp (detected by HPLC) was inhibited by nimodipine, nitrendipine, and TTX, while high K+-evoked release was unchanged by the DHPs or TTX. It is concluded that the reduction in presynaptic Na+ channel permeability might contribute to the cerebral effects of DHPs.

Topics: Animals; Calcium Channel Blockers; Corpus Striatum; Dihydropyridines; In Vitro Techniques; Kinetics; Male; Nerve Endings; omega-Agatoxin IVA; Rats; Rats, Wistar

In the corticotroph-like murine pituitary tumor cell line, AtT-20, adrenocorticotropic hormone release is triggered by corticotropin-releasing hormone and is attenuated by the synthetic adrenal steroid dexamethasone. The precise mechanisms by which dexamethasone inhibits secretion are under investigation. We examined whether dexamethasone can modulate release via regulation of calcium homeostasis. More specifically, we have evaluated the effects of dexamethasone on calcium current, intracellular calcium concentration, and adrenocorticotropic hormone release. Using perforated patch-clamp and calcium imaging with fura PE3/AM, we found that dexamethasone decreases calcium current and intracellular calcium levels. The inhibition of current by dexamethasone is not, however, altered by the calcium channel antagonists nifedipine (L-type) or omega-agatoxin IVA (P/Q-type), despite the presence of these calcium channel subtypes in AtT-20 cells and the exclusive coupling of adrenocorticotropic hormone release to the L-type channel in these cells. We also evaluated the temporal relationship between dexamethasone-mediated inhibition of secretion and calcium influx. Whereas a prolonged (2 h) incubation with dexamethasone inhibits corticotropin-induced release by approximately 40%, a rapid (10 min) incubation (a time interval sufficient for dexamethasone-mediated inhibition of calcium transients) does not inhibit release. These data suggest, therefore, that dexamethasone does, indeed, modulate calcium homeostasis in AtT-20 cells, but that this effect is not responsible for its inhibition of secretion.

Topics: Adrenocorticotropic Hormone; Animals; Calcium; Calcium Channel Blockers; Cell Line; Corticotropin-Releasing Hormone; Dexamethasone; Dihydropyridines; Membrane Potentials; Mice; omega-Agatoxin IVA; Patch-Clamp Techniques; Pituitary Gland; Radioimmunoassay; Spider Venoms; Tumor Cells, Cultured

The properties and modulation by norepinephrine (NE) of voltage-dependent calcium currents were studied in bulbospinal neurons (n = 116) of the rostral ventrolateral medulla (RVLM) using whole cell patch-clamp techniques in neonatal rat brain stem slices. RVLM bulbospinal neurons were identified visually by their location in slices and by the presence of flourescein isothiocyanate-tagged microbeads, which were injected into the spinal cord before the experiment; RVLM neurons were filled with Lucifer yellow during recordings, and the slice was processed for detection of tyrosine hydroxylase immunoreactivity (TH-IR). Thirty-four of 42 recovered cells (81%) were positive for TH-IR, indicating that most recorded cells were C1 neurons. Bulbospinal RVLM neurons expressed a prominent high-voltage-activated (HVA) calcium current, which began to activate at -30 to -40 mV (from a holding potential of -60 or -70 mV), and peaked at approximately 0 mV (0.8 +/- 0.1 nA;mean +/- SE). HVA current comprised predominantly omega-conotoxin GVIA-sensitive, N-type and omega-agatoxin IVA-sensitive, P/Q-type components, with smaller dihydropyridine-sensitive, L-type, and residual current components. Most RVLM bulbospinal neurons (n = 44/52, including 12/14 histologically identified C1 cells) also expressed low-voltage-activated (LVA) calcium current. LVA current began to activate at approximately -60 mV (from a holding potential of -100 mV) and was nearly completely inactivated at -50 mV with a half-inactivation potential of -70 +/- 2 mV. The amplitude of LVA current at -50 mV was 78 +/- 24 pA with Ba2+ and 156 +/- 38 pA with Ca2+ as a charge carrier. NE inhibited HVA current in most bulbospinal RVLM neurons (n = 70/77) with an EC50 of 1.2 muM; NE had no effect on LVA current. Calcium current inhibition by NE was mediated by alpha2-adrenergic receptors (alpha2-ARs) as the effect was mimicked by the selective alpha2-AR agonist, UK-14,304, and blocked by idazoxan, an alpha2-AR antagonist, but unaffected by prazosin and propranolol (alpha1- and beta-AR antagonists, respectively). Most of the NE-sensitive calcium current was N- and P/Q-type. NE-induced inhibition of calcium current evoked by action potential waveforms (APWs) was significantly larger than that evoked by depolarizing steps (34 +/- 2.5 vs. 23 +/- 2.7%; P < 0.05). Although inhibition of calcium current was voltage dependent and partially relieved by strong depolarizations, when calcium currents were evoked with a 10-Hz t

Topics: Action Potentials; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Animals; Animals, Newborn; Brimonidine Tartrate; Calcium; Calcium Channels; Calcium Channels, L-Type; Dihydropyridines; Fluorescent Dyes; Ion Channel Gating; Medulla Oblongata; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Prazosin; Propranolol; Quinoxalines; Rats; Receptors, Adrenergic, alpha-2; Spider Venoms; Tyrosine 3-Monooxygenase

1. Cells derived from a rat pituitary tumour (GC cell line) that continuously release growth hormone behave as endogenous pacemakers. In simultaneous patch clamp recordings and cytosolic Ca2+ concentration ([Ca2+]i) imaging, they displayed rhythmic action potentials (44.7 +/- 2.7 mV, 178 +/- 40 ms, 0.30 +/- 0.04 Hz) and concomitant [Ca2+]i transients (374 +/- 57 nM, 1.0 +/- 0.2 s, 0.27 +/- 0.03 Hz). 2. Action potentials and [Ca2+]i transients were reversibly blocked by removal of external Ca2+, addition of nifedipine (1 microM) or Ni2+ (40 microM), but were insensitive to TTX (1 microM). An L-type Ca2+ current activated at -33.6 +/- 0.4 mV (holding potential (Vh), -40 mV), peaked at -1.8 +/- 1.3 mV, was reduced by nifedipine and enhanced by S-(+)-SDZ 202 791. A T/R-type Ca2+ current activated at -41.7 +/- 2.7 mV (Vh, -80 or -60 mV), peaked at -9.2 +/- 3.0 mV, was reduced by low concentrations of Ni2+ (40 microM) or Cd2+ (10 microM) and was toxin resistant. Parallel experiments revealed the expression of the class E calcium channel alpha1-subunit mRNA. 3. The K+ channel blockers TEA (25 mM) and charybdotoxin (10-100 nM) enhanced spike amplitude and/or duration. Apamin (100 nM) also strongly reduced the after-spike hyperpolarization. The outward K+ tail current evoked by a depolarizing step that mimicked an action potential reversed at -69. 8 +/- 0.3 mV, presented two components, lasted 2-3 s and was totally blocked by Cd2+ (400 microM). 4. The slow pacemaker depolarization (3.5 +/- 0.4 s) that separated consecutive spikes corresponded to a 2- to 3-fold increase in membrane resistance, was strongly Na+ sensitive but TTX insensitive. 5. Computer simulations showed that pacemaker activity can be reproduced by a minimum of six currents: an L-type Ca2+ current underlies the rising phase of action potentials that are repolarized by a delayed rectifier and Ca2+-activated K+ currents. In between spikes, the decay of Ca2+-activated K+ currents and a persistent inward cationic current depolarize the membrane, activate the T/R-type Ca2+ current and initiate a new cycle.

Topics: Action Potentials; Animals; Antisense Elements (Genetics); Apamin; Barium; Biological Clocks; Cadmium; Calcium; Calcium Channel Blockers; Calcium Channels; Charybdotoxin; Computer Simulation; Cytosol; Dihydropyridines; Growth Hormone; Growth Hormone-Releasing Hormone; Nickel; Nifedipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Pituitary Neoplasms; Potassium; Rats; Ryanodine Receptor Calcium Release Channel; Sodium; Spider Venoms; Tetraethylammonium; Tetrodotoxin; Tumor Cells, Cultured

Mice were injected for 30 days with plasma from three patients with Lambert-Eaton Myasthenic Syndrome (LEMS). Recordings were made from the perineurial sheath of motor axon terminals of triangularis sterni muscle preparations. The objective was to characterize pharmacologically the identity of kinetically distinct, defined potential changes associated with motor nerve terminal Ca2+ currents (ICa) that were affected by LEMS autoantibodies. ICa elicited at 0.01 Hz were significantly reduced in amplitude by approximately 35% of control in LEMS-treated nerve terminals. During 10-Hz stimulation, ICa amplitude was unchanged in LEMS-treated motor nerve terminals, but was depressed in control. During 20- or 100-Hz trains, facilitation of ICa occurred in LEMS-treated nerve terminals whereas in control, no facilitation occurred during the trains at 20 Hz and marked depression occurred at 100 Hz. Saturation for amplitude and duration of ICa in control terminals occurred at 2 and 4-6 mM extracellular Ca2+, respectively; in LEMS-treated terminals, the extracellular Ca2+ concentration had to increase by two to three times of control to cause saturation. Amplitude of the two components of ICa observed when the preparation was exposed to 50 microM 3,4-diaminopyridine and 1 mM tetraethylammonium were both reduced by LEMS plasma treatment. The fast component (ICa,s) was reduced by 35%, whereas the slow component (ICa, s) was reduced by 37%. omega-Agatoxin IVA (omega-Aga-IVA; 0.15 microM) and omega-conotoxin-MVIIC (omega-CTx-MVIIC; 5 microM) completely blocked ICa in control motor nerve terminals. The same concentrations of toxins were 20-30% less effective in blocking ICa in LEMS-treated terminals. The residual ICa remaining after treatment with omega-Aga-IVA or omega-CTx-MVIIC was blocked by 10 microM nifedipine and 10 microM Cd2+. Thus LEMS plasma appears to downregulate omega-Aga-IVA-sensitive (P-type) and/or omega-CTx-MVIIC-sensitive (Q-type) Ca2+ channels in murine motor nerve terminals, whereas dihydropyridine (DHP)-sensitive (L-type) Ca2+ channels are unmasked in these terminals. Acute exposure (90 min) of rat forebrain synaptosomes to LEMS immunoglobulins (Igs; 4 mg/ml) did not alter the binding of [3H]-nitrendipine or [125I]-omega-conotoxin-GVIA (-omega-CgTx GVIA) when compared with synaptosomes incubated with an equivalent concentration of control Igs. Conversely, LEMS Igs significantly decreased the Bmax for [3H]-verapamil to approximately 45% of control. The ap

Topics: Animals; Autoantibodies; Binding, Competitive; Calcium; Calcium Channel Blockers; Dihydropyridines; Immunization, Passive; Lambert-Eaton Myasthenic Syndrome; Male; Mice; Mice, Inbred ICR; Motor Neurons; Nifedipine; omega-Agatoxin IVA; omega-Conotoxins; Peptides; Plasma; Potassium Channel Blockers; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Spider Venoms; Synaptosomes; Tetraethylammonium; Verapamil

L-type Ca2+ channels are characterized by their unique sensitivity to organic Ca2+ channel modulators like the 1,4-dihydropyridines (DHPs). To identify molecular motifs mediating DHP sensitivity, we transferred this sensitivity from L-type Ca2+ channels to the DHP-insensitive class A brain Ca2+ channel, BI-2. Expression of chimeras revealed minimum sequence stretches conferring DHP sensitivity including segments IIIS5, IIIS6, and the connecting linker, as well as the IVS5-IVS6 linker plus segment IVS6. DHP agonist and antagonist effects are determined by different regions within the repeat IV motif. Sequence regions responsible for DHP sensitivity comprise only 9.4% of the overall primary structure of a DHP-sensitive alpha 1A/alpha 1S construct. This chimera fully exhibits the DHP sensitivity of channels formed by L-type alpha 1 subunits. In addition, it displays the electrophysiological properties of alpha 1A, as well as its sensitivity toward the peptide toxins omega-agatoxin IVA and omega-conotoxin MVIIC.

Topics: Amino Acid Sequence; Animals; Binding Sites; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Carps; Dihydropyridines; Ion Channel Gating; Isradipine; Molecular Sequence Data; omega-Agatoxin IVA; omega-Conotoxins; Peptide Fragments; Peptides; Protein Structure, Tertiary; Pyrroles; Rabbits; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Spider Venoms

GH3 cell lines stably expressing alpha 1E channel were established and the modulation of this channel by G-protein through membrane-delimited pathways was studied. Alpha 1E channel expressed in GH3 cells showed slowing of activation and reduction of current amplitude by the application of carbachol or somatostatin. Both of these effects caused by these agents were pertussis toxin (PTX) sensitive and voltage dependent. Dialysis of the cell interior with GTP gamma S mimicked the action of these externally applied neurotransmitters, indicating that the alpha 1E channel is modulated by the PTX sensitive G-protein(s) through the membrane-delimited pathway but not by the PTX insensitive pathway that has been observed in alpha 1A channel expressed in GH3 cells. Thus different types of neuronal Ca2+ channels can be modulated not only by a similar mechanism but also by a different mechanism conferring a multilateral regulation of Ca2+ entry through these channels.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Carbachol; Cell Line; Dihydropyridines; Electric Conductivity; Gene Expression; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Neurons; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Peptides; Pertussis Toxin; Pituitary Gland, Anterior; Rats; Somatostatin; Spider Venoms; Virulence Factors, Bordetella

Helothermine (HLTx), a 25.5-kDa peptide toxin isolated from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to be an inhibitor of Ca2+ channels in cerebellar granule cells of newborn rats. Macroscopic currents, carried by 10 mM Ba2+, were measured in whole-cell configuration. The toxin at the saturating dose of 2.5 microM reversibly produced an approximately 67% block of the voltage-dependent Ca2+ current by a fast mechanism of action. The current inhibition and recovery were reached in less than 1 min. Inhibition was concentration-dependent, with a half-effective dose of 0.25 microM. The current block was practically voltage-independent, whereas the steady-state inactivation h infinity was significantly affected by HLTx (approximately 10 mV). The toxin did not affect the activation and inactivation kinetics of the Ca2+ current. Experiments with other Ca2+ channel blockers showed that HLTx abolished omega-cono-toxin GVIA-sensitive Ca2+ currents, as well as omega-Aga-IVA- and dihydropyridine-sensitive Ca2+ currents. These drugs had virtually no effect when HLTx was applied first. The present results indicate that HLTx produce a high-potency blockage of the three pharmacologically distinct Ca2+ current components.

Topics: Animals; Calcium Channel Blockers; Cerebellum; Dihydropyridines; Lizards; Mollusk Venoms; Neurons; Neurotoxins; omega-Agatoxin IVA; omega-Conotoxin GVIA; Peptides; Spider Venoms; Venoms

High voltage-activated Ca2+ currents in rat melanotropic cells consist of a sustained and an inactivating component. In this study the pharmacological properties of the high voltage-activated Ca2+ channels underlying these components are investigated with whole-cell recordings. We report that melanotropes express four pharmacologically distinct high voltage-activated Ca2+ channels. Non-inactivating L-type channels account for 35% of the total high voltage-activated channel population. These channels have a very high affinity for the dihydropyridine nimodipine (EC50 approximately 3 pM). The cone snail toxin omega-conotoxin GVIA irreversibly blocked an inactivating high voltage-activated component which accounted for 26% of the total whole-cell high voltage-activated Ca2+ current. The spider toxin omega-agatoxin IVA reversibly blocked an additional 31% of the total high voltage-activated current. The current blocked by omega-agatoxin IVA was not homogenous and consisted of a sustained component with a high affinity for omega-agatoxin IVA (< 10 nM) and an inactivating current with a low affinity for omega-agatoxin IVA (> 100 nM). Both the omega-agatoxin IVA and omega-conotoxin GVIA-blocked currents were very sensitive to nimodipine and nitrendipine with a half maximal block at 200-500 nM. 10 microM nimodipine blocked 70% of the omega-conotoxin GVIA-sensitive current and 90% of the omega-agatoxin IVA-sensitive current. Thus, omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive high voltage-activated Ca2+ channels in melanotropes have an unusual high affinity for dihydropyridines compared to N-, P-, and Q-type channels in other preparations.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Dihydropyridines; Dose-Response Relationship, Drug; Male; Melanophores; Nimodipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Pituitary Gland; Rats; Rats, Wistar; Spider Venoms

Previous studies have demonstrated multiple components of whole-cell calcium currents in hypoglossal motoneurons (HMs); HMs possess a low-voltage-activated (LVA) current and three types of high-voltage-activated (HVA) calcium currents based on sensitivity to omega-Aga IVA, omega-Conotoxin GVIA (omega-CgTx) and dihydropyridine analogs (DHPs). In the present study, we recorded single-calcium channel activities from HMs using a cell-attached patch-clamp method and found four types of channels that could be discriminated based on kinetics, voltage dependency, DHP sensitivity, and single-channel conductances. The average single-channel conductances with 110 mM barium as a charge carrier were 7, 14, 20, and 28 pS. T-type channels had a single-channel conductance of 7 pS, activated at the most negative potentials for the calcium channels and inactivated during depolarization. L-type channels (DHP-sensitive channels) did not inactivate during depolarization and had a 28-pS single-channel conductance. Based on kinetics and sensitivity to holding potential, it is likely that the channels with conductances of 14 pS and 20 pS represent N-type and P-type channels, respectively. The N-type channel (14 pS) was sensitive to holding potential, showed modal gating, and inactivated during maintained depolarizations, whereas the P-type channel (20 pS) was rather insensitive to holding potential and did not inactivate during depolarization.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Action Potentials; Animals; Calcium Channels; Dihydropyridines; Hypoglossal Nerve; Motor Neurons; Nerve Tissue Proteins; Nifedipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Rats; Rats, Sprague-Dawley; Spider Venoms

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca2+ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of --80 mV, using 10 mM Ba2+ as the charge carrier, only high-voltage-activated (HVA) Ca2+ channels were found. Ba2+ currents (IBa) showed variable sensitivity to dihydropyridine (DHP) Ca2+ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 microM and 10 microM respectively, during short (15-50 ms) depolarizing pulses to 0 mV. The L-type Ca2+ channel agonist Bay K 8644 (1 microM) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of IBa was accompanied by a 20-mV shift in the activation curves for Ca2+ channels towards more hyperpolarizing potentials. Application of the conus toxin omega-conotoxin GVIA (GVIA; 1 microM) blocked 31% of IBa; blockade was irreversible upon removal of the toxin from the extracellular medium. omega-Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of IBa. omega-Conotoxin MVIIC (MVIIC; 5 microM) produced a 36% blockade of IBa; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca2+ channels. The sequential addition of supramaximal concentrations of furnidipine (10 microM), GVIA (1 microM), IVA (100 nM) and MVIIC (3 microM) produced partial inhibition of IBa, which were additive. Our data suggest that the whole cell IBa in rat chromaffin cells exhibits at least four components.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenal Medulla; Animals; Barium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Catecholamines; Cells, Cultured; Dihydropyridines; Female; Ion Channel Gating; Ion Transport; Male; Membrane Potentials; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Patch-Clamp Techniques; Peptides; Rats; Species Specificity; Spider Venoms

The high-voltage-activated (HVA) Ba2+ currents of rat insulinoma RINm5F cells insensitive to dihydropyridines (DHP) and omega-conotoxin GVIA (omega-CTx-GVIA) have been studied for their sensitivity to omega-agatoxin-IVA (omega-Aga-IVA) and omega-CTx-MVIIC. Blockade of HVA currents by omega-Aga-IVA was partial (mean 24%), reversible and saturated around 350 nM (half block approximately 60 nM). Blockade by omega-CTx-MVIIC was more potent (mean 45%), partly irreversible and saturated above 3 microM. The effects of both toxins were additive with that of nifedipine (5 microM) and were more pronounced at positive potentials. omega-Aga-IVA action was additive with that of omega-CTx-GVIA (3 microM) but was largely prevented by cell pre-treatment with omega-CTx-MVIIC (3 microM). In contrast, omega-CTx-MVIIC block was attenuated by omega-CTx-GVIA treatment (approximately 15%), suggesting that omega-CTx-MVIIC blocks the N-type (approximately 15%) and the non-L-, non-N-type channel sensitive to omega-Aga-IVA (approximately 30%). Consistent with this, cells deprived of most non-L-type channels by pre-incubation with omega-CTx-GVIA and omega-CTx-MVIIC exhibited predominant L-type currents that activated at more negative potentials than in normal cells (-30 mV in 5 mM Ba2+) and were effectively depressed by nifedipine (maximal block of 95% from -30 mV to +40 mV). Our results suggest that, besides L- and N-type channels, insulin-secreting RINm5F cells possess also a non-L-, non-N-type channel that contributes significantly to the total current (approximately 30%). Although the pharmacology of this channel is similar to Q-type and alpha 1 class A channels, its range of activation (> -20 mV) and its slow inactivation time course resemble more that of N- and P-type channels. The channel is therefore referred to as "Q-like".

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Electric Stimulation; Insulinoma; Kinetics; Nifedipine; omega-Agatoxin IVA; omega-Conotoxins; Pancreatic Neoplasms; Peptides; Rats; Spider Venoms; Tumor Cells, Cultured

In an attempt to further characterize the type of Ca2+ channels primarily regulating GABA exocytosis, the effects of increasing concentrations of omega CTx MVIIC,-omega-Aga IVA and other Ca2+ channel blockers (nitrendipine, Cd2+ and Ni2+), commonly used for pharmacologically discerning among the various types of Ca2+ channels, were tested on the dissected Ca2+ dependent fraction of the depolarization evoked release of GABA from mouse brain synaptosomes. Our results show that omega-CTx MVIIC inhibits GABA exocytosis with a calculated IC50 of 3 microM and omega-Aga IVA with a calculated IC50 of 50 nM. The divalent cation Cd2+ only diminishes GABA exocytosis at 70 microM, but does not modify this response at lower concentrations (i.e. 1 and 10 microM). Neither nitrendipine (10 microM) nor Ni2+ (100 microM and 500 microM) modified GABA exocytosis. The failure of nitrendipine at a high concentration to inhibit GABA exocytosis discards L-type Ca2+ channels as the main regulators of this response; likewise that of Ni2+ discards Ca2+ channels of the N-type, and the failure of nM concentrations of omega-CTx MVIIC or 500 microM Ni2+, also discards alpha 1A/Q-type Ca2+ channels as the main regulators of the GABA response. On the basis of these results and in particular of the higher potency of omega-Aga IVA than omega-CTx MVIIC, it is concluded that the type of Ca2+ channels that primarily determine the exocytosis of GABA belong to a P-like type of Ca2+ channels.

Topics: Animals; Brain; Cadmium; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Exocytosis; gamma-Aminobutyric Acid; Male; Mice; Mollusk Venoms; Nerve Endings; Nickel; Nitrendipine; omega-Agatoxin IVA; omega-Conotoxins; Peptides; Spider Venoms

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 microM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca2+]i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca2+ influx pathways. Combined incubations with nifedipine 10 microM and 8-Br-cGMP reduced KCl-evoked intracellular Ca2+ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca2+]i transient induced by the L-type Ca2+ channel agonist Bay K 8644 (1 microM) under conditions of low depolarization. Neomycin (0.2 mM) and omega-AgaToxin-IVA (AgTx) (1 microM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca2+ influx pathway in the chromaffin cell.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenal Medulla; Animals; Bucladesine; Calcium; Calcium Channels; Cattle; Cells, Cultured; Cyclic GMP; Dihydropyridines; Neomycin; Nerve Tissue Proteins; omega-Agatoxin IVA; omega-Conotoxin GVIA; Peptides; Potassium Chloride; Second Messenger Systems; Spider Venoms

Diverse types of calcium channels in vertebrate neurons are important in linking electrical activity to transmitter release, gene expression and modulation of membrane excitability. Four classes of Ca2+ channels (T, N, L and P-type) have been distinguished on the basis of their electrophysiological and pharmacological properties. Most of the recently cloned Ca2+ channels fit within this functional classification. But one major branch of the Ca2+ channel gene family, including BII (ref. 15) and doe-1 (ref. 16), has not been functionally characterized. We report here the expression of doe-1 and show that it is a high-voltage-activated (HVA) Ca2+ channel that inactivates more rapidly than previously expressed calcium channels. Unlike L-type or P-type channels, doe-1 is not blocked by dihydropyridine antagonists or the peptide toxin omega-Aga-IVA, respectively. In contrast to a previously cloned N-type channel, doe-1 block by omega-CTx-GVIA requires micromolar toxin and is readily reversible. Unlike most HVA channels, doe-1 also shows unusual sensitivity to block by Ni2+. Thus, doe-1 is an HVA Ca2+ channel with novel functional properties. We have identified a Ca2+ channel current in rat cerebellar granule neurons that resembles doe-1 in many kinetic and pharmacological features.

Topics: Animals; Calcium Channels; Cerebral Cortex; Cloning, Molecular; Dihydropyridines; In Vitro Techniques; Membrane Potentials; Mollusk Venoms; Neurons; Nickel; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Oocytes; Peptides; Peptides, Cyclic; Rabbits; Rats; Skates, Fish; Spider Venoms; Xenopus