calcimycin and chelerythrine

Fluoxetine (FLX), a well-known antidepressant drug under the class of selective serotonin reuptake inhibitor, exerts its action by inhibiting the reuptake of serotonin selectively. In some studies, it has been demonstrated that FLX relaxes the intestinal smooth muscle. In this study, we aimed at studying the signal transduction pathway underlying the muscle relaxation effect of FLX on electrically stimulated rat ileal muscle contraction. To investigate the possible mechanism involved, various antagonists were used. It was found that inhibition with L-NG-nitroarginine methyl ester, ondansetron, GR113808 and bicuculline enhanced the relaxation effect of FLX. However, the effect of FLX was nullified under the presence of atropine, calcium channel modulator (calcium ionophore A23187), and potassium channel blockers (tetraethylammonium chloride, 4-aminopyridine and glybenclamide). Specific pathway-inhibiting antagonists, Y27632 (Rho-kinase inhibitor) and U73122 (phospholipase-C inhibitor) reversed the antagonistic effect of FLX, while ML-9 (myosin light chain kinase inhibitor) and chelerythrine (protein kinase C inhibitor) augmented the FLX-induced inhibition effect. Taken together, we concluded that FLX exerts the inhibitory effect on electric field stimulation response in rat ileal smooth muscle by the inhibition of muscarinic receptors, decrease of intracellular calcium level by inhibiting phospholipase C and opens the potassium channels.

Topics: 4-Aminopyridine; Amides; Animals; Atropine; Azepines; Benzophenanthridines; Bicuculline; Calcimycin; Drug Interactions; Electric Stimulation; Estrenes; Fluoxetine; Glyburide; Ileum; In Vitro Techniques; Indoles; Male; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Ondansetron; Pyridines; Pyrrolidinones; Rats; Signal Transduction; Sulfonamides; Tetraethylammonium

In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated.. The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied.. The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content.. PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry.

Topics: Annexin A5; Benzophenanthridines; Calcimycin; Calcium; Cells, Cultured; Charybdotoxin; Erythrocyte Count; Erythrocytes; Gene Expression Regulation; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Lysophospholipids; Methomyl; Naphthalenes; Phosphatidylserines; Phospholipid Transfer Proteins; Protein Kinase C-alpha; Signal Transduction; Tetradecanoylphorbol Acetate

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Benzophenanthridines; Binding Sites; Butyrylcholinesterase; Catalytic Domain; Cholinesterase Inhibitors; Humans; Isoquinolines; Kinetics; Molecular Docking Simulation; Structure-Activity Relationship

In Schistosoma mansoni, the miracidium-to-primary sporocyst transformation process is associated with many physiological, morphological, transcriptional and biochemical changes. In the present study, we use a medium-throughput small-molecule screen to identify chemical compounds inhibiting or delaying the in vitro transformation of miracidia to the sporocyst stage. The Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) contains 1280 well-characterized chemical compounds with various modes of action including enzyme inhibitors, antibiotics, cell-cycle regulators, apoptosis inducers and GPCR ligands. We identified 47 compounds that greatly reduce or delay this transformation process during a primary screen of live miracidia. The majority of compounds inhibiting larval transformation were from dopaminergic, serotonergic, ion channel and phosphorylation classes. Specifically, we found that dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channel antagonists and a PKC activator significantly reduced in vitro miracidial transformation rates. Many of the targets of these compounds regulate adenylyl cyclase activity, with the inhibition or activation of these targets resulting in increased cAMP levels in miracidia and concomitant blocking/delaying of larval transformation.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Anthelmintics; Benzophenanthridines; Calcimycin; Calcium Channel Blockers; Citalopram; Clomipramine; Colforsin; Cyclic AMP; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Immunohistochemistry; Mice; Schistosoma mansoni; Tetradecanoylphorbol Acetate; Triflupromazine

Proline-rich tyrosine kinase 2 (Pyk2) is activated in neurones following NMDA receptor stimulation via PKC. Pyk2 is involved in hippocampal LTP and acts to potentiate NMDA receptor function. Elevations of intracellular Ca2+ and cAMP levels are key NMDA receptor-dependent triggering events leading to induction of hippocampal LTP. In this study, we compared the ability of A23187 (Ca2+ ionophore) or forskolin (adenylate cyclase activator) to modulate the phosphorylation of Pyk2 in rat hippocampal slices. Using an immunoprecipitation assay, phosphorylated Pyk2 levels were increased following treatment with A23187, levels peaking at around 10 min. Staurosporine, at concentrations inhibiting conventional and novel isoforms of PKC, and chelerythrine, at concentrations inhibiting the atypical PKC isoform PKMxi, were compared for their ability to attenuate the effect of A23187. Exposure of acute hippocampal slices to either chelerythrine or staurosporine completely blocked enhanced phosphorylation of Pyk2 by A23187, suggesting a possible involvement of PKMxi and typical PKCs in Pyk2 activation by Ca2+. In contrast, application of forskolin reduced phosphorylated Pyk2 below basal levels, suggesting that cAMP inhibits Pyk2. These results implicate Ca2+ and multiple forms of PKC in the activation of Pyk2 downstream of NMDA receptors and suggest that cAMP-dependent processes exert a suppressive action on Pyk2.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcimycin; Calcium; Colforsin; Cyclic AMP; Focal Adhesion Kinase 2; Hippocampus; Immunoblotting; Immunoprecipitation; Long-Term Potentiation; Phenanthridines; Phosphorylation; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Staurosporine

This study investigates the exocytic responses of invertebrate hemocytes to pathogen-associated antigens. It demonstrates that a homologue of complement component C3, a key defensive protein of the innate immune system, is expressed by phagocytic hemocytes (non-refractile vacuolated cells) of the tunicate, Styela plicata. C3-like molecules are localized in sub-cellular vesicles and are rapidly exocytosed after stimulation with bacterial, fungal or algal cell surface molecules. Signal transduction analysis indicated that the induced secretion of C3-like molecules is mediated by a G-protein dependent signaling pathway, which modulates tubulin microtubules. All of this evidence indicates that hemocytes can contribute to host defense responses by rapidly exocytosing C3-like proteins at sites of infection.

Topics: Alkaloids; Animals; Benzophenanthridines; Blotting, Western; Calcimycin; Carrageenan; Cell Survival; Cholera Toxin; Colchicine; Colforsin; Complement C3; Cytochalasin D; Cytoplasmic Vesicles; Enzyme-Linked Immunosorbent Assay; Exocytosis; Hemocytes; Immunohistochemistry; Kinetics; Lipopolysaccharides; Mannans; Microscopy, Electron; Phenanthridines; Staurosporine; Tetradecanoylphorbol Acetate; Thapsigargin; Urochordata

Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca(2+), efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca(2+) content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca(2+) entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca(2+) influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca(2+). PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca(2+)-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by omega-agatoxin-TK, a calcium channel blocker specific for Ca(v)2.1 channels. To confirm that a Ca(v)2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the alpha(1A) subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an omega-agatoxin-TK-sensitive, Ca(v)2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.

Topics: Adult; Agatoxins; Alkaloids; Benzophenanthridines; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, N-Type; Calcium Signaling; Cell Size; Diglycerides; Dimethyl Sulfoxide; Enzyme Inhibitors; Erythrocytes; Humans; Ion Transport; Ionophores; Marine Toxins; Okadaic Acid; Osmotic Fragility; Oxazoles; Phenanthridines; Phosphoprotein Phosphatases; Protein Kinase C; Spider Venoms; Staurosporine; Tetradecanoylphorbol Acetate

We investigated the combined effect of 5-hydroxytryptamine (5-HT, serotonin) and calcium ionophore (A23187) on human platelet aggregation. Aggregation, monitored at 37 degrees C using a Dual-channel Lumi-aggregometer, was recorded for 5 min after challenge by a change in light transmission as a function of time. 5-HT (2-200 microM) alone did not cause platelet aggregation, but markedly potentiated A23187 (low dose) induced aggregation. Inhibitory concentration (IC50) values for a number of compounds were calculated as means +/- SEM from dose-response determinations. Synergism between 5-HT (2-5 microM) and A23187 (0.5-2 microM) was inhibited by 5-HT receptor blockers, methysergide (IC50 = 18 microM) and cyproheptadine (IC50 = 20 microM), and calcium channel blockers (verapamil and diltiazem, IC50 = 20 microM and 40 microM respectively). Interpretation of the effects of these blockers is complicated by their lack of specificity. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect at an IC50 value of 9.2 microM. Wortmannin, a phosphatidylinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC50 = 2.6 microM). However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C inhibitor, affected aggregation at concentrations up to 10 microM. We conclude that the synergistic interaction between 5-HT and ionophore may be mediated by activation of PLC/Ca2+ and PI 3-kinase signalling pathways, but definitive proof will require other enzyme inhibitors with greater specificity.

Topics: Adult; Alkaloids; Androstadienes; Benzophenanthridines; Blood Platelets; Calcimycin; Calcium Channel Blockers; Cyproheptadine; Diltiazem; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Enzyme Inhibitors; Estrenes; Female; Genistein; Humans; In Vitro Techniques; Ionophores; Male; Methysergide; Phenanthridines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet Aggregation; Pyrrolidinones; Second Messenger Systems; Serotonin; Type C Phospholipases; Verapamil; Wortmannin

Long-term nitroglycerin (NTG) treatment has been shown to be associated with cross-tolerance to endothelium-dependent vasodilators. It may involve increased production of reactive oxygen species (such as superoxide, O(2)(.-)) that rapidly inactivate the nitric oxide (NO) released from the endothelial cells. It remains to be elucidated, however, whether long-term treatment with NTG alters the activity and expression of the endothelial NO synthase (NOS III) and whether this enzyme can contribute to O(2)(.-) formation. We studied the influence of long-term NTG treatment on the expression of NOS III as assessed by RNase protection assay and Western blot. Tolerance was measured ex vivo in organ chamber experiments with rat aortic rings. O(2)(.-) and NO formation were quantified using lucigenin- and Cypridina luciferin analog-enhanced chemiluminescence as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG (Alzet osmotic minipumps, NTG concentration 10 microg x kg(-1) x min(-1)) for 3 days caused marked tolerance, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, and a significant increase in O(2)(.-)-induced chemiluminescence. Tolerance was associated with a significant increase in NOS III mRNA to 236+/-28% and NOS III protein to 239+/-17%. In control vessels, the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) increased the O(2)(.-)-mediated chemiluminescence, indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. In the setting of tolerance, however, L-NNA decreased steady-state O(2)(.-) levels, indicating the involvement of NOS III in O(2)(.-) formation. Likewise, A23187-induced, NOS III-mediated O(2)(.-) production was more pronounced in tolerant than in control vessels. Vascular NO bioavailability as assessed with ESR spectroscopy using iron-thiocarbamate as a trap for NO was significantly reduced in tolerant vessels. Pretreatment of tolerant tissue in vitro with the protein kinase C (PKC) inhibitors reduced basal and stimulated NOS III-mediated O(2)(.-) production and partially reversed vascular tolerance. These findings suggest that NTG treatment increases the expression of a dysfunctional NOS III gene, leading to increased formation of O(2)(.-) and decreased vascular NO bioavailability. Normalization of NOS III-mediated O(2)(. -) production and improvement of tolerance with PKC inhibition suggests an important role for PKC isoforms in mediating vasc

Topics: Acetylcholine; Alkaloids; Animals; Arginine; Benzophenanthridines; Biological Availability; Calcimycin; Carbazoles; Cloning, Molecular; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Indoles; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroglycerin; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar; RNA, Messenger; Superoxides; Time; Vasodilation

Some investigators have reported previously that phorbol esters inhibit in vitro erythropoietin production stimulated by hypoxia; whereas others have reported that phorbol esters enhanced Epo production during exposure to hypoxia. We have demonstrated in the present experiments that hypoxia significantly increased diacylglycerol levels in cultured human hepatocellular carcinoma (Hep3B) cells. 1-oleoyl-2-acetyl-ras-glycerol (OAG) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), two well-known protein kinase C activators, significantly increased medium levels of erythropoietin as well as erythropoietin messenger RNA levels in normoxic Hep3B cells. A potent protein kinase C inhibitor, chelerythrine chloride, significantly decreased hypoxia-induced increases in medium levels of erythropoietin as well as erythropoietin messenger RNA levels in Hep3B cells. A cis-unsaturated free fatty acid, oleic acid, significantly enhanced OAG-induced medium levels of erythropoietin in normoxic Hep3B cells, whereas a phospholipase A2 inhibitor, mepacrine, significantly decreased hypoxia-induced erythropoietin production in Hep3B cells. These results provide strong support for a positive role for protein kinase C in the hypoxic regulation of erythropoietin production.

Topics: Alkaloids; Benzophenanthridines; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Humans; Ionophores; Liver Neoplasms; Models, Chemical; Oleic Acid; Phenanthridines; Phospholipases A; Phospholipases A2; Protein Kinase C; Quinacrine; Thapsigargin; Tumor Cells, Cultured

We have previously shown that ANG II increases microvascular permeability in normal dog lungs but not after pacing-induced heart failure. This study investigated how ANG II induces permeability in isolated blood-perfused canine lung lobes and what alterations occur during heart failure. In normal lobes, the protein kinase C (PKC) inhibitors staurosporine (500 nM) or chelerythrine (10 microM) did not modify ANG II-induced increases in the capillary filtration coefficient (Kf,c, ml . min-1 . cmH2O-1 . 100 g-1; an index of microvascular permeability), suggesting that PKC is not involved. Thapsigargin (150 nM) was used to stimulate capacitative Ca2+ entry in lobes from control dogs and dogs paced at 245 beats/min for 4 wk to induce heart failure. In control lobes, Kf,c rose after thapsigargin, from 0.06 +/- 0.01 to 0.17 +/- 0.03 ml . min-1 . cmH2O-1 . 100 g-1 (mean +/- SE, P < 0.05) but did not change in the paced group. A Ca2+ ionophore, A-23187, increased Kf,c in both control (10 microM; 0.05 +/- 0.01 to 0.17 +/- 0.05 ml . min-1 . cmH2O-1 . 100 g-1, P < 0.05) and pace (5 microM; 0.06 +/- 0.01 to 0. 21 +/- 0.07 ml . min-1 . cmH2O-1 . 100 g-1, P < 0.05) lobes, indicating that increasing intracellular Ca2+ is sufficient to induce pulmonary microvascular permeability after pacing. We conclude that during heart failure, Ca2+ signaling within the pulmonary microvascular endothelium is altered.

Topics: Alkaloids; Angiotensin II; Animals; Benzophenanthridines; Calcimycin; Calcium; Capillary Permeability; Cardiac Output, Low; Cardiac Pacing, Artificial; Dogs; Endothelium, Vascular; Enzyme Inhibitors; Ionophores; Lung; Phenanthridines; Protein Kinase C; Signal Transduction; Staurosporine; Thapsigargin; Vascular Resistance

To determine whether angiotensin II (Ang II) activates the suicide program of myocytes, primary cultures of adult rat ventricular myocytes were exposed to 10(-9) M of Ang II, for 24 h. Ang II resulted in a five-fold increase in programmed myocyte cell death (PMCD) documented by the terminal deoxynucleotidyl transferase assay and confirmed by DNA agarose gel electrophoresis. Ang II stimulation was associated with translocation of the epsilon and delta isoforms of protein kinase C (PKC) which was coupled with an increase in cytosolic Ca2+ in the cells. The PKC inhibitor chelerythrine abolished Ang II-mediated increases in cytosolic Ca2+ and PMCD. Similarly, pretreatment of cells with the intracellular Ca2+ chelator BAPTA/AM inhibited the formation of DNA strand breaks. Conversely, the Ca2+ ionophore A23187 markedly increased PMCD. Finally, the AT1 receptor antagonist, losartan, completely blocked Ang II-induced PMCD, whereas the AT2 receptor antagonist, PD123319, did not attenuate this phenomenon. In conclusion, ligand binding of AT1 receptors on myocytes triggers PMCD by a mechanism involving PKC-mediated increases in cytosolic calcium, which result in internucleosomal DNA fragmentation.

Topics: Alkaloids; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Apoptosis; Benzophenanthridines; Biphenyl Compounds; Calcimycin; Calcium; Cells, Cultured; Chelating Agents; Cytosol; Egtazic Acid; Enzyme Inhibitors; Imidazoles; Ionophores; Isoenzymes; Losartan; Myocardium; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Tetrazoles

Topics: 3,4-Dihydroxyphenylacetic Acid; Alkaloids; Androstadienes; Aspirin; Benzophenanthridines; Calcimycin; Calcium; Chlorpromazine; Diltiazem; Dopamine; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; GTP-Binding Proteins; Haloperidol; Homovanillic Acid; Humans; Indomethacin; Ion Transport; Ionophores; Phenanthridines; Phentolamine; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet Aggregation; Propranolol; Protein Kinase C; Protein-Tyrosine Kinases; Receptors, Dopamine; Serotonin; Signal Transduction; Verapamil; Wortmannin

We have previously demonstrated that corticotrophin-releasing factor receptor 1 (CRF-R1) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, resulted in a time- and concentration-dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4alpha-phorbol failed to influence significantly CRF-R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down-regulated CRF-R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calcium channels with nifedipine inhibited agonist-mediated down-regulation of CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regulation of CRF-R1 mRNA levels and that calcium is required for agonist-mediated down-regulation of this receptor.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcimycin; Calcium; Carbachol; Cell Line; Corticotropin-Releasing Hormone; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Kinetics; Mice; Nifedipine; Phenanthridines; Protein Kinase C; Receptors, Corticotropin-Releasing Hormone; RNA, Messenger; Tetradecanoylphorbol Acetate; Thapsigargin; Transcription, Genetic

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

Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcimycin; Calcium; Calmodulin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Genistein; Humans; Isoflavones; Male; Mice; Phenanthridines; Phosphorylation; Phosphotyrosine; Protein Kinase Inhibitors; Proteins; Seminal Plasma Proteins; Spermatozoa; Staurosporine

Modulation of protein kinase C (PKC) activity in basophils (B) can influence IgE-mediated histamine release (HR). The present study investigated the effects of chelerythrine, which inhibits isolated PKC (IC50 0.7 microM), on different activation pathways in B. Fc epsilon RI-mediated HR was strongly inhibited by chelerythrine (86.5 +/- 5.4%, 5 microM, n = 11). TPA-induced mediator release was also suppressed: 77.1 +/- 8.5% inhibition (7.5 microM). HR due to non-immunological stimuli (A23187, FMLP) was strongly inhibited by chelerythrine. Previously, other selective PKC-inhibitors have been shown to potentiate IgE-mediated HR from B suggesting a negative modulatory role of PKC, whereas non-specific inhibitors such as staurosporine inhibited HR. Chelerythrine might therefore be less selective for PKC. This may be indicated by the fact that chelerythrine inhibits PKC at its catalytic domain, which is homologous with other protein kinases.

Topics: Alkaloids; Basophils; Benzophenanthridines; Calcimycin; Histamine Release; Humans; Immunoglobulin E; In Vitro Techniques; N-Formylmethionine Leucyl-Phenylalanine; Phenanthridines; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate