kn-62 and chelerythrine

Nifedipine, a blocker of L-type Ca(2+)-channels, increased quantal content of endplate potentials in newly formed nerve-muscle synapses, while R 24571 (calmodulin inhibitor) and KN 62 (inhibitor of calmodulin-dependent kinase II) did not change it. KN 62 suppressed the increase in quantal content of endplate potentials evoked by nifedipine. Similar to nifedipine, chelerythrine and bisindolylmaleimide I (blockers of protein kinase C) increased quantal content of endplate potentials. In the presence of chelerythrine, nifedipine lost its ability to facilitate secretion of neurotransmitter. 4-Aminopyridine, a blocker of voltage-gated potassium channels, increased quantal content of endplate potentials. In the presence of chelerythrine, 4-aminopyridine induced no additional increase in the quantal content of endplate potentials. In its turn, chelerythrine induced no extra facilitation of secretion in the presence of 4-aminopyridine. It is hypothesized that Ca(2+)-dependent inhibition of secretion results from suppression of calmodulin-dependent kinase II and activation of protein kinase C, which potentiates the work of voltage-gated K(+)-channels.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 4-Aminopyridine; Acetylcholine; Animals; Benzophenanthridines; Calcium Channels, L-Type; Calcium-Calmodulin-Dependent Protein Kinases; Excitatory Postsynaptic Potentials; Imidazoles; Indoles; Maleimides; Mice; Muscle, Skeletal; Nifedipine; Potassium Channels, Voltage-Gated; Protein Kinase C; Synapses

1 Extracellular ATP can activate a cation-selective channel/pore on human B-lymphocytes, known as the P2X7 receptor. Activation of this receptor is linked to PLD stimulation. We have used ATP-induced 86Rb+ (K+) efflux to examine the effect of benzophenanthridine alkaloids on P2X7 channel/pore function in human B-lymphocytes. 2 Both ATP and the nucleotide analogue 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) induced an 86Rb+ efflux, which was completely inhibited by the isoquinoline derivative 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine (KN-62), a potent P2X7 receptor antagonist. 3 The benzophenanthridine alkaloid chelerythrine, a potent PKC inhibitor, inhibited the ATP-induced 86Rb+ efflux by 73.4+/-3.5% and with an IC50 of 5.6+/-2.3 microm. Similarly, other members of this family of compounds, sanguinarine and berberine, blocked the ATP-induced 86Rb+ efflux by 58.8+/-4.8 and 61.1+/-8.0%, respectively. 4 Concentration-effect curves to ATP estimated an EC50 value of 78 microm and in the presence of 5 and 10 microm chelerythrine this increased slightly to 110 and 150 microm, respectively, which fits a noncompetitive inhibitor profile for chelerythrine. 5 Chelerythrine at 10 microm was effective at inhibiting the ATP-induced PLD stimulation in B-lymphocytes by 94.2+/-21.9% and the phorbol 12-myristate 13-acetate-induced PLD stimulation by 68.2+/-7.4%. 6 This study demonstrates that chelerythrine in addition to PKC inhibition has a noncompetitive inhibitory action on the P2X7 receptor itself.

Topics: Adenosine Triphosphate; Alkaloids; B-Lymphocytes; Benzophenanthridines; Berberine; Enzyme Activation; Enzyme Inhibitors; Humans; Isoquinolines; Kinetics; Phenanthridines; Phospholipase D; Protein Kinase C; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Rubidium Radioisotopes; Tetradecanoylphorbol Acetate; Time Factors

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

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Amides; Animals; Benzamides; Benzophenanthridines; Benzopyrans; Butadienes; Cell Line; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Indoles; Inhibitory Concentration 50; Isoquinolines; Lithium; Magnesium; Nitriles; Phenanthridines; Phosphorylation; Potassium Chloride; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Sirolimus; Substrate Specificity; Sulfonamides

The effects of cocaine on glycine-induced Cl- current (I(GLY)) of single neurons, freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Cocaine depressed I(GLY) in a concentration-dependent manner, with an IC50 of 0.78 mM. Preincubation with 1 mM cocaine alone had no effect on I(GLY), suggesting that resting glycine channels are insensitive to cocaine. The depression of I(GLY) by cocaine was independent of membrane voltage. Internal cell dialysis with 1 mM cocaine failed to modify I(GLY). Because the depression of I(GLY) was noncompetitive, cocaine may act on the glycine receptor-chloride ionophore complex at a site distinct from that to which glycine binds. The cocaine suppression of I(GLY) was unaffected by 1 microM tetrodotoxin and 1 microM strychnine. Blockers of protein kinase C (Chelerythrine), kinase A (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl, (H-89)) and Ca-calmodulin-dependent kinase (1-[N,O-bis(5-isoquinoline-sulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine (KN-62)) were also ineffective, which suggests that these phosphorylating mechanisms do not modulate cocaine-induced suppressant action on I(GLY). This extracellular, strychnine-independent depression of I(GLY) may contribute to cocaine-induced seizures.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Anesthetics, Local; Animals; Benzophenanthridines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Chloride Channels; Cocaine; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Glycine; Glycine Agents; Hippocampus; Isoquinolines; Kainic Acid; Membrane Potentials; Neurons; Patch-Clamp Techniques; Phenanthridines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Strychnine; Sulfonamides; Tetrodotoxin

We investigated roles of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein II (CAM II) kinase activities in the maintenance of axonal transport in cultured isolated mouse dorsal root ganglion (DRG) cells. Video-enhanced microscopic recordings revealed that the PKC inhibitor chelerythrine (1 microM) reduced anterograde and retrograde axonal transport, while the CAM II kinase inhibitor KN-62 (10 microM) had no effect. Morphological observation showed that neurite growth was prevented by the presence of chelerythrine (1 microM). From these results, we conclude that PKC activity is required to maintain axonal transport and thereby neurite growth.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Axonal Transport; Benzophenanthridines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Enzyme Inhibitors; Ganglia, Spinal; Male; Mice; Mice, Inbred C57BL; Neurites; Neurons, Afferent; Phenanthridines; Protein Kinase C

In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and 45Ca2+ uptake. Treatment of chromaffin cells with 10 microM K-252a, 50 microM H-7, or 50 microM H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca2+/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC50 value of 0.32 microM), but had no effect on cell morphology. The reduction of secretory function induced by 1 microM KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated 45Ca2+ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated 45Ca2+ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca2+ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenal Medulla; Alkaloids; Animals; Benzophenanthridines; Biological Transport; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Catecholamines; Cattle; Cells, Cultured; Enzyme Inhibitors; Indole Alkaloids; Isoquinolines; Phenanthridines; Piperazines; Protein Kinase C; Staurosporine; Substrate Specificity

To characterize the effects of the cellular events associated with contraction on atrial natriuretic factor (ANF) secretion, primary neonatal rat atrial myocytes were electrically paced to contract while being monitored for ANF release, cytoplasmic calcium, phosphoinositide hydrolysis, and protein kinase C activation. Similar measurements were also carried out in the presence of endothelin-1 (ET) for comparison of contraction-related and hormone-stimulated ANF secretion. Pacing (6-8 Hz) immediately increased ANF secretion by 3-5-fold and the time-averaged cytoplasmic calcium concentration (as monitored with indo-1 fluorescence) varied with pace frequency in a similar manner, suggesting that cytoplasmic calcium may play a key role in pace-induced ANF secretion. Furthermore, nifedipine and ryanodine, which inhibited the contractile calcium transients, inhibited pace-induced ANF release, whereas Bay K 8644 increased both the calcium transients and ANF secretion. Pace-induced ANF release was also completely inhibited by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK) but was not inhibited by chelerythrine, a protein kinase C-selective inhibitor. Pace-induced ANF release averaged 40% of that elicited by ET which is known to require both PKC and CaMK for maximal effects on ANF secretion. The effects of pacing and ET on ANF secretion were approximately additive. In contrast to pacing, ET strongly stimulated phosphoinositide hydrolysis, activated PKC, and did not increase cytoplasmic calcium. Thus, regulation of ANF secretion by contraction rate depends primarily on the contractile calcium transients and CaMK and is independent of PKC.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Alkaloids; Analysis of Variance; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cytoplasm; Endothelins; Fluorescent Dyes; Heart; Heart Atria; Indoles; Isoquinolines; Kinetics; Myocardial Contraction; Myocardium; Phenanthridines; Phorbol 12,13-Dibutyrate; Phosphatidylinositols; Phosphoproteins; Piperazines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Spectrometry, Fluorescence