piperidines and chelerythrine

Prazosin is an α1 adrenoceptor antagonist used in pharmacotherapy for the treatment of hypertension. Prazosin alters lipid metabolism in vivo, but the involved mechanism is not fully understood. In this study, we investigated the mechanism underlying the alteration of lipid metabolism. We show that the prazosin-stimulated release of hepatic triacylglyceride lipase (HTGL) from primary cultured rat hepatocytes involved Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) activation.. Primary cultured rat hepatocytes were incubated with prazosin and other agents. The hepatocytes were used in the CaMK-II and protein kinase A (PKA) activity assay. The supernatant was used in the HTGL activity assay and western blotting.. Prazosin-stimulated HTGL release was suppressed by the inositol triphosphate receptor inhibitor xestospongin C and by the calmodulin inhibitor trifluoperazine but not by the protein kinase C inhibitor chelerythrine chloride or a diacylglycerol kinase inhibitor (R59949). Furthermore, the calmodulin-dependent protein kinase II (CaMK-II) activity in prazosin-treated hepatocytes increased in a time- and dose-dependent manner. The cAMP-dependent PKA activity of prazosin-stimulated hepatocytes was suppressed by a phospholipase C (PLC) inhibitor (U-73122), trifluoperazine, and a CaMK-II inhibitor (KN-93).. These results suggested that prazosin-stimulated HTGL release from hepatocytes was caused by activation of PKA associated with stimulation of CaMK-II activity through a signal cascade from PLC.

Topics: Animals; Benzophenanthridines; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Estrenes; Hepatocytes; Lipoprotein Lipase; Macrocyclic Compounds; Male; Oxazoles; Piperidines; Prazosin; Primary Cell Culture; Pyrrolidinones; Quinazolinones; Rats; Sulfonamides; Time Factors; Trifluoperazine

The flagellate Trypanosoma brucei causes sleeping sickness in humans and nagana in animals. Only a few drugs are registered to treat trypanosomiasis, but those drugs show severe side effects. Also, because some pathogen strains have become resistant, new strategies are urgently needed to combat this parasitic disease. An underexplored possibility is the application of combinations of several trypanocidal agents, which may potentiate their trypanocidal activity in a synergistic fashion. In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei. Alkaloids were selected that affect different molecular targets: berberine and chelerythrine (intercalation of DNA), piperine (induction of apoptosis), vinblastine (inhibition of microtubule assembly), emetine (intercalation of DNA, inhibition of protein biosynthesis), homoharringtonine (inhibition of protein biosynthesis), and digitonin (membrane permeabilization and uptake facilitation of polar compounds). Most combinations resulted in an enhanced trypanocidal effect. The addition of digitonin significantly stimulated the activity of almost all alkaloids against trypanosomes. The strongest effect was measured in a combination of digitonin with vinblastine. The highest dose reduction indexes (DRI) were measured in the two-drug combination of digitonin or piperine with vinblastine, where the dose of vinblastine could be reduced 9.07-fold or 7.05-fold, respectively. The synergistic effects of mutual combinations of alkaloids and of alkaloids with digitonin present a new avenue to treat trypanosomiasis but one which needs to be corroborated in future animal experiments.

Topics: Alkaloids; Animals; Benzodioxoles; Benzophenanthridines; Berberine; Digitonin; Drug Combinations; Drug Synergism; Emetine; Harringtonines; Homoharringtonine; Models, Theoretical; Piperidines; Polyunsaturated Alkamides; Trypanocidal Agents; Trypanosoma brucei brucei; Vinblastine

Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited binding of [(3)H]SR141716A to mouse brain membranes (IC50s: <1µM). Piperonyl butoxide and (S)-methoprene were less potent (IC50s: 21 and 63µM respectively). Benzophenanthridines and piperonyl butoxide were more selective towards brain CB1 receptors versus spleen CB2 receptors. All compounds reduced Bmax of [(3)H]SR141716A binding to CB1 receptors, but only methoprene and piperonyl butoxide increased Kd (3-5-fold). Benzophenanthridines increased the Kd of [(3)H]CP55940 binding (6-fold), but did not alter Bmax. (S)-methoprene increased the Kd of [(3)H]CP55940 binding (by almost 4-fold) and reduced Bmax by 60%. Piperonyl butoxide lowered the Bmax of [(3)H]CP55940 binding by 50%, but did not influence Kd. All compounds reduced [(3)H]SR141716A and [(3)H]CP55940 association with CB1 receptors. Combined with a saturating concentration of SR141716A, only piperonyl butoxide and (S)-methoprene increased dissociation of [(3)H]SR141716A above that of SR141716A alone. Only piperonyl butoxide increased dissociation of [(3)H]CP55940 to a level greater than CP55940 alone. Binding results indicate predominantly allosteric components to the study compounds action. 4-Aminopyridine-(4-AP-) evoked release of l-glutamate from synaptosomes was partially inhibited by WIN55212-2, an effect completely neutralized by AM251, (S)-methoprene and piperonyl butoxide. With WIN55212-2 present, benzophenanthridines enhanced 4-AP-evoked l-glutamate release above 4-AP alone. Modulatory patterns of l-glutamate release (with WIN-55212-2 present) align with previous antagonist/inverse agonist profiling based on [(35)S]GTPγS binding. Although these compounds exhibit lower potencies compared to many classical CB1 receptor inhibitors, they may have potential to modify CB1-receptor-dependent behavioral/physiological outcomes in the whole animal.

Topics: Animals; Benzophenanthridines; Benzoxazines; Binding Sites; Brain; Cyclohexanols; Glutamic Acid; Isoquinolines; Male; Methoprene; Mice; Morpholines; Naphthalenes; Piperidines; Piperonyl Butoxide; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Spleen; Synaptosomes

Cardiomyopathy is associated with cardiac Na(+) channel downregulation that may contribute to arrhythmias. Previously, we have shown that elevated intracellular NADH causes a decrease in cardiac Na(+) current (I(Na)) signaled by an increase in mitochondrial reactive oxygen species (ROS). In this study, we tested whether the NADH-mitochondria ROS pathway was involved in the reduction of I(Na) in a nonischemic cardiomyopathic model and correlated the findings with myopathic human hearts. Nonischemic cardiomyopathy was induced in C57BL/6 mice by hypertension after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and salt water substitution. Sham operated mice were used as controls. After six weeks, heart tissue and ventricular myocytes isolated from mice were utilized for whole cell patch clamp recording, NADH/NAD(+) level measurements, and mitochondrial ROS monitoring with confocal microscopy. Human explanted hearts were studied using optical mapping. Compared to the sham mice, the arterial blood pressure was higher, the left ventricular volume was significantly enlarged (104.7±3.9 vs. 87.9±6.1 μL, P<0.05), and the ejection fraction was reduced (37.1±1.8% vs. 49.4±3.7%, P<0.05) in DOCA mice. Both the whole cell and cytosolic NADH level were increased (279±70% and 123±2% of sham, respectively, P<0.01), I(Na) was decreased (60±10% of sham, P<0.01), and mitochondrial ROS overproduction was observed (2.9±0.3-fold of sham, P<0.01) in heart tissue and myocytes of myopathic mice vs. sham. Treatment of myocytes with NAD(+) (500 μM), mitoTEMPO (10 μM), chelerythrine (50 μM), or forskolin (5 μM) restored I(Na) back to the level of sham. Injection of NAD(+) (100mg/kg) or mitoTEMPO (0.7 mg/kg) twice (at 24h and 1h before myocyte isolation) to animals also restored I(Na). All treatments simultaneously reduced mitochondrial ROS levels to that of controls. CD38 was found to transduce the extracellular NAD(+) signal. Correlating with the mouse model, failing human hearts showed a reduction in conduction velocity that improved with NAD(+). Nonischemic cardiomyopathy was associated with elevated NADH level, PKC activation, mitochondrial ROS overproduction, and a concomitant decrease in I(Na). Reducing mitochondrial ROS by application of NAD(+), mitoTEMPO, PKC inhibitors, or PKA activators, restored I(Na). NAD(+) improved conduction velocity in human myopathic hearts.

Topics: Action Potentials; ADP-ribosyl Cyclase 1; Animals; Benzophenanthridines; Cardiomyopathies; Colforsin; Down-Regulation; Heart Conduction System; Humans; In Vitro Techniques; Membrane Glycoproteins; Membrane Potentials; Mice; Mice, Inbred C57BL; Mitochondria, Heart; Myocytes, Cardiac; NAD; NAV1.5 Voltage-Gated Sodium Channel; Organophosphorus Compounds; Oxidative Stress; Patch-Clamp Techniques; Piperidines; Reactive Oxygen Species

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal smooth muscle. Under current clamping, ICCs had a mean resting membrane potential of -58 ± 3 mV and externally applied ET produced membrane depolarization in a dosedependent manner. These effects were reduced by intracellular GDP beta S. A comparison of the concentration-dependent membrane depolarizations on pacemaker potentials to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1≥ET-2≥ET-3 in cultured murine small intestinal ICCs. The pretreatment with Ca(2+)-free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the ET-1 induced membrane depolarizations. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the ET-1 induced effects on pacemaker potentials. Pretreatment with BQ-123 (ET(A )receptor antagonist) or BQ-788 (ET(B )receptor antagonist) blocked the ET-1 induced effects on pacemaker potentials in cultured murine small intestinal ICCs. However, pretreatment with BQ-788 selectively did not block the ET-1 induced effects on pacemaker potentials in cultured murine large intestinal ICCs. Also, only externally applied selective ET(B )receptor agonist, IRL 1620 did not show any influence on pacemaker potentials in cultured murine large intestine ICCs. RT-PCR results indicated the presence of the ET(A )and ET(B )receptor in ICCs. These results suggested that ET-1 modulates pacemaker potentials through ET(A )and ET(B )receptor activation in murine small intestinal ICCs and ET(A )receptor activation in murine large intestinal ICCs by external Ca(2+) influx and internal Ca(2+) release via protein kinase C or cyclooxygenase-independent mechanism. Therefore, the ICCs are targets for ET and their interaction can affect intestinal motility.

Topics: Animals; Benzophenanthridines; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Endothelin-1; Endothelin-2; Endothelin-3; Interstitial Cells of Cajal; Intestine, Large; Intestine, Small; Membrane Potentials; Mice; Mice, Inbred BALB C; Naproxen; Oligopeptides; Patch-Clamp Techniques; Peptides, Cyclic; Piperidines; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Thapsigargin

Release of stored calcium ions during activation of ryanodine receptors with ryanodine or caffeine elevates the mean amplitude of spontaneous miniature end-plate potentials. Blockade of these receptors with selective antagonists abolishes this effect. Preliminary loading of the motor nerve terminals with intracellular calcium buffer EGTA-AM, but not with BAPTA-AM, also completely prevented the ryanodine-induced increment of miniature end-plate potential amplitude probably induced by the release of stored calcium. Vesamicol, a selective blocker of acetylcholine transport into vesicles, prevented the ryanodine-induced increment of the mean amplitude of miniature end-plate potentials. This increment was 2-fold more pronounced after preliminary blockade of protein kinase C with chelerythrine and was completely abolished by blockade of protein kinase A with H-89.

Topics: Acetylcholine; Animals; Benzophenanthridines; Caffeine; Calcium; Cations, Divalent; Cholinergic Antagonists; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Isoquinolines; Mice; Miniature Postsynaptic Potentials; Neuromuscular Junction; Piperidines; Presynaptic Terminals; Protein Kinase C; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sulfonamides; Tissue Culture Techniques

This investigation focused primarily on the interaction of two benzophenanthridine alkaloids (chelerythrine and sanguinarine), piperonyl butoxide and (S)-methoprene with G-protein-coupled cannabinoid CB(1) receptors of mouse brain in vitro. Chelerythrine and sanguinarine inhibited the binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at low micromolar concentrations (IC(50)s: chelerythrine 2.20 μM; sanguinarine 1.10 μM). The structurally related isoquinoline alkaloids (berberine and papaverine) and the phthalide isoquinoline ((-)-β-hydrastine) were either inactive or considerably below IC(50) at 30 μM. Chelerythrine and sanguinarine antagonized CP-55940-stimulated binding of [(35)S] GTPγS to the G-protein (IC(50)s: chelerythrine 2.09 μM; sanguinarine 1.22 μM). In contrast to AM251, both compounds strongly inhibited basal binding of [(35)S]GTPγS (IC(50)s: chelerythrine 10.06 μM; sanguinarine 5.19μM). Piperonyl butoxide and S-methoprene inhibited the binding of [(3)H]CP-55940 (IC(50)s: piperonyl butoxide 8.2 μM; methoprene 16.4 μM), and also inhibited agonist-stimulated (but not basal) binding of [(35)S]GTPγS to brain membranes (IC(50)s: piperonyl butoxide 22.5 μM; (S)-methoprene 19.31 μM). PMSF did not modify the inhibitory effect of (S)-methoprene on [(3)H]CP-55940 binding. Our data suggest that chelerythrine and sanguinarine are efficacious antagonists of G-protein-coupled CB(1) receptors. They exhibit lower potencies compared to many conventional CB(1) receptor blockers but act differently to AM251. Reverse modulation of CB(1) receptor agonist binding resulting from benzophenanthridines engaging with the G-protein component may explain this difference. Piperonyl butoxide and (S)-methoprene are efficacious, low potency, neutral antagonists of CB(1) receptors. Certain of the study compounds may represent useful starting structures for development of novel/more potent G-protein-coupled CB(1) receptor blocking drugs.

Topics: Alkaloids; Animals; Benzophenanthridines; Brain; Drug Interactions; Inhibitory Concentration 50; Isoquinolines; Male; Methoprene; Mice; Piperidines; Piperonyl Butoxide; Protein Binding; Pyrazoles; Receptor, Cannabinoid, CB1

Neurotensin (NT) is a neuropeptide involved in the modulation of nociception. We have investigated the actions of NT on cultured postnatal rat spinal cord dorsal horn (DH) neurons. NT induced an inward current associated with a decrease in membrane conductance in 46% of the neurons and increased the frequency of glutamatergic miniature excitatory synaptic currents in 37% of the neurons. Similar effects were observed in acute slices. Both effects of NT were reproduced by the selective NTS1 agonist JMV449 and blocked by the NTS1 antagonist SR48692 and the NTS1/NTS2 antagonist SR142948A. The NTS2 agonist levocabastine had no effect. The actions of NT persisted after inactivation of G(i/o) proteins by pertussis toxin but were absent after inactivation of protein kinase C (PKC) by chelerythrine or inhibition of the MAPK (ERK1/2) pathway by PD98059. Pre- and postsynaptic effects of NT were insensitive to classical voltage- and Ca(2+) -dependent K(+) channel blockers. The K(+) conductance inhibited by NT was blocked by Ba(2+) and displayed no or little inward rectification, despite the presence of strongly rectifying Ba(2+) -sensitive K(+) conductance in these neurons. This suggested that NT blocked two-pore domain (K2P) background K(+) -channels rather than inwardly rectifying K(+) channels. Zn(2+) ions, which inhibit TRESK and TASK-3 K2P channels, decreased NT-induced current. Our results indicate that in DH neurons NT activates NTS1 receptors which, via the PKC-dependent activation of the MAPK (ERK1/2) pathway, depolarize the postsynaptic neuron and increase the synaptic release of glutamate. These actions of NT might modulate the transfer and the integration of somatosensory information in the DH.

Topics: Animals; Benzophenanthridines; Excitatory Postsynaptic Potentials; Glutamic Acid; Neurotensin; Oligopeptides; Patch-Clamp Techniques; Pertussis Toxin; Piperidines; Posterior Horn Cells; Potassium Channels; Pyrazoles; Quinolines; Rats; Rats, Sprague-Dawley; Receptors, Neurotensin; Signal Transduction; Synapses; Synaptic Transmission

This study investigated whether KMUP-1, a xanthine-based derivative, inhibits L-type Ca(2+) currents (I(Ca,L)) in rat basilar artery smooth muscle cells (RBASMCs). We used whole cell patch-clamp recording to monitor Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels (LTCCs). Under voltage-clamp conditions, holding at -40 mV, KMUP-1 (1, 3, 10 μM) inhibited I(Ba) in a concentration-dependent manner and its IC(50) value was 2.27 ± 0.45 μM. A high concentration of KMUP-1 (10 μM) showed without modifying the I(Ba) current-voltage relationship. On the other hand, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM) increase I(Ba) was inhibited by KMUP-1. Pretreatment with the PKC inhibitor chelerythrine (5 μM) intensified KMUP-1-inhibited I(Ba). However, the Rho kinase inhibitor Y-27632 (30 μM) failed to affect the I(Ba) inhibition by KMUP-1. In light of these results, we suggest that KMUP-1 inhibition of LTCCs in concentration- and voltage-dependent manners in RBASMCs may be due, at least in part, to its modulation of the PKC pathway.

Topics: Animals; Barium Compounds; Basilar Artery; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels, L-Type; Chlorides; Female; Membrane Potentials; Myocytes, Smooth Muscle; Patch-Clamp Techniques; Piperidines; Protein Kinase C; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Signal Transduction; Xanthines

Verrucotoxin is the major component of venom from the stonefish (Synanceia verrucosa). Stings from the dorsal spines of the stonefish produce intensive pain, convulsions, hypotension, paralysis, respiratory weakness and collapse of the cardiovascular system, occasionally leading to death. It has been reported that verrucotoxin might modulate ATP-sensitive K+ (KATP) current in frog atrial fibers. However, the mechanism by which verrucotoxin acts on KATP current remains unclear. In this study, we examined whether verrucotoxin inhibited KATP current in guinea pig ventricular myocytes, using the patch clamp method. Verrucotoxin suppressed KATP current induced by pinacidil (KATP channel opener) in a concentration-dependent manner, with a half maximum concentration of 16.3 microg/ml. The effect of verrucotoxin on KATP current was suppressed by atropine (1 microM), a muscarinic receptor antagonist, or by 4-diphenylacetoxy-N-methylpiperidine (100 nM), a muscarinic M3 receptor antagonist. Furthermore, the effect of verrucotoxin on KATP current was attenuated by the protein kinase C (PKC) inhibitor chelerythrine (10 microM) and calphostin C (10 microM), yet not by the cAMP-dependent protein kinase (PKA) inhibitor H-89 (0.5 microM). These results suggest that verrucotoxin inhibits KATP current through the muscarinic M3 receptor-PKC pathway. These findings enhance our understanding of the toxic effects of verrucotoxin from the stonefish.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Atropine; Benzophenanthridines; Dose-Response Relationship, Drug; Female; Fish Venoms; Glycoproteins; Guinea Pigs; Heart Ventricles; In Vitro Techniques; Ion Channel Gating; Membrane Potentials; Muscarinic Antagonists; Myocytes, Cardiac; Patch-Clamp Techniques; Pinacidil; Piperidines; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Protein Kinase Inhibitors; Receptor, Muscarinic M3; Signal Transduction; Time Factors

Experiments were conducted to characterize the effects of oxytocin (OT) and vasopressin (VP) on epithelial cells isolated from human (1 degree HVD) and porcine (1 degree PVD) vas deferens and an immortalized epithelial cell line derived from porcine vas deferens (PVD9902 cells). Cultured monolayers were assessed in modified Ussing flux chambers and the OT- or VP-induced change in short circuit current (I(SC)) was recorded. All cell types responded to basolateral OT or VP with a transient increase in I(SC) that reached a peak of 3-5 microA cm(-2). Concentration-response curves constructed with 1 degree PVD and PVD9902 cells revealed that the apparent K(D) (k(app)) for OT was approximately 100-fold less than the k(app) for VP. Amplicons for the OT receptor (OXTR) and vasopressin type 2 and type 1a receptors (AVPR2 and AVPR1A) were generated with RT-PCR and the identification of each amplicon confirmed by sequence analysis. A selective antagonist for OXTR and AVPR1A fully blocked the effects of OT and partially blocked the effects of VP when assessed in both 1 degree PVD and PVD9902 monolayers. APVR2 antagonists blocked the effects of low (< or =30 nM) but not high concentrations of VP, indicating that VP was affecting both AVPR2 and a second receptor subtype, likely OXTR or AVPR1A. Experiments employing chelerythrine demonstrated that OT stimulation of vas deferens monolayers requires PKC activity. Alternatively, VP (but not OT) increased the accumulation of cytosolic cAMP in vas deferens epithelial cells. Results from this study demonstrate that OT and VP can modulate ion transport across vas deferens epithelia by independent mechanisms. OT and VP have the potential to acutely change the environment to which sperm are exposed and thus, have the potential to affect male fertility.

Topics: Alkaloids; Animals; Anions; Antidiuretic Hormone Receptor Antagonists; Benzophenanthridines; Cell Line; Cyclic AMP; Electrophysiology; Epithelial Cells; Humans; Ion Transport; Male; Oxytocin; Piperidines; Quinolones; Receptors, Oxytocin; Receptors, Vasopressin; Reverse Transcriptase Polymerase Chain Reaction; Swine; Vas Deferens; Vasopressins

To examine whether the protective effect of remifentanil preconditioning (RPC) on postischemic hearts is mediated by protein kinase (PKC) activation in comparison with ischemic preconditioning (IPC).. Male Sprague-Dawley rats were anesthetized and their chests were opened. The experiment was performed with chelerythrine (CHE, 2 mg/kg), GF109203X (0.05 mg/kg) protein kinase C (PKC) inhibitors administered before RPC (remifentanil 6 microg x kg(-1) x min(-1) x 3 cycle) or IPC, respectively. Infarct size (IS), as a percentage of the area at risk (AAR), was determined by triphenyltetrazolium staining.. In groups subjected to IPC and RPC the IS/AAR were significantly reduced (IS/AAR from 52.7%+/-5.5% to 12.9%+/-3.4%, P<0.01 vs CON and 16.2%+/-6.4%, P<0.01 vs CON), respectively. CHE and GF, both PKC inhibitors, administered 5 min before RPC or IPC completely abolished the cardioprotective effect of RPC (IS/AAR: CHE+RPC 51.2%+/-5.0%, GF+RPC 53.6%+/-6.1%, P>0.05 vs CON) or IPC (CHE+IPC 53.7%+/-4.3%, GF+IPC 54.1%+/-6.2%, P>0.05 vs CON). The difference was not significant in any of the hemodynamic parameters between control and treatment groups during ischemia and reperfusion.. Remifentanil confers myocardial protection against ischemic injury through a mechanism that is similar to IPC and involves PKC activation.

Topics: Alkaloids; Animals; Benzophenanthridines; Cardiotonic Agents; Hemodynamics; Indoles; Ischemic Preconditioning, Myocardial; Male; Maleimides; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phenanthridines; Piperidines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Remifentanil

The modulatory activity of two xanthones (3,4-dihydroxyxanthone and 1-formyl-4-hydroxy-3-methoxyxanthone) on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was evaluated using an in vivo yeast phenotypic assay. Both xanthones caused an effect compatible with PKC inhibition, similar to that elicited by known PKC inhibitors (chelerythrine and NPC 15437). PKC inhibition caused by xanthones was confirmed using an in vitro kinase assay. The yeast phenotypic assay revealed that xanthones present differences on their potency towards the distinct PKC isoforms tested. It is concluded that 3,4-dihydroxyxanthone and 1-formyl-4-hydroxy-3-methoxyxanthone may become useful PKC inhibitors and xanthone derivatives can be explored to develop new isoform-selective PKC inhibitors.

Topics: Alkaloids; Arachidonic Acid; Benzophenanthridines; Enzyme Inhibitors; Immunoblotting; Immunochemistry; Indicators and Reagents; Isoenzymes; Phenanthridines; Phenotype; Phosphatidylserines; Piperidines; Plasmids; Protein Kinase C; Saccharomyces cerevisiae; Tetradecanoylphorbol Acetate; Xanthines

We have previously shown that the atypical antipsychotic drug clozapine facilitates N-methyl-D-aspartate (NMDA)- and electrically evoked responses in pyramidal cells of the medial prefrontal cortex (mPFC). In the present study, we investigated the role of protein kinase C (PKC) in the action of clozapine. Bath administration of the PKC activator phorbol-12-myristate 13-acetate (PMA), but not the inactive isomer 4alpha-PMA, significantly enhanced the NMDA-evoked inward current and electrically evoked excitatory postsynaptic currents. Chelerythrine, a selective blocker of PKC, completely prevented the potentiating action produced by either clozapine or PMA on these currents in the mPFC cells. Intracellular injection of the PKC inhibitor PKC-I, but not the control substance PKC-S, through the recording electrode totally blocked clozapine's potentiating effect, indicating that a post-synaptic expressed PKC is critically involved in the augmenting action of clozapine on NMDA-evoked currents. Of the PKC inhibitor PKC-I, but not the control substance PKC-S, through the recording electrode totally blocked clozapine's potentiating effect, indicating that a post-synaptic expressed PKC is critically involved in the augmenting action of clozapine on NMDA-evoked currents. To further test the role of PKC in mediating the augmenting action of clozapine, we performed experiments in PKCgamma mutant and wild-type mice. In contrast to results in pyramidal cells from rats or wild-type mice, neither clozapine nor PMA was able to potentiate NMDA-induced currents in the mPFC from the PKCgamma mutant mice. Taken together, these results suggest that the PKC signal transduction pathway is critically involved in the facilitating action of clozapine on the NMDA-induced responses in pyramidal cells of the mPFC.

Topics: Alkaloids; Animals; Benzophenanthridines; Clozapine; Drug Interactions; Electric Stimulation; Electrophysiology; Enzyme Activators; Enzyme Inhibitors; Evoked Potentials; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Fluorobenzenes; GABA Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Mice; Mice, Knockout; Mice, Mutant Strains; N-Methylaspartate; Peptide Fragments; Phenanthridines; Phorbols; Piperidines; Protein Kinase C; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Serotonin Antagonists; Tetradecanoylphorbol Acetate; Time Factors

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 aim of the present study was to compare the potency of a series of widely used PKC inhibitors acting either at the regulatory (NPC 15437, tamoxifen and D-sphingosine) or at the catalytic domain (Ro 32-0432, chelerythrine and rottlerin) on individual mammalian PKC isoforms of the classical (alpha and betaI), novel (delta and eta) and atypical (zeta) PKC families, using the yeast phenotypic assay, in order to determine their isoform-selectivity. The PKC inhibitors studied presented differences in their ability to reduce the effect of the appropriate PKC activator (estimated as EC50 ratios) which was interpreted as an index of PKC inhibitory potency. In general, the more marked inhibition was observed on novel PKC isoforms, particularly on PKC-eta. This study indicates promising isoform-selectivity of some PKC inhibitors, namely NPC 15437 for PKC-eta or rottlerin for both novel PKC isoforms. It also suggests that the PKC domain involved in the inhibition does not seem to be relevant for the potency and isoform-selectivity of PKC inhibitors.

Topics: Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Catalysis; Enzyme Inhibitors; Escherichia coli; Indoles; Isoenzymes; Phenanthridines; Piperidines; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Protein Kinase C-delta; Pyrroles; Saccharomyces cerevisiae; Sphingosine; Tamoxifen; Yeasts

Human urotensin II-(1-11) and its N-terminally shortened analogues, human urotensin II-(4-11)-OH and human urotensin II-(4-11)-NH2 are potent vasoconstrictor peptides in isolated rat thoracic aorta. Human urotensin II-induced tonic aorta ring contractions are inhibited by the Ca2+ channel antagonists, verapamil, nitrendipine and diltiazem; D609 (Tricyclodecan-9-yl-xanthogenate, K), selective inhibitor of phosphatidylcholine-specific phospholipase C and partially by phospholipase C inhibitor U-73122 [1-[6-((17ss-3 Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-25-dione] and a selective inhibitor of phosphatidyl-inositol-specific phospholipase C-ET-18-OCH3 (Edelfosine,1-O-octadecyl-2O-methyl-rac-glycero-3-phosphorylcholine); protein kinase C inhibitors, chelerythrine and NPC-15437 [S-2,6-diamino-N-[[1-(1-oxotridecyl)-2-piperidinyl]methyl]-hexanamide dihydrochloride]; tyrosine kinase inhibitors, genistein and tyrphostin B42 and Rho-kinase inhibitor HA-1077 [1-(5-isoquinolinylsulfonyl)-homopiperazine dihydrochloride]. This indicates that human urotensin II-induced tonic contractions of the rat aorta are mediated by phospholipase C, protein kinase C, tyrosine kinases and Rho-kinase related pathways. In the high K+ medium, human urotensin II induces dose-dependent phasic oscillations of aortic rings. These are inhibited by Ca2+ channel antagonists, the phospholipase C inhibitor, U-73122 and protein kinase C inhibitors, chelerythrine and NPC-15437, indicating that human urotensin II-induced phasic oscillations of the rat aorta are mediated by phospholipase C and protein kinase C-dependent pathways. Given their close structural similarity, several somatostatin analogues, importantly containing DCys5 and DTrp7 and expressing different degrees of somatostatin receptor antagonist activity, were tested for possible inhibitory effects on human urotensin II-induced contractions of the rat aorta rings. Pre-incubation of rat aorta rings in the presence of somatostatin analogues, which are preferentially sst2 specific binders: PRL-2882; PRL-2903 and PRL-2915 at micro-molar concentrations significantly blocked the development of human urotensin II-induced tonic contractions. Somatostatin receptor antagonists dose-dependently inhibited human urotensin II-induced Ca2+ transients in rat thoracic aorta rings. These somatostatin receptor antagonists displayed moderate affinities for recombinant rat and human urotensin II receptor binding sites. The data support the

Topics: Alkaloids; Animals; Aorta, Thoracic; Benzophenanthridines; Calcium Channel Blockers; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Genistein; Humans; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Peptide Fragments; Peptides, Cyclic; Phenanthridines; Piperidines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Somatostatin; rho-Associated Kinases; Tyrphostins; Urotensins; Vasoconstriction

The present study was designed to examine the roles of protein kinase C (PKC) and phosphodiesterase (PDE) in modulating the action of kappa receptor stimulation on cAMP accumulation in isolated iris-ciliary bodies (ICBs) of New Zealand White rabbits. The kappa receptor agonist, (+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL), and the PKC activator, phorbol 12,13-dibutyrate (PDBu), both caused a concentration-dependent inhibition of forskolin-stimulated cAMP production. The inhibitory effect of BRL on cAMP levels was significantly reduced in the presence of the selective kappa receptor antagonist, norbinaltorphimine (10(-6) M), but the effect of PDBu was not, thus supporting the involvement of kappa-opioid receptors in the response to BRL. In the presence of 3-isobutyl-1-methylxanthine or rolipram (10(-5) M), the inhibitory effect of BRL or PDBu (10(-6) M) on cyclic AMP accumulation was abolished. In the presence of the selective PKC antagonist, chelerythrine (10(-6) M), the inhibitory effect of PDBu or BRL (10(-6) M) was significantly reduced. Direct measurement of PDE activity demonstrated the ability of BRL and PDBu (10(-6) M) to augment the activity of these enzymes. Preincubation of ICBs with rolipram (10(-5) M) or chelerythrine (10(-6) M) caused significant reversal of both BRL- and PDBu-induced increases in PDE activity. These results indicate that stimulation of PKC and PDE4 activity is part of the complex mechanism whereby kappa-opioid receptor agonists reduce levels of cAMP in the rabbit ICB. This mechanism of action could contribute to the ability of kappa-opioid agonists to suppress aqueous flow rate and to lower intraocular pressure.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; Alkaloids; Animals; Benzophenanthridines; Ciliary Body; Colforsin; Cyclic AMP; Cyclic Nucleotide Phosphodiesterases, Type 4; In Vitro Techniques; Iris; Naltrexone; Narcotic Antagonists; Phenanthridines; Phorbol 12,13-Dibutyrate; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperidines; Protein Kinase C; Pyrrolidines; Rabbits; Receptors, Opioid, kappa; Rolipram

Tachykinins have been suggested to play a significant role in the mammalian striatum, at least in part by the control of acetylcholine release from cholinergic interneurons. In the present study, we have examined the ability of known tachykinin agonists and antagonists to modulate the activity of these interneurons in mouse striatal slices. Using whole-cell patch-clamp recordings, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide each produced a dose-dependent depolarization of visually identified cholinergic interneurons that was retained under conditions designed to interrupt synaptic transmission. The nature of these neurons and the expression of multiple tachykinin receptors was confirmed using single-cell reverse transcriptase-polymerase chain reaction analysis. Using in vitro superfusion techniques, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide, respectively, each produced a dose-dependent increase in acetylcholine release, the selectivity of which was confirmed using the neurokinin-1, neurokinin-2 and neurokinin-3 receptor antagonists SR140333, GR94800 and SR142801 (100 nM). U73122 (10 microM), a phospholipase C inhibitor, blocked [sar9,Met(O2)11]substance P- and senktide-induced acetylcholine release, but had no effect on [beta-ala8]neurokinin A(4-10)-induced release. The protein kinase C inhibitors chelerythrine and Ro-31-8220 (both 1 microM) significantly inhibited responses induced by all three agonists. These findings indicate that tachykinins modulate the activity of mouse striatal cholinergic interneurons. Furthermore, neurokinin-2 receptors are shown to perform a role in mouse that has not been identified previously in other species.

Topics: 2-Amino-5-phosphonovalerate; Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Choline O-Acetyltransferase; Corpus Striatum; Electrophysiology; Enzyme Inhibitors; Estrenes; Excitatory Amino Acid Antagonists; In Situ Hybridization; Indoles; Interneurons; Membrane Potentials; Mice; Mice, Inbred C57BL; Neurokinin A; Oligopeptides; omega-N-Methylarginine; Peptide Fragments; Phenanthridines; Phosphodiesterase Inhibitors; Piperidines; Pyrrolidinones; Quinoxalines; Quinuclidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Tachykinin; RNA, Messenger; Signal Transduction; Substance P; Tachykinins; Tetrodotoxin; Tritium

Increased protein kinase C activity has been linked to diabetic vascular complications in the retina and kidney, which were attenuated by protein kinase C antagonist treatment. Neuropathy has a vascular component, therefore, the aim was to assess whether treatment with WAY151 003 or chelerythrine, inhibitors of protein kinase C regulatory and catalytic domains respectively, could correct nerve blood flow, conduction velocity, Na(+),K(+)-ATPase, and glutathione deficits in diabetic rats.. Diabetes was induced by streptozotocin. Sciatic nerve conduction velocity was measured in vivo and sciatic endoneurial perfusion was monitored by microelectrode polarography and hydrogen clearance. Glutathione content and Na(+),K(+)-ATPase activity were measured in extracts from homogenised sciatic nerves.. After 8 weeks of diabetes, sciatic blood flow was 50 % reduced. Two weeks of WAY151 003 (3 or 100 mg/kg) treatment completely corrected this deficit and chelerythrine dose-dependently improved nerve perfusion. The inhibitors dose-dependently corrected a 20 % diabetic motor conduction deficit, however, at high doses ( > 3.0 mg/kg WAY151003; > 0.1 mg/kg chelerythrine) conduction velocity was reduced towards the diabetic level. Sciatic Na(+),K(+)-ATPase activity, 42 % reduced by diabetes, was partially corrected by low but not high dose WAY151 003. In contrast, only a very high dose of chelerythrine partially restored Na(+),K(+)-ATPase activity. A 30 % diabetic deficit in sciatic glutathione content was unchanged by protein kinase C inhibition. The benefits of WAY151 003 on blood flow and conduction velocity were blocked by nitric oxide synthase inhibitor co-treatment.. Protein kinase C contributes to experimental diabetic neuropathy by a neurovascular mechanism rather than through Na(+),K(+)-ATPase defects.

Topics: Alkaloids; Animals; Benzophenanthridines; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Enzyme Inhibitors; Male; Neural Conduction; Phenanthridines; Piperidines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Sciatic Nerve; Sodium-Potassium-Exchanging ATPase