kt-5720 and chelerythrine

Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by progressive and selective death of dopaminergic neurons. Multifunctional neuropeptide orexin-A is involved in many biological events of the body. It has been shown that orexin-A has protective effects in neurodegenerative disease such as PD. However, its cellular mechanisms have not yet been fully clarified. Here, we investigated the intracellular signaling pathway of orexin-A neuroprotection in 6-hydroxydopamine (6-OHDA)-induced SH-SY5H cells damage as an in vitro model of PD. The cells were incubated with 150 μM 6-OHDA, and the viability was examined by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Mitochondrial membrane potential and intracellular calcium were measured by fluorescent probes. Western blotting was also used to determine cyclooxygenase type 2 (COX-2), nuclear factor erythroid 2 related factor 2 (Nrf2), and HSP70 protein levels. The data showed that 6-OHDA has decreasing effects on cell viability, Nrf2, and HSP70 protein expression and increases the level of mitochondrial membrane potential, intracellular calcium, and COX-2 protein. Orexin-A (500 pM) significantly attenuated the 6-OHDA-induced cell damage. Furthermore, Orexin-A significantly prevented the mentioned effects of 6-OHDA on SH-SY5Y cells. Orexin 1 receptor antagonist (SB3344867), PKC, and PI3-kinase (PI3K) inhibitors (chelerythrin and LY294002, respectively) could suppress the orexin-A neuroprotective effect. In contrast, blockage of PKA by a selective inhibitor (KT5720) had no effects on the orexin protection. The results suggest that orexin-A protective effects against 6-OHDA-induced neurotoxicity are performed via its receptors, PKC and PI3K signaling pathways.

Topics: Benzophenanthridines; Calcium; Carbazoles; Cell Line, Tumor; Cell Survival; Chromones; Cyclooxygenase 2; HSP70 Heat-Shock Proteins; Humans; Intracellular Space; Membrane Potential, Mitochondrial; Morpholines; Neuroblastoma; Neuroprotective Agents; Neurotoxins; NF-E2-Related Factor 2; Orexin Receptors; Orexins; Oxidopamine; Phosphatidylinositol 3-Kinases; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Signal Transduction

Recent efforts to identify the molecules that are involved in the maintenance of long-term memories in mammals have focused attention on atypical isoforms of protein kinase C (PKC). Inhibition of these kinases by either the general PKC inhibitor, chelerythrine, or the more specific inhibitor, zeta inhibitory peptide (ZIP), can abolish both long-term potentiation in the hippocampus and as well as spatial, fear, appetitive, and sensorimotor memories. These inhibitors can also abolish long-term facilitation and long-term sensitization in the mollusk Aplysia californica. We have extended these results to an insect, the cockroach Leucophaea maderae. We show that systemic injections of either chelerythrine or ZIP erase long-term olfactory memories in the cockroach, but have no effect on memory acquisition during conditioning. We also show that inhibition of either protein kinase A (PKA) or protein synthesis can block memory acquisition but neither has an effect on the memory once it is formed. The results suggest that sustaining memories in insects requires the persistent activity of one or more isoforms of PKC and point to a strong evolutionary conservation of the molecular mechanisms that underlie the persistence of long-term memories in the central nervous system.

Topics: Animals; Benzophenanthridines; Carbazoles; Cell-Penetrating Peptides; Cockroaches; Conditioning, Psychological; Lipopeptides; Memory, Long-Term; Protein Kinase Inhibitors; Protein Synthesis Inhibitors; Pyrroles

Synapses express different forms of plasticity that contribute to different forms of memory, and both memory and plasticity can become labile after reactivation. We previously reported that a persistent form of nonassociative long-term facilitation (PNA-LTF) of the sensorimotor synapses in Aplysia californica, a cellular analog of long-term sensitization, became labile with short-term heterosynaptic reactivation and reversed when the reactivation was followed by incubation with the protein synthesis inhibitor rapamycin. Here we examined the reciprocal impact of different forms of short-term plasticity (reactivations) on a persistent form of associative long-term facilitation (PA-LTF), a cellular analog of classical conditioning, which was expressed at Aplysia sensorimotor synapses when a tetanic stimulation of the sensory neurons was paired with a brief application of serotonin on 2 consecutive days. The expression of short-term homosynaptic plasticity [post-tetanic potentiation or homosynaptic depression (HSD)], or short-term heterosynaptic plasticity [serotonin-induced facilitation or neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFa)-induced depression], at synapses expressing PA-LTF did not affect the maintenance of PA-LTF. The kinetics of HSD was attenuated at synapses expressing PA-LTF, which required activation of protein kinase C (PKC). Both PA-LTF and the attenuated kinetics of HSD were reversed by either a transient blockade of PKC activity or a homosynaptic, but not heterosynaptic, reactivation when paired with rapamycin. These results indicate that two different forms of persistent synaptic plasticity, PA-LTF and PNA-LTF, expressed at the same synapse become labile when reactivated by different stimuli.. Activity-dependent changes in neural circuits mediate long-term memories. Some forms of long-term memories become labile and can be reversed with specific types of reactivations, but the mechanism is complex. At the cellular level, reactivations that induce a reversal of memory must evoke changes in neural circuits underlying the memory. What types of reactivations induce a labile state at neural connections that lead to reversal of different types of memory? We find that a critical neural connection in Aplysia, which is modified with different stimuli that mediate different types of memory, becomes labile with different types of reactivations. These results provide insights for developing strategies in alleviating maladaptive memories accompanying anxiety disorders.

Topics: Animals; Aplysia; Benzophenanthridines; Biophysics; Carbazoles; Cells, Cultured; Conditioning, Classical; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; FMRFamide; Ganglia, Sensory; Long-Term Potentiation; Nerve Net; Patch-Clamp Techniques; Pyrroles; Sensory Receptor Cells; Serotonin; Synapses; Time Factors

It has been reported that training in behavioral tasks modifies the ability to induce long-term potentiation (LTP) in an N-methyl-D-aspartate receptor (NMDAR)-dependent manner. This receptor leads to calcium entry into neuronal cells, promoting the activation of protein kinases as protein kinase A (PKA) and protein kinase C (PKC), which contribute significantly to the formation of different types of memories and play a pivotal role in the expression of LTP. Our previous studies involving the insular cortex (IC) have demonstrated that induction of LTP in the basolateral amygdaloid nucleus (BLA)-IC projection prior to conditioned taste aversion (CTA) training enhances the retention of this task. Recently, we showed that CTA training triggers a persistent impairment in the ability to induce subsequent synaptic plasticity on the BLA-IC pathway in a protein synthesis-dependent manner, but the underlying molecular mechanisms remain unclear. In the present study we investigated whether the blockade of NMDAR, as well as the inhibition of PKC and PKA affects the CTA-dependent impairment of the IC-LTP. Thus, CTA-trained rats received high frequency stimulation in the Bla-IC projection in order to induce LTP 48 h after the aversion test. The NMDAR antagonist CPP and the specific inhibitors for PKC (chelerythrine) and PKA (KT-5720) were intracortically administered during the acquisition session. Our results show that the blockade of NMDAR and the inhibition of PKC activity prevent the CTA memory-formation as well as the IC-LTP impairment. Nevertheless, PKA inhibition prevents the memory formation of taste aversion but produces no interference with the CTA-dependent impairment of the IC-LTP. These findings reveal the differential roles of protein kinases on CTA-dependent modification of IC-LTP enhancing our understanding of the effects of memory-related changes on synaptic function.

Topics: Animals; Avoidance Learning; Benzophenanthridines; Carbazoles; Cerebral Cortex; Cyclic AMP-Dependent Protein Kinases; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Long-Term Potentiation; Male; Piperazines; Protein Kinase C; Pyrroles; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Taste

Although up-regulation of β-adrenergic receptors (β-ARs) occurs after long-term use of their antagonists in various tissues, the available data are little on mechanisms of β-AR up-regulation induced by their continuous blockade. The present study attempted to clarify mechanisms of β-AR up-regulation using mouse cerebral cortical neurons continuously exposed to nadolol (10 nM), a non-selective β-AR antagonist, for 24 h. Nadolol dose-dependently induced both subtypes of β-ARs, β₁- and β₂-ARs, which were not suppressed by protein A kinase inhibition with KT5720. On the other hand, blockade of α₁-ARs, which are immunohistochemically confirmed to be co-localized with β-ARs in the same neurons, significantly inhibited only β₁-AR up-regulation and the expression of β₂-ARs did not alter. In addition, phenylephrine, an agonist specific to α₁-ARs up-regulated β₁-ARs, but not β₂-ARs. Under the conditions with β-AR up-regulation, the level of phosphorylated protein kinase Cα (pPKCα) increased, which is significantly suppressed by prazosin, an α1-AR antagonist. Furthermore, nadolol decreased the degradation of mRNA of β₁-ARs, but not β₂-ARs. These results indicate that the nadolol-induced β₁-AR up-regulation is mediated via PKC-relating pathway via α₁-AR activation with stabilizing β₁-AR mRNA and that the increased expression of β₂-ARs is regulated by pathways different from those for β₁-AR expression.

Topics: Adrenergic Agents; Adrenergic beta-Antagonists; Analysis of Variance; Animals; Benzophenanthridines; Carbazoles; Cells, Cultured; Cerebral Cortex; Dopamine beta-Hydroxylase; Embryo, Mammalian; Enzyme Inhibitors; Mice; Mice, Inbred ICR; Nadolol; Neurons; Pyrroles; Receptors, Adrenergic, alpha-1; Receptors, Adrenergic, beta-1; Signal Transduction; Time Factors; Up-Regulation

Ventricular tachyarrhythmias are often precipitated by physical or emotional stress, indicating a link between increased adrenergic stimulation and cardiac ion channel activity. Human ether-a-go-go related gene (hERG) potassium channels conduct the rapid component of delayed rectifier potassium current, I(kr), a crucial component for action potential repolarization. To evaluate the correlation between increased alpha(1)-adrenergic activity and the rapid component of cardiac I(kr), whole-cell patch-clamp recording was performed in isolated guinea-pig ventricular myocytes. Stimulation of alpha(1)-adrenoceptors using phenylephrine (0.1 nM-100 microM) reduced I(kr) current in a dose-dependent manner at 37 degrees C. Phenylephrine (0.1 microM) reduced I(kr) current to 66.83+/-3.16%. Chelerythrine (1 microM), a specific inhibitor of protein kinase C (PKC) completely inhibited the changes in I(kr) trigged by 0.1 microM phenylephrine. KT5720 (2.5 microM), a specific inhibitor of protein kinase A (PKA) partially inhibited the current decrease induced by 0.1 microM phenylephrine. Both chelerythrine and KT5720 drastically reduced the phenylephrine-induced effects, indicating possible involvement of PKC and PKA in the alpha(1)-adrenergic inhibition of I(kr). Our data suggest a link between I(kr) and the alpha(1)-adrenoceptor, involving activation of PKC and PKA in arrhythmogenesis.

Topics: Animals; Benzophenanthridines; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Guinea Pigs; Heart Ventricles; Kinetics; Myocytes, Cardiac; Patch-Clamp Techniques; Phenylephrine; Potassium; Potassium Channels, Inwardly Rectifying; Protein Kinase C; Pyrroles; Receptors, Adrenergic, alpha-1

Opioids can induce analgesia and also hyperalgesia in humans and in animals. It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. However, the exact mechanism(s) underlying opioid-induced hyperalgesia has not yet been clarified. Here, we have investigated cellular events involved in low-dose morphine hyperalgesia in male Wistar rats. The data showed that morphine (0.01 microg i.t.) could elicit hyperalgesia as assessed by the tail-flick test. G(alphas) mRNA and protein levels increased significantly following exposure to the hyperalgesic dose of morphine. Furthermore, morphine at an analgesic dose (20 microg i.t.) significantly decreased cAMP levels in the dorsal half of the lumbar spinal cord, whereas the tissue cAMP levels were not affected by morphine treatment at a hyperalgesic dose. Intrathecal administration of nifedipine, an L-type calcium channel blocker, antagonized the hyperalgesia induced by the low-dose of morphine. Furthermore, pretreatment with the selective protein kinase C (PKC) inhibitor chelerytrine resulted in prevention of the morphine-induced hyperalgesia. KT 5720, a specific inhibitor of protein kinase A (PKA), did not show any effect on low-dose morphine-induced hyperalgesia. These results indicate a role for G(alphas), the PLC-PKC pathway, and L-type calcium channels in intrathecal morphine-induced hyperalgesia in rats. Activation of ordinary G(alphas) signaling through cAMP levels did not appear to play a major role in the induction of hyperalgesia by low-dose of morphine.

Topics: Alkaloids; Analgesics, Opioid; Animals; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels, L-Type; Carbazoles; Cyclic AMP; Dose-Response Relationship, Drug; Gene Expression; GTP-Binding Proteins; Hyperalgesia; Immunoblotting; Indoles; Male; Morphine; Nifedipine; Polymerase Chain Reaction; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Rats; Rats, Wistar; RNA, Messenger; Spinal Cord

Incubation with serum modulates the transporters that regulate intracellular pH (pH(i)) in articular chondrocytes, upregulating acid extrusion by Na(+)-H(+) exchange (NHE). There is stimulation of NHE1, together with induction of NHE3 activity. These isoforms exhibit differential responses to components of mechanical load experienced by chondrocytes during joint loading. The identity of the component(s) of serum responsible is unknown. A possibility, however, is insulin-like growth factor-1 (IGF-1), present in normal cartilage and found at enhanced levels in osteoarthritic tissue. In the present study, the effects of IGF-1 on pH(i) regulation have been characterized using fluorescence measurements of bovine articular chondrocytes, and the sensitivity of pH(i) regulation to hyperosmotic shock and raised hydrostatic pressure determined. For cells isolated in the absence of IGF-1, pH(i) recovery following acidification was predominantly mediated by NHE1. Recovery was enhanced when cells were incubated for 18 h with 20 ng mL(-1) IGF; this effect represented increased acid extrusion by NHE1, supplemented by NHE3 activity. NHE3 activity was not detected in IGF-1-treated cells that had been incubated with the protein synthesis inhibitor cycloheximide, although NHE1 activity was unaffected. In the absence of IGF-1, suspension in hyperosmotic solutions or raised hydrostatic pressure enhanced pH(i) recovery of acidified cells. This response was missing in cells incubated with IGF-1. Unresponsiveness to hyperosmotic shock represented inhibition of NHE3 activity, and was prevented using the protein kinase A inhibitor KT5720. For raised hydrostatic pressure, a decrease in NHE1 activity was responsible, and was prevented by the protein kinase C inhibitor chelerythrine.

Topics: Animals; Benzophenanthridines; Carbazoles; Cartilage, Articular; Cation Transport Proteins; Cattle; Cells, Cultured; Chondrocytes; Cycloheximide; Drug Combinations; Enzyme Inhibitors; Hydrogen-Ion Concentration; Hydrostatic Pressure; Insulin-Like Growth Factor I; Male; Osmotic Pressure; Protein Synthesis Inhibitors; Pyrroles; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers

The effects of (+/-)3,4-methylenedioxyamphetamine (MDA) were studied in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. The RP4 neuron generated spontaneous action potentials. Extracellular or intracellular application of MDA elicited action potential bursts of the central RP4 neuron. The action potential bursts elicited by MDA were not blocked when neurons were immersed in high-Mg2+ solution, Ca2+-free solution, nor after continuous perfusion with atropine, d-tubocurarine, propranolol, prazosin, haloperidol, sulpiride or methiothepin. Notably, the induction of action potential bursts was blocked by pretreatment with protein kinase C (PKC) inhibitors, chelerythrine and Ro 31-8220, but not by protein kinase A (PKA) inhibitors, KT-5720 and H89, nor by the phospholipase C (PLC) inhibitor, U73122. PKC activators, i.e., phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acety-sn-glycerol (OAG; a membrane-permeant DAG analog), facilitate the induction of action potential bursts elicited by MDA. Voltage-clamp studies revealed that MDA decreased the delayed rectifying K+ current (I(KD)) of the RP4 neuron. Further, although Ro 31-8220 did not affect the I(KD), Ro 31-8220 decreased the inhibitory effect of MDA on the I(KD). These results suggest that the generation of action potential bursts elicited by MDA was not due to (1) the synaptic effects of neurotransmitters, (2) the cholinergic, adrenergic, dopaminergic or serotoninergic receptors of the excitable membrane. Instead, the MDA-elicited action potential bursts are closely related to PKC activity and the inhibitory effects on the I(KD).

Topics: Action Potentials; Alkaloids; Animals; Benzophenanthridines; Calcium; Carbazoles; Electrophysiology; Enzyme Inhibitors; Estrenes; Ganglia, Invertebrate; Hallucinogens; In Vitro Techniques; Indoles; Isoquinolines; Magnesium; N-Methyl-3,4-methylenedioxyamphetamine; Neurons; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Protein Kinase C; Pyrroles; Pyrrolidinones; Snails; Sulfonamides; Tetradecanoylphorbol Acetate

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.

Topics: Adenosine Triphosphate; Alkaloids; Aminoimidazole Carboxamide; Animals; Antimycin A; Benzophenanthridines; Brefeldin A; Carbazoles; Cell Hypoxia; Cells, Cultured; Connexin 43; Cycloheximide; Deoxyglucose; Flavonoids; Imidazoles; Indoles; JNK Mitogen-Activated Protein Kinases; Maleimides; Myocardial Contraction; Myocytes, Cardiac; Okadaic Acid; Ouabain; Phenanthridines; Phosphorylation; Potassium Cyanide; Protein Processing, Post-Translational; Pyridines; Pyrroles; Rats; Recombinant Fusion Proteins; Ribonucleotides; Staurosporine; Tacrolimus; Tetradecanoylphorbol Acetate

Brain-derived neurotrophic factor (BDNF) plays fundamental roles in synaptic plasticity in rat hippocampus. Recently, using rat hippocampal slices, we found that BDNF induces activation of calcium/calmodulin-dependent protein kinase 2 (CaMKII), a critical mediator of synaptic plasticity. CaMKII in turn activates the p38 subfamily of mitogen-activated protein kinases (MAPK) and its downstream effector, MAPK-activated protein kinase 2 (MAPKAPK-2). Herein, we determined whether some kinases of this pathway connect BDNF to the cyclic AMP response element -binding protein (CREB), a transcription factor also involved in plasticity and survival. Crude cytosolic and nuclear fractions were prepared from hippocampal slices of adult rat, and then kinase involvement in CREB phosphorylation was studied with a combination of pharmacologic inhibition and antibody depletion. In addition, the regional localization of this signaling pathway was immunohistochemically investigated. We show that: (i). the BDNF-stimulated CaMKII cascade phosphorylates the key positive regulatory site of CREB via its end MAPKAPK-2 component; (ii). this process appears to be highly localized in the outermost cell layer of the dentate gyrus. The present findings suggest that CaMKII is involved in neurotrophic-dependent activation of CREB in the dentate gyrus. Such a signaling process could be important for controlling synaptic plasticity in this major area for the afferent inputs to the hippocampal formation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Androstadienes; Animals; Antibodies; Benzophenanthridines; Benzopyrans; Blotting, Western; Brain-Derived Neurotrophic Factor; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Carbazoles; Cell Nucleus; Chromones; Cyclic AMP Response Element-Binding Protein; Cytosol; Enzyme Inhibitors; Flavonoids; Hippocampus; Imidazoles; Immunohistochemistry; In Vitro Techniques; Indoles; Intracellular Signaling Peptides and Proteins; Male; Morpholines; Naphthalenes; Phenanthridines; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Pyridines; Pyrroles; Rats; Signal Transduction; Time Factors; Tyrphostins; Wortmannin

Serotonin (5-hydroxytryptamine [5-HT]) receptors are located in peripheral tissues as well as in the central nervous system. Serotonin receptors mediate positive inotropic and chronotropic effects in atria. The aim of this study was to investigate physiological role of endogenous serotonin on the regulation of atrial natriuretic peptide (ANP) secretion from the atria.. An isolated perfused nonbeating rat atrial model was used. Changes in atrial volume induced by increasing intra-atrial pressure were measured. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance.. Serotonin, an endogenous 5-HT receptor agonist, caused concentration-dependent suppressions of stretch-induced ANP secretion, which were less pronounced than those caused by alpha-methyl-5-HT maleate, a 5-HT(2) receptor selective agonist. The suppression of stretch-induced ANP secretion due to serotonin and alpha-methyl-5-HT maleate was attenuated by ketanserin, a 5-HT(2) receptor antagonist, and accentuated by RS23597-190, a 5-HT(4) receptor antagonist. The suppressive effect of serotonin on ANP secretion was attenuated by neomycin, staurosporine, and chelerythrine. In contrast, 2-[1-(4-piperonyl)piperazinyl]benzothiazole, a 5-HT(4) receptor selective agonist, caused an accentuation of stretch-induced ANP secretion, which was completely blocked by RS23597-190 and SB203186 HCl but not by ketanserin. This effect was not affected by MDL12330, KT-5720, or H-89. The intracellular Ca(2+) concentration in single atrial myocytes was not changed by serotonin and agonist for either 5-HT(2) or 5-HT(4) receptor.. These results suggest that atrial 5-HT(2) and 5-HT(4) receptor agonists have opposite actions on the regulation of ANP secretion and the suppressive effect of serotonin on the ANP secretion may act through 5-HT(2) receptor and phospholipase C pathway.

Topics: Adenylyl Cyclase Inhibitors; Alkaloids; Aminobenzoates; Animals; Atrial Natriuretic Factor; Benzophenanthridines; Benzothiazoles; Calcium; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Depression, Chemical; Dose-Response Relationship, Drug; Heart; Heart Atria; Imines; Indoles; Isoquinolines; Ketanserin; Male; Myocytes, Cardiac; Neomycin; para-Aminobenzoates; Perfusion; Phenanthridines; Piperazines; Piperidines; Protein Kinase C; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; Receptors, Serotonin, 5-HT4; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Staurosporine; Sulfonamides; Thiazoles; Type C Phospholipases

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Alkaloids; Animals; Benzophenanthridines; Carbazoles; Chloride Channels; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Estrenes; In Vitro Techniques; Indoles; Membrane Potentials; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Phenanthridines; Portal Vein; Prazosin; Propranolol; Pyrroles; Pyrrolidinones; Rabbits; Tetradecanoylphorbol Acetate

Antigen processing in B cells is initiated by antigen binding to the surface B cell antigen receptor (BCR). The BCR is a signaling receptor which also functions to endocytose bound antigen for subsequent intracellular processing and presentation with class II molecules. Previously, using subcellular fractionation, we showed that although the surface BCR constitutively traffics from the cell surface to the class II peptide-loading compartment (IIPLC), cross-linking the BCR regulates trafficking, resulting in a more rapid movement of the BCR to the IIPLC (Song et al., 1995, J. Immunol. 155, 4255). The rate of degradation of both the BCR and the bound antigen was also accelerated following BCR cross-linking. Here we provide evidence that the effect of cross-linking the BCR on antigen processing is in part dependent on signal cascades initiated by the BCR. We show that the protein kinase inhibitors Genistein and Chelerythrine, which block BCR signaling, reduce BCR-enhanced antigen processing in a dose-dependent manner. The kinase inhibitors have a small effect on the rate of internalization of the BCR and antigen following BCR cross-linking and significantly decrease the accelerated trafficking to the IIPLC. The increased rate of degradation of the BCR and antigen induced by BCR cross-linking is also decreased by the kinase inhibitors. BCR signaling does not appear to have a global effect on intracellular membrane trafficking as cross-linking the BCR did not alter the rate of trafficking of newly synthesized class II molecules to the IIPLC. Thus, the signaling function of the BCR appears to play a significant role in regulating discrete steps in the intracellular antigen processing pathway.

Topics: Alkaloids; Animals; Antigen Presentation; Benzophenanthridines; Carbazoles; Genistein; Histocompatibility Antigens Class II; Indoles; Mice; Phenanthridines; Protein Kinases; Pyrroles; Rabbits; Receptors, Antigen, B-Cell