h-89 and chelerythrine

The efficacy of protein kinase inhibitors (PKIs) has been shown in clinical assays for cancer, but as isolated agents, they only have a modest effect. One of the most important characteristics of mitogen-activated PKIs is their ability to decrease the apoptotic threshold of cancer cells, sensitizing them to the action of other antiapoptotic agents. The secretory clusterin protein is an inhibitor of apoptosis with a cytoprotective function. We describe the use of clusterin-specific antisense oligonucleotides and siRNA to sensitize breast carcinoma cells to several PKIs. MCF-7 and MDA-MB-231 cells were treated with antisense oligonucleotide or siRNA to clusterin and the following PKIs: H-89, chelerythrine and genistein. The three inhibitors used in this study upregulated clusterin expression and treatments that included antisense oligonucleotide or siRNA to clusterin reduced the number of viable cells more effectively than did treatment with the drugs alone. Therefore, treatment with such combinations may benefit patients with breast cancer.

Topics: Benzophenanthridines; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Clusterin; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Female; Genistein; Humans; Isoquinolines; Oligonucleotides, Antisense; Protein Kinase Inhibitors; RNA, Small Interfering; Sulfonamides; Thionucleotides

Chronic amphetamine intake leads to neurogenic bladder and chronic urinary retention. The mechanism underlying persistent urinary retention is unclear. The pelvic-urethral reflex (PUR) is essential for the urethra to develop sufficient resistance to maintain urine continence, an important function of the urinary system. Recent studies on PUR activities have indicated that repetitive/tetanic stimulation of the pelvic afferent fibers induces spinal reflex potentiation (SRP) in PUR activities, which further increases urinary retention. In this study, results showed that test stimulation (TS, 1/30 Hz) evoked a baseline reflex activity, while repetitive stimulation (RS, 1 Hz) induced reflex potentiation in the external urethral sphincter. Intrathecal d-amphetamine (AMPH, 30 μM) did not but higher AMPH concentration (100 μM) induced SRP in TS-induced reflex activity. H89 (10 μM, a protein kinase A inhibitor), but not chelerythrine chloride (CTC, 10 μM, a protein kinase C inhibitor), prevented the 100 μM AMPH-elicited SRP. At 30 μM, forskolin, an activator of adenylyl cyclase, elicited SRP. The co-administration of 10 μM forskolin and 30 μM AMPH induced SRP in TS-induced reflex activity. These results implied that the repetitive/tetanic stimulation of the pelvic afferent fibers could induce SRP in PUR activities, so that the urethra can produce sufficient resistance and played a significant role in urinary retention. Findings in this study demonstrated that amphetamine could induce bladder dysfunction by triggering protein kinase A activation, and provide a practical basis for the development of treatment for amphetamine-associated urinary retention.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Amidines; Amphetamine-Related Disorders; Animals; Benzophenanthridines; Central Nervous System Stimulants; Chronic Disease; Colforsin; Dextroamphetamine; Excitatory Amino Acid Agonists; Female; Glutamic Acid; Isoquinolines; N-Methylaspartate; Oxidants; Protein Kinase Inhibitors; Rats, Wistar; Reflex; Spinal Cord; Sulfonamides; Urinary Retention; Urination; Valine

During intestinal inflammation TNFα levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNFα inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNFα on sugar transport and to identify the intracellular mechanisms involved.. Caco-2 cells were grown on culture plates and pre-incubated during different periods with various TNFα concentrations before measuring the apical uptake of galactose, α-methyl-glucoside (MG) or fructose for 15 min. To elucidate the signaling pathway implicated, cells were pre-incubated for 30min with the PKA inhibitor H-89 or the PKC inhibitor chelerythrine, before measuring the sugar uptake. The expression in the apical membrane of the transporters implicated in the sugars uptake process (SGLT1 and GLUT5) was determined by Western blot.. TNFα inhibited 0.1mM MG uptake after pre-incubation of the cells for 6-48h with the cytokine and in the absence of cytokine pre-incubation. In contrast, 5mM fructose uptake was stimulated by TNFα only after long pre-incubation times (24 and 48 h). These effects were mediated by the binding of the cytokine to its specific receptor TNFR1, present in the apical membrane of the Caco-2 cells. Analysis of the expression of the MG and fructose transporters at the brush border membrane of the cells, after 24h pre-incubation with the cytokine, revealed decrease on the amount of SGLT1 and increase on the amount of GLUT5 proteins. Short-term inhibition of MG transport by TNFα was not modified by H-89 but was blocked by chelerythrine.. SGLT1 and GLUT5 expression in the plasma membrane is regulated by TNFα in the human epithelial cell line Caco-2 cells, leading to alteration on sugars transport, suggesting that TNFα could be considered as a physiological local regulator of nutrients absorption in response to an intestinal inflammatory status.

Topics: Benzophenanthridines; Biological Transport; Caco-2 Cells; Cell Line; Fructose; Galactose; Glucose Transporter Type 5; Humans; Inflammation; Intestinal Mucosa; Isoquinolines; Methylglucosides; Monosaccharide Transport Proteins; Protein Kinase Inhibitors; Sodium-Glucose Transporter 1; Sulfonamides; Tumor Necrosis Factor-alpha

Salmonid fish sperm motility is known to be suppressed in millimolar concentrations of extracellular K(+), and dilution of K(+) upon spawning triggers cAMP-dependent signaling for motility initiation. In a previous study, however, we demonstrated that suspending sperm in a 10% glycerol solution and subsequent dilution into a low-osmotic solution induced motility independently of extracellular K(+) and cAMP. In the present study, we further investigated the glycerol-induced motility mechanism. We found that treatment with solutions consisting of organic or inorganic ions, as well as glycerol, induced sperm motility in an osmolarity-dependent manner. Elimination of intracellular Ca(2+) by BAPTA-AM significantly inhibited glycerol-treated sperm motility, whereas removal of extracellular Ca(2+) by EGTA did not. Monitoring intracellular Ca(2+), using fluo-4, revealed that intracellular Ca(2+) increased when sperm were suspended in hypertonic solutions, and a subsequent dilution into a hypotonic solution led to a decrease in intracellular Ca(2+) concomitant with motility initiation. In addition, upon dilution of sperm into a hypertonic glycerol solution prior to demembranation, the motility of demembranated sperm was reactivated in the absence of cAMP. The motility recovery suggests that completion of axonemal maturation occurred during exposure to a hypertonic environment. As a result, it is likely that glycerol treatment of sperm undergoing hypertonic shock causes mobilization of intracellular Ca(2+) from the intracellular Ca(2+) store and also causes maturation of axonemal proteins for motility initiation. The subsequent dilution into a hypotonic solution induces a decrease in intracellular Ca(2+) and flagellar movement. This novel mechanism of sperm motility initiation seems to act in a salvaging manner for the well-known K(+)-dependent pathway.

Topics: Animals; Benzophenanthridines; Calcium; Egtazic Acid; Estrenes; Flagella; Glycerol; Hypertonic Solutions; Isoquinolines; Male; Naphthalenes; Osmotic Pressure; Pyrrolidinones; Salmonidae; Sperm Motility; Sulfonamides

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

Ischemic episodes lead to profound functional and structural alterations of the gastrointestinal tract which may contribute to disorders of intestinal motility. Enhancement of glutamate overflow and the consequent activation of NMDA (N-methyl-D-aspartate) receptors may participate to such changes by modulating different enteric neurotransmitter systems, including cholinergic motor pathways.. The molecular mechanism/s underlying activation of NMDA receptors in the guinea pig ileum were investigated after glucose/oxygen deprivation (in vitro ischemia) and during reperfusion.. The number of ileal myenteric neurons positive for NR1, the functional subunit of NMDA receptors, and its mRNA levels were unchanged after in vitro ischemia/reperfusion. In these conditions, the protein levels of NR1, and of its phosphorylated form by protein kinase C (PKC), significantly increased in myenteric neurons, whereas, the levels of NR1 phosphorylated by protein kinase A (PKA) did not change, with respect to control values. Spontaneous glutamate overflow increased during in vitro ischemia/reperfusion. In these conditions, the NMDA receptor antagonists, D(-)-2-amino-5-phosphonopentanoic acid [(D)-AP5] (10 μmol L(-1)) and 5,7-dichlorokynurenic acid (5,7-diClKyn acid) (10 μmol L(-1)) and the PKC antagonist, chelerythrine (1 μmol L(-1)), but not the PKA antagonist, H-89 (1 μmol L(-1)), were able to significantly depress the increased glutamate efflux.. The present data suggest that in the guinea pig ileum during in vitro ischemia/reperfusion, NR1 protein levels increase. Such event may rely upon posttranscriptional events involving NR1 phosphorylation by PKC. Increased NR1 levels may, at least in part, explain the ability of NMDA receptors to modulate a positive feedback on ischemia/reperfusion-induced glutamate overflow.

Topics: 2-Amino-5-phosphonovalerate; Animals; Benzophenanthridines; Guinea Pigs; Ileum; In Vitro Techniques; Isoquinolines; Kynurenic Acid; Male; Models, Animal; Myenteric Plexus; Phosphorylation; Protein Kinase C; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Sulfonamides

Thyroid hormones (THs) play a crucial role in the maturation and functioning of mammalian central nervous system. Thyroxine (T4) and 3, 3', 5-L-triiodothyronine (T3) are well known for their genomic effects, but recently attention has been focused on their non genomic actions as modulators of neuronal activity. In the present study we report that T4 and T3 reduce, in a non competitive manner, GABA-evoked currents in rat hippocampal cultures with IC₅₀s of 13±4μM and 12±3μM, respectively. The genomically inactive compound rev-T3 was also able to inhibit the currents elicited by GABA. Blocking PKC or PKA activity, chelating intracellular calcium, or antagonizing the integrin receptor αVβ3 with TETRAC did not affect THs modulation of GABA-evoked currents. THs affect also synaptic activity in hippocampal and cortical cultured neurons. T3 and T4 reduced to approximately 50% the amplitude and frequency of spontaneous inhibitory synaptic currents (sIPSCs), without altering their decay kinetic. Tonic currents evoked by low GABA concentrations were also reduced by T3 (40±5%, n=14), but not by T4. Similarly, T3 decreased currents elicited by low concentrations of THIP, a low affinity GABAA receptor agonist that preferentially activates extrasynaptic receptors, whereas T4 was ineffective. Thus, our data demonstrate that T3 and T4 selectively affect GABAergic phasic and tonic neurotransmission. Since THs concentrations can be regulated at the level of the synapses these data suggest that the network activity of the whole brain could be differently modulated depending on the relative amount of these two hormones. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Topics: Animals; Benzophenanthridines; Calcium; Cells, Cultured; Chelating Agents; Dose-Response Relationship, Drug; Egtazic Acid; Evoked Potentials; Hippocampus; Isoquinolines; Neurons; Patch-Clamp Techniques; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Sulfonamides; Thyroid Hormones; Thyroxine; Triiodothyronine

Gonadotrophin-releasing hormone (GnRH) is a hypothalamic hormone transported by the hypophyseal portal bloodstream to the pituitary gland, where it binds to GnRH receptors. However, GnRH receptors are expressed in multiple extrapituitary tissues, although their physiological relevance is not fully understood. GnRH agonists are employed extensively in steroid deprivation therapy, especially to suppress testosterone in prostate cancer. Because GnRH agonist treatment is associated with increased coronary heart disease and myocardial infarction, we investigated the impact of GnRH on cardiomyocyte contractile function. Cardiomyocytes were isolated from mouse hearts and mechanical and intracellular Ca(2+) properties were evaluated, including peak shortening amplitude (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90) ), maximal velocity of shortening/relengthening (± dLdt), electrically-stimulated rise in Fura-2 fluorescence intensity (ΔFFI) and Ca(2+) decay. GnRH (1 ng/ml) increased PS, ± dL/dt, resting FFI and ΔFFI, and prolonged TPS, TR(90) and Ca(2+) decay time, whereas 1 pg/ml GnRH affected all these cardiomyocyte variables, except TPS, resting FFI and ΔFFI. A concentration of 1 fg/ml GnRH and the GnRH cleavage product, GnRH-[1-5] (300 pg/ml), had no effect on any cardiomyocyte parameter. The 1 pg/ml GnRH-elicited responses were attenuated by the GnRH receptor antagonist cetrorelix (10 μm), the protein kinase A (PKA) inhibitor H89 (1 μm) but not the protein kinase C inhibitor chelerythrine chloride (1 μm). These data revealed that GnRH is capable of regulating cardiac contractile function via a GnRH receptor/PKA-dependent mechanism. If present in the human heart, dysfunction of such a system may play an important role in cardiac pathology observed in men treated with GnRH agonists for prostate cancer.

Topics: Animals; Anti-Bacterial Agents; Benzophenanthridines; Calcium; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Gonadotropin-Releasing Hormone; Hormone Antagonists; Humans; Isoquinolines; Male; Mice; Myocardial Contraction; Myocytes, Cardiac; Protein Kinase C; Sulfonamides

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)](i) transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH.

Topics: Animals; Benzophenanthridines; Calcium; Calcium Channels, T-Type; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Electrophysiological Phenomena; Isoquinolines; Leydig Cells; Luteinizing Hormone; Male; Mice; Peptides; Protein Kinase C; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate

Previous studies have suggested that bilirubin can potentiate GABA/glycinergic synaptic transmission in lateral superior olivary nucleus neurons, but the cellular mechanism has not been defined. The present study evaluated the possible roles of protein kinase A (PKA) and C (PKC) in bilirubin potentiation of GABA/glycinergic synaptic transmission in rat ventral cochlear nucleus (VCN) neurons. VCN neurons were acutely isolated from postnatal 10-12-day-old (P10-12) rats and were voltage-clamped in whole-cell mode. Miniature inhibitory postsynaptic currents (mIPSC) frequencies, but not amplitude, were increased by bilirubin. Forskolin (PKA activator) and H-89 (PKA inhibitor) also individually increased mIPSCs frequency, with an additional increase induced by co-incubation with bilirubin and H-89. Pretreatment with forskolin blocked bilirubin potentiation. mIPSC frequency was not altered by phorbol 12,13-diacetate (PKC activator), but mIPSC frequency was increased following co-application of bilirubin. The mIPSC frequency was increased by chelerythrine (PKC inhibitor), and then further increased after the addition of bilirubin. Neither H-89, forskolin, nor PDA, nor their co-application with bilirubin affected mIPSC amplitudes of GABA-activated (I(GABA))/glycine-activated (I(gly)) currents, suggesting a presynaptic locus of activity. Chelerythrine decreased the mIPSC amplitudes and I(GABA)/I(gly), suggesting a postsynaptic locus of activity. These data suggest that both PKA and PKC can modulate GABA and glycine release in rat VCN neurons. Bilirubin facilitates transmitter release via presynaptic PKA activation, which might provide insight into the cellular mechanism underlying bilirubin-induced hearing dysfunction.

Topics: Animals; Animals, Newborn; Antioxidants; Benzophenanthridines; Bilirubin; Cochlear Nucleus; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; gamma-Aminobutyric Acid; Glycine; In Vitro Techniques; Isoquinolines; Neural Inhibition; Neurons; Patch-Clamp Techniques; Phorbol Esters; Protein Kinase C; Rats; Signal Transduction; Sulfonamides; Synaptic Transmission

The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLX's positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular Ca(2+) without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-delta, decreases in PKC-alpha and -beta(II), but no effect on PKC-epsilon. Inhibition of PKC with chelerythrine chloride or PKC-delta with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects.

Topics: Actin Cytoskeleton; Animals; Benzophenanthridines; Blotting, Western; Ca(2+) Mg(2+)-ATPase; Cardiotonic Agents; Enzyme Inhibitors; Female; Heart; In Vitro Techniques; Isometric Contraction; Isoquinolines; Mice; Mice, Inbred C57BL; Myocardium; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Relaxin; Signal Transduction; Sulfonamides

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

Inhibition of Qo site of mitochondrial complex III under hypoxia has received attention, but its downstream pathways remain unclear. We used Qo site inhibitor myxothiazol to mimic the inhibition of the Qo site of complex III and studied the effects of the inhibition of this site on persistent and transient sodium currents and neuron excitability in rat hippocampal CA1 cells. The results showed myxothiazol apparently increased persistent sodium currents but with a weak effect on transient sodium currents; the effect of myxothiazol on persistent sodium currents was blocked by protein kinase C inhibitor and superoxide scavengers, but not by hydrogen peroxide scavenger and hydroxyl radical formation inhibitor; myxothiazol could increase the activity of protein kinase C and neuron excitability. These results suggest that the inhibition of Qo site of mitochondrial complex III increases persistent sodium currents via superoxide production and protein kinase C activation.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Alkaloids; Animals; Antifungal Agents; Benzophenanthridines; Cyclic AMP-Dependent Protein Kinases; Electron Transport Complex I; Electron Transport Complex III; Enzyme Activation; Enzyme Inhibitors; Free Radical Scavengers; Glycine; Hippocampus; Hydroxyl Radical; Hypoxia, Brain; Isoquinolines; Metalloporphyrins; Methacrylates; Mitochondria; Patch-Clamp Techniques; Phenanthridines; Protein Kinase C; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Sodium; Sulfhydryl Compounds; Sulfonamides; Superoxides; Thiazoles

The effect of hyposmotic shock on exocytosis was examined in isolated hepatocytes of turbot, a marine flatfish, using the molecular probe FM1-43. Sudden exposure to a reduced osmolality caused an increase in cell exocytic activity related to the osmotic gradient between intra- and extracellular fluids. Cytoskeletal microtubules could contribute to this hyposmotic-induced exocytosis since colchicine inhibited the process. Protein kinase C, phosphatidylinositol-3 kinase, phospholipases A2, C and D could constitute key enzymes in the mechanism since their inhibition by specific agents altered the hyposmotic-induced exocytic activity. Moreover, arachidonic acid and derivates from the 5-lipoxygenase pathway as well as calcium could participate in the process. As regulatory volume decrease (RVD) exhibited by turbot hepatocytes following hyposmotic stimulation involves similar features, a potential role of exocytosis in volume regulation is suggested. In particular, exocytosis could serve RVD by contributing to ATP release since this latter process similarly appeared to be phospholipase D-dependent and related to the osmotic gradient. This study provides the first evidence of a volume-sensitive exocytosis that could aim at volume constancy in a marine teleost fish cell type.

Topics: 1-Butanol; Alkaloids; Androstadienes; Animals; Benzophenanthridines; Cell Membrane; Colchicine; Cytochalasin B; Cytoskeleton; Estrenes; Exocytosis; Flatfishes; Fluorescent Dyes; Hepatocytes; Indomethacin; Ionomycin; Isoquinolines; Masoprocol; Osmotic Pressure; Phenanthridines; Pyridinium Compounds; Pyrrolidinones; Quaternary Ammonium Compounds; Quinacrine; Signal Transduction; Sulfonamides; Thapsigargin; Wortmannin

In trout hepatocytes, hypertonicity and cytosolic acidification are known to stimulate Na+/H+ exchanger (NHE) activity, which contributes to recovery of cell volume and intracellular pH (pHi), respectively. The present study investigated the signalling mechanisms underlying NHE activation under these conditions. Exposing trout hepatocytes to cariporide, a specific inhibitor of NHE-1, decreased baseline pHi, completely blocked the hypertonicity-induced increase of pHi and reduced the hypertonicity-induced proton secretion by 80%. Changing extracellular pH (pHe) above and below normal values, and allowing cells to adjust pHi accordingly, significantly delayed alkalinization during hypertonic exposure, whereas following an acid load an enhanced pHi recovery with increasing pHe was seen. Chelating Ca2+, and thereby preventing the hypertonicity-induced increase in intracellular Ca2+ ([Ca2+]i), significantly diminished hypertonic elevation of pHi, indicating that Ca2+ signalling might be involved in NHE activation. A reduction in alkalinization and proton secretion was also observed in the presence of the protein kinase A (PKA) inhibitor H-89 or the calmodulin (CaM) inhibitor calmidazolium. A complete inhibition of hypertonic- and acidification-induced changes of pHi concurrent with an increase in hypertonically induced proton efflux was seen with the protein kinase C (PKC) inhibitor chelerythrine. Recovery of pHi following sodium propionate addition was reduced by more than 60% in the presence of cariporide, was sensitive to PKA inhibition, and tended to be reduced by CaM inhibition. In conclusion, we showed that NHE-1 is the main acid secretion mechanism during hypertonicity and recovery following acid loading. In addition, Ca2+-, PKA- and CaM-dependent pathways are involved in NHE-1 activation for recovery of cell volume and pHi. On the other hand, PKC appeared to have an impact on NHE-independent pathways affecting intracellular acid-base homeostasis.

Topics: Alkaloids; Animals; Benzophenanthridines; Calmodulin; Enzyme Inhibitors; Guanidines; Hepatocytes; Homeostasis; Hydrogen-Ion Concentration; Imidazoles; Intracellular Signaling Peptides and Proteins; Isoquinolines; Oncorhynchus mykiss; Osmotic Pressure; Phenanthridines; Phosphorylation; Propionates; Signal Transduction; Sodium-Hydrogen Exchangers; Sulfonamides; Sulfones

The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y(2) and P2Y(4) receptor agonist, stimulated ATP release from Schwann cells in a dose-dependent manner with an ED(50) of 0.24 microm. UTP-stimulated ATP release occurs through P2Y(2) receptors as it was blocked by suramin which inhibits P2Y(2) but not P2Y(4) receptors. Furthermore, positive immunostaining of P2Y(2) receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y(4) receptors, did not significantly stimulate ATP release. UTP-stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP(3) formation inhibitor lithium chloride, the cell membrane-permeable Ca(2+) chelator BAPTA-AM and the endoplasmic reticulum Ca(2+)-dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi-complex inhibitor brefeldin A, microtubule disruption with nocodazole, F-actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP-stimulated ATP release is mediated by activation of P2Y(2) receptors that initiate an IP(3)-Ca(2+) cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Animals, Newborn; Benzophenanthridines; Botulinum Toxins; Botulinum Toxins, Type A; Brefeldin A; Calcium; Cyclic AMP-Dependent Protein Kinases; Cytochalasin D; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Interactions; Estrenes; Furosemide; Glyburide; Glycyrrhetinic Acid; Guanosine Triphosphate; Immunohistochemistry; Isoquinolines; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Phenanthridines; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Synthesis Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Schwann Cells; Sciatic Nerve; Sulfonamides; Suramin; Thapsigargin; Time Factors; Type C Phospholipases; Uridine Triphosphate

Hypotonic shock rapidly inhibits Cl(-) secretion by chloride cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic solutions, yet the mechanism for the inhibition and its recovery are not known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to hypotonic shock in the presence of a variety of chemicals: a general protein kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks PKC alpha and beta subtypes, H-89 that blocks PKA, SB203580 that blocks p38 mitogen-activated protein kinase (MAPK), as well as serine/threonine protein phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a blocker of volume-activated anion channels (VSOAC). Chelerythrine has no effect on hypotonic inhibition but blocked the recovery, indicating PKC involvement in stimulation. Gö6976 had little effect, suggesting that PKC alpha and PKC beta subtypes are not involved. H-89 did not block hypotonic inhibition but decreased the recovery, indicating PKA may be involved in the recovery and overshoot (after restoration of isotonic conditions). SB203580 significantly enhanced the decrease in current by hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic inhibition. Okadaic acid increased the steady state current, slowed the hypotonic inhibition but made the decrease in current larger; also the recovery and overshoot were completely blocked. Hypotonic stress rapidly and transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 micromol l(-1)) inhibited Cl(-) secretion that was high, increased Cl(-) secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes: (1) activation of NKCC and

Topics: Alkaloids; Animals; Benzophenanthridines; Blotting, Western; Chlorides; Cyclic AMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Electrophysiology; Epithelium; Focal Adhesion Protein-Tyrosine Kinases; Fundulidae; Imidazoles; Immunohistochemistry; Ion Channels; Isoquinolines; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Nova Scotia; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phenanthridines; Protein Kinase C; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyridines; Saline Solution, Hypertonic; Sulfonamides; Tamoxifen; Water-Electrolyte Balance

With the nystatin-perforated whole-cell patch-clamp recording technique, the modulatory effects of adenosine on GABA-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (laminae I and II) of the rat spinal dorsal horn. The results showed that: (1) GABA acted on GABA(A) receptor and elicited inward Cl(-) currents (I(GABA)) at a holding potential (V(H)) of -40 mV; (2) adenosine suppressed GABA-induced Cl(-) current with affecting neither the reversal potential of I(GABA) nor the apparent affinity of GABA to its receptor; (3) N6-cyclo-hexyladenosine, a selective A(1) adenosine receptor agonist, mimicked the suppressing effect of adenosine on I(GABA), whereas 8-cyclopentyl-1,3-dipropylxanthine, a selective A(1) adenosine receptor antagonist, blocked the suppressing effect of adenosine; (4) chelerythrine, an inhibitor of protein kinase C, reduced the suppressing effect of adenosine on I(GABA); (5) pretreatment with 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester, a Ca(2+) chelator, did not affect adenosine-induced suppression of I(GABA). The results indicate that: (1) the suppression of adenosine on I(GABA) is mediated by adenosine A(1) receptor and through a Ca(2+)-independent protein kinase C transduction pathway; (2) the interactions between adenosine and GABA might be involved in the modulation of nociceptive information transmission at spinal cord level.

Topics: Adenosine; Alkaloids; Analgesics; Animals; Animals, Newborn; Benzophenanthridines; Bicuculline; Chelating Agents; Diglycerides; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Electric Conductivity; Enzyme Inhibitors; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Isoquinolines; Lithium; Membrane Potentials; Muscimol; Neural Inhibition; Patch-Clamp Techniques; Phenanthridines; Posterior Horn Cells; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Spinal Cord; Sulfonamides; Xanthines

Effects of norepinephrine (NE) on the glycine-mediated inhibitory response were investigated in neurons acutely dissociated from the rat spinal dorsal horn, using nystatin perforated patch recording mode under voltage-clamp conditions. NE reversibly and concentration dependently facilitated Cl(-) current induced by 3 x 10(-5) M glycine. NE neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. This effect could be mimicked by clonidine (10(-7) M) and blocked by yohimbine (10(-6) M), respectively. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89), an inhibitor of protein kinase A, effectively mimicked the effect of NE on glycine response, whereas chelerythrine (an inhibitor of protein kinase C) failed. NE further enhanced glycine response even in the presence of chelerythrine or stearoylcarnitine chloride (another inhibitor of protein kinase C) or chelerythrine together with stearoylcarnitine chloride. The present results suggest that alpha2-adrenoceptor is involved in the potentiation of NE on glycine response in freshly isolated spinal dorsal horn neurons. Activation of alpha2-adrenoceptor down-regulates the activity of protein kinase A that results in the potentiation of the glycinergic inhibitory effects within the spinal dorsal horn.

Topics: Alkaloids; Animals; Benzophenanthridines; Clonidine; Dose-Response Relationship, Drug; Drug Combinations; Electric Conductivity; Enzyme Inhibitors; Glycine; Isoquinolines; Neurons; Norepinephrine; Nystatin; Patch-Clamp Techniques; Phenanthridines; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-2; Sulfonamides; Yohimbine

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

Human sperm capacitation involves complex signal transduction mechanisms during which double phosphorylation of the threonine-glutamine-tyrosine motif (P-Thr-Glu-Tyr-P) occurs in some sperm proteins. The objective of this study was to investigate the regulation of this process. Fetal cord serum ultrafiltrate (FCSu), follicular fluid ultrafiltrate (FFu), progesterone and a combination of N(6),2'-O-dibutyryl cAMP (dbcAMP; cell permeant analogue of cAMP) and 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase inhibitor) were used as inducers of capacitation alone or in combination with inhibitors of protein kinase A (H89), protein kinase C (chelerythrine), protein tyrosine kinase (tyrphostin A47, PP2) and of dual specificity kinase (MEK-like kinases; PD98059). The level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation induced by FCSu, FFu and progesterone was regulated by a similar signal transduction pathway and involved receptor type protein tyrosine kinase and dual specificity kinase (MEK or MEK-like) but not protein kinase A or C. However, the level of P-Thr-Glu-Tyr-P in these sperm proteins during capacitation induced by dbcAMP+IBMX was mainly mediated through protein kinase A and C and receptor type protein tyrosine kinase, but not by dual specificity kinase. In conclusion, human sperm capacitation induced by some biological and pharmacological agents is regulated through very different signal transduction pathways.

Topics: 1-Methyl-3-isobutylxanthine; Alkaloids; Amino Acid Motifs; Benzophenanthridines; Bucladesine; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Fetal Blood; Flavonoids; Follicular Fluid; Humans; Isoquinolines; Male; MAP Kinase Kinase Kinase 1; Phenanthridines; Phosphorylation; Progesterone; Protein Kinase C; Protein Serine-Threonine Kinases; Seminal Plasma Proteins; Signal Transduction; Sperm Capacitation; Sulfonamides; Tyrosine; Tyrphostins

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl(-) absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V(te), at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress. The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (V(te) and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V(te) and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of V(te) and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine. It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.

Topics: Actins; Alkaloids; Anguilla; Animals; Azepines; Benzophenanthridines; Bridged Bicyclo Compounds, Heterocyclic; Bumetanide; Cell Size; Colchicine; Cytochalasins; Cytoskeleton; Enzyme Inhibitors; Hypertonic Solutions; Hypotonic Solutions; Intestinal Mucosa; Ion Transport; Isoquinolines; Marine Toxins; Microtubules; Naphthalenes; Osmotic Pressure; Oxazoles; Phenanthridines; Phosphorylation; Protein Serine-Threonine Kinases; Sulfonamides; Thiazoles; Thiazolidines

Intracellular recordings were obtained from rat presumed jaw-closing motoneurons in slice preparations to investigate the involvement of the serotonin(7) (5-HT(7)) receptors in serotonergic inhibition of the postspike medium-duration afterhyperpolarization (mAHP) and enhancement of the afterdepolarization (ADP). 5-HT-induced suppression of the mAHP and enhancement of the ADP were mimicked by application of the 5-HT(1A/7) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and antagonized by the 5-HT(2/6/7) receptor antagonist clozapine, whereas the 5-HT(2) receptor agonist alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) did not affect the mAHP and ADP. 8-OH-DPAT-induced attenuation of the mAHP and enhancement of the ADP were also antagonized by clozapine and another 5-HT(2/6/7) receptor antagonist ritanserin, whereas the 5-HT(1A) receptor antagonist pindolol failed to block the 8-OH-DPAT-induced effects on the mAHP and ADP. 8-OH-DPAT-induced suppression of the mAHP and enhancement of the ADP were also antagonized by a protein kinase A (PKA) inhibitor H89, whereas 8-OH-DPAT could inhibit the mAHP and enhance the ADP in the presence of a protein kinase C (PKC) inhibitor chelerythrine. The 8-OH-DPAT-induced suppression of the mAHP was enhanced under raised [Ca(2+)](o) and this enhancement was reduced by chelerythrine. It is suggested that the 5-HT(7) receptors are involved in 5-HT-induced attenuation of the mAHP and enhancement of the ADP through activation of PKA, and the attenuation of mAHP through the 5-HT(7) receptors is enhanced under raised [Ca(2+)](o) by PKC activation.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Action Potentials; Alkaloids; Animals; Benzophenanthridines; Brain Stem; Calcium; Clozapine; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Enzyme Inhibitors; Isoquinolines; Jaw; Membrane Potentials; Motor Neurons; Phenanthridines; Pindolol; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; Ritanserin; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Sulfonamides

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha, which can sequester betagamma-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the betagamma-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.

Topics: Alkaloids; Benzophenanthridines; Blotting, Northern; Cell Differentiation; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gs; Heterotrimeric GTP-Binding Proteins; Humans; Isoquinolines; Mitogen-Activated Protein Kinases; Mutation; Osteoblasts; Osteosarcoma; Parathyroid Hormone-Related Protein; Phenanthridines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Proteins; ras Proteins; Signal Transduction; Sulfonamides; Transfection; Tumor Cells, Cultured

To determine the role of membrane transporters in intracellular pH (pH(i)) regulation under conditions of low microenvironmental O(2), we monitored pH(i) in isolated single CA1 neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O(2) deprivation or anoxia (PO(2) approximately equal to 0 Torr), a large increase in pH(i) was seen in CA1 neurons in HEPES buffer, but a drop in pH(i), albeit small, was observed in the presence of HCO(3)(-). Ionic substitution and pharmacological experiments showed that the large anoxia-induced pH(i) increase in HEPES buffer was totally Na(+) dependent and was blocked by HOE-694, strongly suggesting the activation of the Na(+)/H(+) exchanger (NHE). Also, this pH(i) increase in HEPES buffer was significantly smaller in Na(+)/H(+) exchanger isoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pH(i) increase following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pH(i) increase in the absence of HCO. We conclude that 1) O(2) deprivation activates Na(+)/H(+) exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pH(i) during low-O(2) states in neurons.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acids; Alkaloids; Animals; Benzophenanthridines; Cell Hypoxia; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Guanidines; Hippocampus; Hydrogen-Ion Concentration; Hypoxia, Brain; In Vitro Techniques; Isoquinolines; Male; Mice; Mice, Neurologic Mutants; Neurons; Oxygen; Phenanthridines; Protein Kinase C; Sodium; Sodium-Hydrogen Exchangers; Sulfonamides; Sulfones

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

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both L- and T-type voltage-gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H89 blocked this increase in both L- and T-type Ca2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12, 13-dibutyrate (1 micromol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19-36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca2+ currents via cAMP/PKA, but decreases voltage-gated K+ currents via the PKC signalling system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcium Channels; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Growth Hormone-Releasing Hormone; Humans; Isoquinolines; Naphthalenes; Patch-Clamp Techniques; Phenanthridines; Phorbol 12,13-Dibutyrate; Pituitary Gland; Potassium Channels; Protein Kinase C; Sheep; Signal Transduction; 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

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

Topics: Alkaloids; Animals; Benzophenanthridines; Biological Transport; Cells, Cultured; Cerebral Cortex; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Fetus; Glutamic Acid; Isoquinolines; Kinetics; Neurons; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar; Sulfonamides; Tetradecanoylphorbol Acetate

A perforated patch recording method was used to determine the effects of genistein (Gen), a protein tyrosine kinase (PTK) inhibitor, on basal L-type Ca2+ current (ICa,L) in feline atrial myocytes. Gen (50 microM) elicited biphasic changes in ICa,L: an initial inhibition (-55 +/- 4%; phase 1) followed by a secondary stimulation (34 +/- 9%; phase 2) of ICa,L. Withdrawal of Gen elicited a further potentiation of ICa,L (152 +/- 19%; phase 3) above control (n = 46). In general, phase 1 inhibition and phase 3 potentiation varied directly with Gen concentration, and phase 2 stimulation exhibited biphasic concentration-dependent changes compared with control. When cells were dialyzed using a ruptured patch recording method, Gen elicited only inhibition of ICa,L; phases 2 and 3 were abolished. Vanadate (1 mM), an inhibitor of protein tyrosine phosphatase, abolished both Gen-induced inhibition and stimulation of ICa,L. Daidzein (50 microM), a weakly active analog of Gen, exerted no significant effects on ICa,L, and withdrawal of daidzein failed to potentiate ICa,L. In a few cells, Gen elicited a prominent vanadate-sensitive stimulation of ICa,L in the absence of any significant inhibition of ICa,L. Gen-induced changes in ICa,L were unaffected by either 100 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM) or 1 microM ryanodine, agents that alter intracellular Ca2+; 4 microM H-89 or 50 microM Rp diastereomer of adenosine 3',5'-monophosphothioate (RP-cAMPS), inhibitors of protein kinase A (PKA); 0.1 microM calphostin C or 2 microM chelerythrine, inhibitors of protein kinase C (PKC); or 100 microM NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthase. We conclude that in feline atrial myocytes, Gen acts via membrane-bound PTKs to inhibit ICa,L and via cytosolic PTKs to stimulate ICa,L. Gen-induced changes in ICa,L are not related to changes in intracellular Ca2+ or to secondary interactions with either PKA, PKC, or NO signaling pathways. These results indicate that in atrial myocytes ICa,L is regulated by two independent and competing PTK signaling mechanisms.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cats; Cells, Cultured; Cyclic AMP; Egtazic Acid; Enzyme Inhibitors; Female; Genistein; Heart; Heart Atria; Isoquinolines; Kinetics; Male; Membrane Potentials; Myocardium; omega-N-Methylarginine; Phenanthridines; Protein Kinase Inhibitors; Ryanodine; Sulfonamides; Thionucleotides

The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P < 0.001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP-protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP-PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP-PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP-PKA pathway in ovine somatotrophs during the action of GRF or GHRP.

Topics: Alkaloids; Animals; Benzophenanthridines; Carcinogens; Cell Membrane; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Growth Hormone; Growth Hormone-Releasing Hormone; Isoquinolines; Naphthalenes; Oligopeptides; Phenanthridines; Pituitary Gland; Protein Kinase C; Second Messenger Systems; Sheep; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate; Time Factors

Mammalian circadian rhythms originate in the hypothalamic suprachiasmatic nuclei (SCN), from which rhythmic neural activity can be recorded in vitro. Application of neurochemicals can reset this rhythm. Here we determine cellular correlates of the phase-shifting properties of neuropeptide Y (NPY) on the hamster circadian clock in vitro. Drug or control treatments were applied to hypothalamic slices containing the SCN on the first day in vitro. The firing rates of individual cells were sampled on the second day in vitro. Control slices exhibited a peak in firing rate in the middle of the day. Microdrop application of NPY to the SCN phase advanced the time of peak firing rate. This phase-shifting effect of NPY was not altered by block of sodium channels with tetrodotoxin or block of calcium channels with cadmium and nickel, consistent with a direct postsynaptic site of action. Pretreatment with the glutamate receptor antagonists (DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium) also did not alter phase shifts to NPY. Blocking GABAA receptors with bicuculline (Bic) had effects only at very high (millimolar) doses of Bic, whereas blocking GABAB receptors did not alter effects of NPY. Phase shifts to NPY were blocked by pretreatment with inhibitors of protein kinase C (PKC), suggesting that PKC activation may be necessary for these effects. Bathing the slice in low Ca2+/high Mg2+ can block phase shifts to NPY, possibly via a depolarizing action. A depolarizing high K+ bath can also block NPY phase shifts. The results are consistent with direct action of NPY on pacemaker neurons, mediated through a signal transduction pathway that depends on activation of PKC.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Alkaloids; Animals; Benzophenanthridines; Bicuculline; Calcium Channel Blockers; Cations, Divalent; Cell Communication; Circadian Rhythm; Cricetinae; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; GABA Antagonists; Indoles; Ion Channels; Isoquinolines; Male; Maleimides; Mesocricetus; Naphthalenes; Nerve Tissue Proteins; Neuropeptide Y; Phenanthridines; Phorbol Esters; Protein Kinase C; Receptors, GABA-A; Receptors, GABA-B; Receptors, Glutamate; Signal Transduction; Sulfonamides; Suprachiasmatic Nucleus; Tetrodotoxin

Using a balanced conditioned place preference (CPP) paradigm, we studied the role of protein kinases A (PKA) and C (PKC) on the acquisition, consolidation and expression of cocaine place conditioning. H7, a non-selective inhibitor of protein kinases, was administered intracerebroventricularly at 1 and 10 micrograms/10 microliters. The higher dose significantly reduced the time spent by rats in the cocaine compartment when given immediately after each conditioning session (consolidation), whereas it had no effect when administered before cocaine during the training phase (acquisition) or before testing for place preference in the absence of cocaine (expression). The same effect was found on administering immediately after each training session 3 micrograms/10 microliters chelerythrine, a selective PKC inhibitor, or 10 micrograms/10 microliters H89, a selective PKA inhibitor, suggesting that both kinases contribute to the consolidation of stimulus-reward association which determines rats' behavior in the cocaine CPP. Changes in the activity of PKA and PKC may thus be part of the cascade of events that contribute to enhancing synaptic responses in the consolidation phase of cocaine CPP and determine rats' behavior associated with the memory of the rewarding effect of cocaine during cocaine CPP expression. These findings may have implications for the study of cocaine 'craving' and relapse.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Benzophenanthridines; Cocaine; Conditioning, Operant; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Injections, Intraventricular; Isoquinolines; Male; Motor Activity; Narcotics; Phenanthridines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Sulfonamides

The cellular regulation mechanism of Na-K-Cl cotransport was studied in dispersed acinar cells of the guinea pig nasal gland by a microfluorimetric imaging method using the Na(+)-sensitive dye sodium-binding benzofuran isophthalate. Addition of 1 micron acetylcholine (ACh) induced an immediate increase in intracellular Na+ concentration ([Na+]i) by 36.7 +/- 9.9 mM, which was almost completely abolished by the addition of atropine. The increased [Na+]i after cholinergic stimulation was due to the external (Cl-)-dependent cotransport system (about 80% of the total Na+ influx) and the dimethyl amiloride-sensitive (Na+)-H+ exchange system (of about 20%). The ACh-induced increase in [Na+]i was dependent on extracellular Ca2+ and was prevented by pretreatment with 8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate or O-O'-bis(2-aminophenyl)ethyleneglycol-N, N, N', N'-tetraacetic acid tetraacetoxymethylester. Addition of 1 microns ionomycin mimicked the ACh-induced increase in [Na+]i which was dependent on external Cl-. Moreover, both a calmodulin antagonist trifluoperazine and a myosin light chain kinase inhibitor ML-7 reduced the ACh-induced response in [Na+]i. However, the following treatment did not affect the basal [Na+]i nor the ACh-induced increase in [Na+]i: (i) addition of dibutyryl cAMP, 8-Br-cGMP, or phorbol 12-myristate 13-acetate, (ii) pretreatment of protein kinase inhibitors, H-89, H-8, H-7 or chelerythrine, (iii) prevention of cytosolic Cl- efflux by the addition of diphenylamine-2-carboxylic acid or, (iv) prevention of cytosolic K+ efflux by the addition of charybdotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Biological Transport; Bucladesine; Bumetanide; Calcium; Calmodulin; Carrier Proteins; Charybdotoxin; Cholinergic Fibers; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Exocrine Glands; Furosemide; Guinea Pigs; Ionomycin; Ionophores; Isoquinolines; Nasal Cavity; Phenanthridines; Protein Kinase Inhibitors; Protein Kinases; Second Messenger Systems; Signal Transduction; Sodium; Sodium-Potassium-Chloride Symporters; Sulfonamides; Tetradecanoylphorbol Acetate; Trifluoperazine; Vasoactive Intestinal Peptide