h-89 and adenosine-3--5--cyclic-phosphorothioate

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

Topics: Cell Line; Cyclic AMP; Epidermal Growth Factor; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Genes, Immediate-Early; Humans; Isoquinolines; Mitogens; Reproducibility of Results; Ribosomal Protein S6 Kinases, 90-kDa; Sulfonamides; Tetradecanoylphorbol Acetate; Thionucleotides

Acute ethanol overdose can induce dysfunction of cerebellar motor regulation and cerebellar ataxia. In this study, we investigated the effect of ethanol on facial stimulation-evoked inhibitory synaptic responses in cerebellar Purkinje cells (PCs) in urethane-anesthetized mice, using in vivo patch-clamp recordings. Under voltage-clamp conditions, ethanol (300 mM) decreased the amplitude, half-width, rise time and decay time of facial stimulation-evoked outward currents in PCs. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent, with an IC50 of 148.5 mM. Notably, the ethanol-induced inhibition of facial stimulation-evoked outward currents were significantly abrogated by cannabinoid receptor 1 (CB1) antagonists, AM251 and O-2050, as well as by the CB1 agonist WIN55212-2. Moreover, the ethanol-induced inhibition of facial stimulation-evoked outward currents was prevented by cerebellar surface perfusion of the PKA inhibitors H-89 and Rp-cAMP, but not by intracellular administration of the PKA inhibitor PKI. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activating presynaptic CB1 receptors via the PKA signaling pathway. These findings suggest that ethanol overdose impairs sensory information processing, at least in part, by inhibiting GABA release from molecular layer interneurons onto PCs.

Topics: Action Potentials; Animals; Benzoxazines; Central Nervous System Depressants; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dronabinol; Ethanol; gamma-Aminobutyric Acid; Isoquinolines; Membrane Potentials; Mice; Mice, Inbred ICR; Morpholines; Naphthalenes; Patch-Clamp Techniques; Physical Stimulation; Piperidines; Purkinje Cells; Pyrans; Pyrazoles; Receptor, Cannabinoid, CB1; Reflex; Signal Transduction; Sulfonamides; Thionucleotides

Melatonin secretion from the pineal gland is triggered by norepinephrine released from sympathetic terminals at night. In contrast, cholinergic and parasympathetic inputs, by activating nicotinic cholinergic receptors (nAChR), have been suggested to counterbalance the noradrenergic input. Here we investigated whether adrenergic signaling regulates nAChR channels in rat pinealocytes. Acetylcholine or the selective nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) activated large nAChR currents in whole cell patch-clamp experiments. Norepinephrine (NE) reduced the nAChR currents, an effect partially mimicked by a β-adrenergic receptor agonist, isoproterenol, and blocked by a β-adrenergic receptor antagonist, propranolol. Increasing intracellular cAMP levels using membrane-permeable 8-bromoadenosine (8-Br)-cAMP or 5,6-dichlorobenzimidazole riboside-3',5'-cyclic monophosphorothioate (cBIMPS) also reduced nAChR activity, mimicking the effects of NE and isoproterenol. Further, removal of ATP from the intracellular pipette solution blocked the reduction of nAChR currents, suggesting involvement of protein kinases. Indeed protein kinase A inhibitors, H-89 and Rp-cAMPS, blocked the modulation of nAChR by adrenergic stimulation. After the downmodulation by NE, nAChR channels mediated a smaller Ca(2+) influx and less membrane depolarization from the resting potential. Together these results suggest that NE released from sympathetic terminals at night attenuates nicotinic cholinergic signaling.

Topics: Acetylcholine; Animals; Calcium; Cells, Cultured; Cyclic AMP; Down-Regulation; Isoquinolines; Male; Nicotinic Agonists; Norepinephrine; Phosphorylation; Pineal Gland; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Sulfonamides; Thionucleotides

The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Benzoquinones; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Genistein; Humans; In Vitro Techniques; Isoquinolines; Lactams, Macrocyclic; Leupeptins; Male; Oligopeptides; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein-Tyrosine Kinases; Rifabutin; Sperm Capacitation; Spermatozoa; Sulfonamides; Thionucleotides

Channels formed by the gap junction protein connexin36 (Cx36) contribute to the proper control of insulin secretion. We investigated the impact of chronic hyperlipidemia on Cx36 expression in pancreatic beta-cells. Prolonged exposure to the saturated free fatty acid palmitate reduced the expression of Cx36 in several insulin-secreting cell lines and isolated mouse islets. The effect of palmitate was fully blocked upon protein kinase A (PKA) inhibition by H89 and (Rp)-cAMP, indicating that the cAMP/PKA pathway is involved in the control of Cx36 expression. Palmitate treatment led to overexpression of the inducible cAMP early repressor (ICER-1gamma), which bound to a functional cAMP-response element located in the promoter of the CX36 gene. Inhibition of ICER-1gamma overexpression prevented the Cx36 decrease, as well as the palmitate-induced beta-cell secretory dysfunction. Finally, freshly isolated islets from mice undergoing a long term high fat diet expressed reduced Cx36 levels and increased ICER-1gamma levels. Taken together, these data demonstrate that chronic exposure to palmitate inhibits the Cx36 expression through PKA-mediated ICER-1gamma overexpression. This Cx36 down-regulation may contribute to the reduced glucose sensitivity and altered insulin secretion observed during the pre-diabetic stage and in the metabolic syndrome.

Topics: Animals; Cell Line, Tumor; Connexins; Cyclic AMP; Cyclic AMP Response Element Modulator; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus; Diet, Atherogenic; Dietary Fats; Down-Regulation; Gap Junction delta-2 Protein; Gap Junctions; Insulin; Insulin Secretion; Insulin-Secreting Cells; Isoquinolines; Metabolic Syndrome; Mice; Palmitic Acid; Protein Kinase Inhibitors; Rats; Sulfonamides; Thionucleotides

We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

Topics: Animals; Caco-2 Cells; Carbazoles; Cell Fractionation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Humans; Indoles; Intestines; Isoquinolines; Membrane Proteins; Microscopy, Confocal; Occludin; Octoxynol; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Pyrroles; RNA, Messenger; Rotavirus; Sulfonamides; Thionucleotides; Tight Junctions; Transcription, Genetic; Zonula Occludens-1 Protein; Zonula Occludens-2 Protein

The stimulatory effect of insulin-like growth factor I (IGF-I) on myelin basic protein (MBP) expression, a parameter for oligodendrocyte development, is mediated by the MAPK and PI3K signaling pathways. We have previously shown that the second messenger cAMP inhibits IGF-I-induced MAPK activation as well as MBP expression. We also showed that the PKA inhibitor Rp-cAMPS reverted the cAMP effect on IGF-I-induced MBP without affecting the cAMP effect on IGF-I-induced MAPK activation. Here we report that, in contrast to Rp-cAMPS, H89 (a PKA inhibitor structurally non-related to Rp-cAMPS) enhances both the inhibitory effect of cAMP on IGF-I-induced MBP expression and the inhibitory effect of cAMP on IGF-I-induced MAPK activation. Likewise, H89 is capable of inhibiting the IGF-I-induced MAPK activation in the absence of PKA stimulation. Thus, we hypothesize that an unspecific action of H89 on a target located upstream MAPK could account for the discrepancies between the effects elicited by Rp-cAMPS and H89.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Differentiation; Cells, Cultured; Culture Media; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Immunohistochemistry; Insulin-Like Growth Factor I; Isoquinolines; Mitogen-Activated Protein Kinases; Myelin Basic Protein; Oligodendroglia; Phosphatidylinositol 3-Kinases; Rats; Rats, Sprague-Dawley; Substrate Specificity; Sulfonamides; Thionucleotides

Cardiac voltage-gated L-type Ca channels (Ca(V)) are multiprotein complexes, including accessory subunits such as Ca(V)beta2 that increase current expression. Recently, members of the Rad and Gem/Kir-related family of small GTPases have been shown to decrease current, although the mechanism remains poorly defined. In this study, we evaluated the contribution of the L-type Ca channel alpha-subunit (Ca(V)1.2) to Ca(V)beta2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies Ca(V)beta2-Rem inhibition of Ca channel current. We first tested the effect of Rem on Ca(V)1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with Ca(V)1.2 reduces Ba current expression under basal conditions, and Ca(V)beta2a coexpression enhances Rem block of Ca(V)1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 microM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without Ca(V)beta2a. To test whether the H-89 action was a consequence of the phosphorylation status of Ca(V)1.2, we examined Rem regulation of the PKA-insensitive Ca(V)1.2 serine 1928 (S1928) to alanine mutation (Ca(V)1.2-S1928A). Ca(V)1.2-S1928A current was not inhibited by Rem and when coexpression with Ca(V)beta2a was not completely blocked by Rem coexpression, suggesting that the phosphorylation of S1928 contributes to Rem-mediated Ca channel modulation. As a model for native Ca channel complexes, we tested the ability of Rem overexpression in HIT-T15 cells and embryonic ventricular myocytes to interfere with native current. We find that native current is also sensitive to Rem block and that H-89 pretreatment relieves the ability of Rem to regulate Ca current. We conclude that Rem is capable of regulating L-type current, that release of Rem block is modulated by cellular kinase pathways, and that the Ca(V)1.2 COOH terminus contributes to Rem-dependent channel inhibition.

Topics: Animals; Calcium; Calcium Channels, L-Type; Cell Line; Cricetinae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; Humans; Isoquinolines; Mesocricetus; Mice; Mice, Inbred ICR; Monomeric GTP-Binding Proteins; Myocytes, Cardiac; Patch-Clamp Techniques; Phosphorylation; Protein Kinase Inhibitors; Sulfonamides; Thionucleotides

It has previously been suggested that ACTH and ACTH-related peptides may act as paracrine modulators of insulin secretion in the islets of Langerhans. We have, therefore, examined the expression and function of the ACTH receptor (the melanocortin 2 receptor, MC2-R) in human and mouse primary islet tIssue and in the MIN6 mouse insulinoma cell line. Mouse MC2-R mRNA was detected in both MIN6 cells and mouse islet tIssue by PCR amplification of cDNA. In perifusion experiments with MIN6 pseudo-islets, a small, transient increase in insulin secretion was obtained when ACTH(1-24) (1 nM) was added to medium containing 2 mM glucose (control) but not when the medium glucose content was increased to 8 mM. Further investigations were performed using static incubations of MIN6 cell monolayers; ACTH(1-24) (1 pM-10 nM) provoked a concentration-dependent increase in insulin secretion from MIN6 monolayer cells that achieved statistical significance at concentrations of 1 and 10 nM (150 +/- 13.6% basal secretion; 187 +/- 14.9% basal secretion, P<0.01). Similar responses were obtained with ACTH(1-39). The phosphodiesterase inhibitor IBMX (100 microM) potentiated the responses to sub-maximal doses of ACTH(1-24). Two inhibitors of the protein kinase A (PKA) signaling pathway, Rp-cAMPS (500 microM) and H-89 (10 microM), abolished the insulin secretory response to ACTH(1-24) (0.5-10 nM). Treatment with 1 nM ACTH(1-24) caused a small, statistically significant increase in intracellular cAMP levels. Secretory responses of MIN6 cells to ACTH(1-24) were also influenced by changes in extracellular Ca2+ levels. Incubation in Ca2+-free buffer supplemented with 0.1 mM EGTA blocked the MIN6 cells' secretory response to 1 and 10 nM ACTH(1-24). Similar results were obtained when a Ca2+ channel blocker (nitrendipine, 10 microM) was added to the Ca2+-containing buffer. ACTH(1-24) also evoked an insulin secretory response from primary tIssues. The addition of ACTH(1-24) (0.5 nM) to perifusions of mouse islets induced a transient increase in insulin secretion at 8 mM glucose. Perifused human primary islets also showed a secretory response to ACTH(1-24) at basal glucose concentration (2 mM) with a rapid initial spike in insulin secretion followed by a decline to basal levels. Overall the results demonstrate that the MC2-R is expressed in beta-cells and suggest that activation of the receptor by ACTH initiates insulin secretion through the activation of PKA in association with Ca2+ influx into

Topics: 1-Methyl-3-isobutylxanthine; Adrenocorticotropic Hormone; Animals; Calcium Channel Blockers; Cell Line; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Isoquinolines; Mice; Nitrendipine; Paracrine Communication; Phosphodiesterase Inhibitors; Receptor, Melanocortin, Type 2; RNA, Messenger; Stimulation, Chemical; Sulfonamides; Thionucleotides

Antipsychotic drugs regulate gene transcription in striatal neurons by blocking dopamine D2-like receptors. Little is known about the underlying changes in chromatin structure, including covalent modifications at histone N-terminal tails that are epigenetic regulators of gene expression. We show that treatment with D2-like antagonists rapidly induces the phosphorylation of histone H3 at serine 10 and the acetylation of H3-lysine 14 in bulk chromatin from striatum and in nuclei of striatal neurons. We find that, in vivo, D2-like antagonist-induced H3 phospho-acetylation is inhibited by the NMDA receptor antagonist MK-801 and by the protein kinase A (PKA) inhibitor Rp-adenosine 3c',5c'-cyclic monophosphorothioate triethylammonium salt but increased by the PKA activator Sp-adenosine 3c',5c'-cyclic monophosphorothioate triethylammonium salt. Furthermore, in dissociated striatal cultures which lack midbrain and cortical pre-synaptic inputs, H3 phospho-acetylation was induced by glutamate, L-type Ca2+ channel agonists and activators of cAMP-dependent PKA but inhibited by NMDA receptor antagonists or PKA antagonists. The dual modification, H3pS10-acK14, was enriched at genomic sites with active transcription and showed the kinetics of the early response. Together, these results suggest that histone modifications and chromatin structure in striatal neurons are dynamically regulated by dopaminergic and glutamatergic inputs converging on the cellular level. Blockade of D2-like receptors induces H3 phospho-acetylation, H3pS10-acK14, through cAMP-dependent PKA, and post-synaptic NMDA receptor signaling.

Topics: Acetylation; Animals; Animals, Newborn; Blotting, Southern; Blotting, Western; Chromatin Assembly and Disassembly; Corpus Striatum; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dizocilpine Maleate; Dopamine Agents; Dopamine Antagonists; Dopamine D2 Receptor Antagonists; Drug Administration Routes; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Genes, fos; Glial Fibrillary Acidic Protein; Glutamic Acid; Haloperidol; Histones; Immunohistochemistry; In Vitro Techniques; Indoles; Isoquinolines; Male; Methylation; Mice; Neurons; Phosphopyruvate Hydratase; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Dopamine D2; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Thionucleotides; Time Factors

Local perfusion of the dorsal root ganglion (DRG) with tumor necrosis factor alpha (TNF-alpha) in rats induces cutaneous hypersensitivity to mechanical stimuli. Thus we investigated the cellular mechanisms of TNF-alpha-induced mechanical hyperalgesia. The L(4) and L(5) DRGs with the sciatic nerves attached were excised from rats for in vitro dorsal root microfilament recording. After baseline recording for 15 min, TNF-alpha (0.001, 0.01, 0.1, or 1 ng/ml) was applied to the DRG for 15 min, followed by washout for at least 30 min. Alternatively, H-89 or Rp-cAMPS, two specific cAMP-dependent protein kinase (PKA) inhibitors, was added to the perfusion solution for 15 min prior to TNF-alpha application. TNF-alpha (1 ng/ml) induced neuronal discharges in 67% (14/21) of C fibers and 27% (4/15) of Abeta fibers when applied topically to the DRG. Acute TNF-alpha application not only evoked discharges in silent fibers, but also enhanced ongoing activity of spontaneously active fibers and increased neuronal sensitivity to electrical stimulation of the peripheral nerves. H-89 (10 microM) and Rp-cAMPS (100 microM) each completely blocked the TNF-alpha-evoked response in most C and Abeta fibers tested but did not affect fiber conductivity. Our results demonstrates that exogenous inflammatory cytokines such as TNF-alpha can elicit a PKA-dependent response in sensory neurons and thus strongly suggest that endogenous TNF-alpha may contribute to the development of certain pathological pain states.

Topics: Administration, Topical; Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Enzyme Inhibitors; Ganglia, Spinal; Isoquinolines; Male; Nerve Fibers; Neural Conduction; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Sulfonamides; Thionucleotides; Time Factors; Tumor Necrosis Factor-alpha

This study assessed the effects of drugs which manipulate the cAMP system on afterdischarges (ADs) induced in the CA1 region of rat hippocampal slices. The adenylate cyclase activator forskolin (50 microM) and the phosphodiesterase inhibitor rolipram (0.1 and 1 microM) enhanced AD generation. These effects were reversed by the cAMP-dependent protein kinase inhibitors H-89 (5 microM) and Rp-cAMPS (100 microM). These findings suggest that AD generation can be modulated through cAMP generation and the subsequent activation of the cAMP-dependent protein kinase.

Topics: Action Potentials; Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Enzyme Activators; Enzyme Inhibitors; Epilepsy; Hippocampus; Isoquinolines; Male; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Rolipram; Seizures; Sulfonamides; Thionucleotides

The species-specific sound production of acoustically communicating grasshoppers can be stimulated by pressure injection of both nicotinic and muscarinic agonists into the central body complex and a small neuropil situated posterior and dorsal to it. To determine the role of muscarinic acetylcholine receptors (mAChRs) in the control of acoustic communication behavior and to identify the second-messenger pathways affected by mAChR-activation, muscarinic agonists and membrane-permeable drugs known to interfere with specific mechanisms of intracellular signaling pathways were pressure injected to identical sites in male grasshopper brains. Repeated injections of small volumes of muscarine elicited stridulation of increasing duration associated with decreased latencies. This suggested an accumulation of excitation over time that is consistent with the suggested role of mAChRs in controlling courtship behavior: to provide increasing arousal leading to higher intensity of stridulation and finally initiating a mating attempt. At sites in the brain where muscarine stimulation was effective, stridulation could be evoked by forskolin, an activator of adenylate cyclase (AC); 8-Br-cAMP-activating protein kinase A (PKA); and 3-isobuty-1-methylxanthine, leading to the accumulation of endogenously generated cAMP through inhibition of phosphodiesterases. This suggested that mAChRs mediate excitation by stimulating the AC/cAMP/PKA pathway. In addition, muscarine-stimulated stridulation was inhibited by 2'-5'-dideoxyadenonsine and SQ 22536, two inhibitors of AC; H-89 and Rp-cAMPS, two inhibitors of PKA; and by U-73122 and neomycin, two agents that inhibit phospholipase C (PLC) by independent mechanisms. Because the inhibition of AC, PKA, or PLC by various individually applied substances entirely suppressed muscarine-evoked stridulation in a number of experiments, activation of both pathways, AC/cAMP/PKA and PLC/IP(3)/diacylglycerine, appeared to be necessary to mediate the excitatory effects of mAChRs. With these studies on an intact "behaving" grasshopper preparation, we present physiological relevance for mAChR-evoked excitation mediated by sequential activation of the AC- and PLC-initiated signaling pathways that has been reported in earlier in vitro studies.

Topics: Acetylcholine; Adenine; Adenylyl Cyclases; Animal Communication; Animals; Brain; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dideoxyadenosine; Diglycerides; Enzyme Inhibitors; Estrenes; Grasshoppers; Inositol 1,4,5-Trisphosphate; Isoquinolines; Muscarine; Muscarinic Agonists; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Receptors, Muscarinic; Second Messenger Systems; Sphingosine; Sulfonamides; Thapsigargin; Thionucleotides; Type C Phospholipases

Muscarinic acetylcholine receptors exert slow and prolonged synaptic effects in both vertebrate and invertebrate nervous systems. Through activation of G proteins, they typically decrease intracellular cAMP levels by inhibition of adenylate cyclase or stimulate phospholipase C and the turnover of inositol phosphates. In insects, muscarinic receptors have been credited with two main functions: inhibition of transmitter release from sensory neuron terminals and regulation of the excitability of motoneurons and interneurons. Our pharmacological studies with intact and behaving grasshoppers revealed a functional role for muscarinic acetylcholine receptors as being the basis for specific arousal in defined areas of the brain, underlying the selection and control of acoustic communication behavior. Periodic injections of acetylcholine into distinct areas of the brain elicited songs of progressively increasing duration. Coinjections of the muscarinic receptor antagonist scopolamine and periodic stimulations with muscarine identified muscarinic receptor activation as being the basis for the underlying accumulation of excitation. In contrast to reports from other studies on functional circuits, muscarinic excitation was apparently mediated by activation of the adenylate cyclase pathway. Stimulation of adenylate cyclase with forskolin and of protein kinase A with 8-Br-cAMP mimicked the stimulatory effects of muscarine whereas inhibition of adenylate cyclase with SQ22536 and of protein kinase A with H-89 and Rp-cAMPs suppressed muscarine-stimulated singing behavior. Activation of adenylate cyclase by muscarinic receptors has previously been reported from studies on membrane preparations and heterologous expression systems, but a physiological significance of this pathway remained to be demonstrated in an in vivo preparation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Acetylcholine; Adenine; Adenylyl Cyclases; Animal Communication; Animals; Arousal; Brain; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Female; Grasshoppers; GTP-Binding Proteins; Insect Proteins; Isoquinolines; Male; Muscarine; Muscarinic Agonists; Muscarinic Antagonists; Nerve Tissue Proteins; Receptors, Muscarinic; Second Messenger Systems; Species Specificity; Sulfonamides; Thionucleotides

Major adaptations after chronic exposure to morphine include an up-regulation of the adenosine 3',5'-monophosphate pathway. Acute opioids, via mu-opioid receptors, disinhibit midbrain serotonergic neurons by suppressing inhibitory GABAergic transmission in the dorsal raphe nucleus and adjacent periaqueductal gray. This study examined whether chronic morphine induces a compensatory increase in GABA inputs to 5-hydroxytryptamine neurons and whether this was associated with an up-regulation of the adenosine 3',5'-monophosphate pathway. The firing rate of serotonergic neurons was reduced in brain slices from morphine-dependent rats, an effect reversed by the GABA(A) antagonist bicuculline. The reduction in firing rate was accompanied by an increased frequency of spontaneous GABAergic inhibitory postsynaptic currents, indicating increased GABA tone in the slice. The increase in GABA tone in brain slices from dependent rats was associated with increased induction of inhibitory postsynaptic currents by the adenylyl cyclase activator forskolin, suggesting an up-regulation of the adenosine 3',5'-monophosphate pathway. Indeed, chronic morphine increased levels of adenylyl cyclase VIII (but not of adenylyl cyclase I, III or V) immunoreactivity in the dorsal raphe nucleus area. Two adenosine 3',5'-monophosphate-mediated mechanisms for the increase in GABA tone were discerned. The first, which predominated when impulse-flow was blocked by tetrodotoxin, involves protein kinase A since it was sensitive to protein kinase A inhibitors. The second, seen when impulse-flow was intact (i.e. absence of tetrodotoxin), was insensitive to protein kinase A inhibitors but was suppressed by ZD7288, a blocker of hyperpolarizing-activated Ih channels which are directly activated by adenosine 3',5'-monophosphate. We conclude that chronic morphine induces an up-regulation of the adenosine 3',5'-monophosphate pathway in GABAergic inputs to serotonergic cells, resulting in an increase in spontaneous and impulse-flow dependent GABA release. These changes would lead to an increase in GABA tone and subsequently to the reported decrease in serotonergic activity during opiate withdrawal.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Cardiovascular Agents; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; In Vitro Techniques; Interneurons; Isoquinolines; Male; Membrane Potentials; Morphine; N-Methylaspartate; Neural Inhibition; Periaqueductal Gray; Phenylephrine; Pyrimidines; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Serotonin; Substance Withdrawal Syndrome; Sulfonamides; Sympathomimetics; Tetrazoles; Tetrodotoxin; Thionucleotides

The effects of cAMP accumulation evoked by acetylcholine (ACh) stimulation were studied in rat submandibular acinar cells by observing the exocytotic events, swelling of intercellular canaliculi (IC) and intracellular Ca2+ concentration ([Ca2+]i), which were monitored using an optical microscope. ACh stimulation evoked transient increases followed by sustained increases in the frequency of exocytotic events and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation evoked sustained increases in these parameters. BAPTA treatment reduced the frequency of exocytotic events evoked by 5 microM ACh in the absence of extracellular Ca2+, and further addition of Rp-cAMPS or H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining ACh-evoked responses (50 %). Addition of PKA inhibitors in the presence of extracellular Ca2+ reduced the frequency of exocytotic events evoked by 500 microM ACh in non-BAPTA-loaded cells. However, IC swelling evoked by 5 microM ACh was not affected by addition of PKA inhibitors, and was eliminated in BAPTA-loaded cells perfused with Ca2+-free solution. These results indicate that the IC swelling is regulated by [Ca2+]i and the frequency of exocytotic events is regulated by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89 inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells. Biochemical analysis revealed that ACh stimulation increased the cAMP content in perfused submandibular glands. These results indicate that ACh stimulates the accumulation of cAMP in submandibular acinar cells and that this accumulation of cAMP modulates Ca2+-regulated exocytosis.

Topics: Acetylcholine; Animals; Body Fluids; Calcium; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Exocytosis; Intracellular Membranes; Isoproterenol; Isoquinolines; Male; Osmolar Concentration; Rats; Rats, Wistar; Submandibular Gland; Sulfonamides; Thionucleotides

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.

Topics: Acetylcholine; Adrenergic beta-Agonists; Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Gene Expression; Heart Atria; Ion Channel Gating; Isoproterenol; Isoquinolines; Membrane Potentials; Muscle Fibers, Skeletal; Myocardium; Oocytes; Phosphorylation; Potassium Channels; Potassium Channels, Inwardly Rectifying; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Sulfonamides; Thionucleotides; Vasodilator Agents; Xenopus laevis

This study investigates the possible modulation of platelet-derived growth factor-(PDGF, 20 ng/ml)-induced DNA synthesis in bovine coronary artery smooth muscle cells by the protein kinase A inhibitors Rp-adenosine-3',5'-cyclic phosphorothioate (Rp-cAMPS, 0. 03-10 microM) and ¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl¿ (H-89, 0.01-1 microM). Rp-cAMPS concentration dependently enhanced PDGF-induced DNA synthesis. In contrast, no potentiation of PDGF-induced DNA synthesis was seen with H-89. However, H-89 but not Rp-cAMPS, inhibited p42/p44 mitogen-activated protein kinase phosphorylation. Thus, Rp-cAMPS, but not H-89, unmasks inhibitory actions of protein kinase A on PDGF-induced mitogenesis of vascular smooth muscle cells. Low specificity may limit the use of H-89 as protein kinase A inhibitor.

Topics: Animals; Cattle; Cells, Cultured; Coronary Vessels; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Isoquinolines; Mitogen-Activated Protein Kinase 1; Mitogens; Muscle, Smooth, Vascular; Phosphorylation; Platelet-Derived Growth Factor; Sulfonamides; Thionucleotides

The aim of this study was to investigate the exact mechanism of interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in isolated smooth muscle cells and smooth muscle strips of the pig gastric fundus. In isolated smooth muscle cells, the maximal relaxant effect of VIP (10(-9) M) was inhibited by 94% by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NA, 10(-4) M) and by 85% by the inducible NOS (iNOS)-selective inhibitor N-(3-(aminomethyl)-benzyl)acetamide (1400W; 10(-6) M). The relaxant effect of VIP was reduced by more than 70% by the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10(-6) M), the glucocorticoid dexamethasone (10(-5) M) and three protein kinase A inhibitors: (R)-p-cyclic adenosine-3', 5'-monophosphothioate ((R)-p-cAMPS; 10(-6) M), ¿(8R,9S, 11S)-(-)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a, g]cycloocta[cde]-trin-den-1-one¿ (KT5720; 10(-6) M) and N-(2-(p-bromo-cinnamylamino)ethyl))-5-isoquinoline sulfonamide dihydrochloride (H-89; 10(-5) M). In contrast, no influence of the NOS inhibitors, ODQ, dexamethasone, nor the protein kinase A inhibitors could be observed on the relaxant effect of VIP in smooth muscle strips. These data demonstrate that the experimental method completely changes the influence of NOS inhibitors on the relaxant effect of VIP in the pig gastric fundus. The isolation procedure of the smooth muscle cells might induce iNOS that can be activated by VIP.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Amidines; Animals; Arginine; Benzylamines; Carbazoles; Colforsin; Cyclic AMP; Dexamethasone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gastric Fundus; In Vitro Techniques; Indoles; Isoquinolines; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Oxadiazoles; Protein Kinase Inhibitors; Pyrroles; Quinoxalines; Sulfonamides; Swine; Thionucleotides; Vasoactive Intestinal Peptide

It has previously been shown that when boar spermatozoa are incubated in a modified Krebs-Ringer bicarbonate (mKRB), head-to-head agglutination occurs in many cells. The aim of the present study was to investigate the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and serum albumin on sperm agglutination and to discuss a possible mechanism for sperm agglutination. Spermatozoa were collected from four mature boars, washed and incubated in mKRB. After a 1-h incubation, a sample of each sperm suspension was smeared gently on a separate glass slide, dried and stained in a phosphate-buffered solution of Giemsa to assess the percentage of head-to-head agglutinated cells in each suspension. In the samples incubated in mKRB, approximately 50% of the spermatozoa were agglutinated with one another at the acrosome. However, the percentages of head-to-head agglutinated spermatozoa were greatly reduced by a lack of calcium chloride in mKRB, but were recovered by the addition of dibutyryl cAMP (dbcAMP, a cAMP analogue) in a dose-dependent manner between 1 and 1000 microM. Addition of 3-isobutyl-1-methylxanthine (IBMX, 100 and 500 microM) instead of dbcAMP also significantly increased the percentages of head-to-head agglutinated spermatozoa. Moreover, the effects of adding dbcAMP were attenuated by treatment with Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt (0.25-1.0 mM, a cAMP antagonist) or H-89 (5 microM, a protein kinase-A inhibitor), but were enhanced by treatment with okadaic acid (500 nM) and calyculinA (500 nM) (inhibitors of protein serine/threonine phosphatase). In sperm samples incubated in mKRB containing 0.1% polyvinyl alcohol (mKRB-P) or mKRB-P lacking calcium chloride and supplemented with 1 mM dbcAMP, a lack of bovine serum albumin (BSA) resulted in a significant decrease in the percentages of head-to-head agglutinated spermatozoa. Addition of porcine serum albumin (PSA, 1-4 mg mL(-1)) or methyl-beta-cyclodextrin (MBC, 5-10 mg mL(-1)) instead of BSA was as effective as BSA (4 mg mL(-1)) in enhancing sperm agglutination. However, the effects of BSA (4 mg mL(-1)) or MBC (5 mg mL(-1)) were reduced by pre-mixing these reagents with cholesterol 3-sulfate (a cholesterol analogue, 5 microg mL(-1) for BSA and 375 microg mL(-1) for MBC). In addition, a protein 'anti-agglutinin' inhibiting sperm agglutination, was extracted from spermatozoa incubated with serum albumin or MBC and detected by sodium dodecyl sulfate-polyacrylamide gel el

Topics: 1-Methyl-3-isobutylxanthine; Animals; beta-Cyclodextrins; Bucladesine; Cells, Cultured; Culture Media; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclodextrins; Enzyme Inhibitors; Glycoproteins; Isoquinolines; Male; Marine Toxins; Oxazoles; Phosphoprotein Phosphatases; Serum Albumin; Signal Transduction; Sperm Agglutination; Sperm Capacitation; Sperm Motility; Spermatozoa; Sulfonamides; Swine; Thionucleotides

Recent studies of beta-adrenergic receptor (beta-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to beta2-AR versus beta1-AR stimulation.. The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to beta-AR subtype stimulation. In addition, many of these parameters and L-type Ca2+ current (ICa) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by beta1-AR stimulation could explain the resultant modulation of cardiac function, substantial beta2-AR-mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that beta2-AR-stimulated increases in both ICa and contraction were abolished by PKA inhibition. Thus, the beta2-AR-directed cAMP/PKA signaling modulates sarcolemmal L-type Ca2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins.. These results indicate that the dissociation of beta2-AR signaling from cAMP regulatory systems is only apparent and that beta2-AR-stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.

Topics: Actin Cytoskeleton; Adrenergic Agents; Animals; Calcium-Binding Proteins; Cardiotonic Agents; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dogs; Heart; Imidazoles; Isoquinolines; Muscle Relaxation; Myocardial Contraction; Myocardium; Norepinephrine; Phosphorylase a; Phosphorylase b; Phosphorylase Kinase; Phosphorylation; Protein Processing, Post-Translational; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Reserpine; Sarcolemma; Second Messenger Systems; Sulfonamides; Thionucleotides; Troponin C

Long-term facilitation of neurotransmission by monoaminergic systems is implicated in the cellular mechanism of memory and learning-related processes at invertebrate synapses. Using whole-cell recording and rat cerebellar slices, we have examined whether mammalian monoamine-containing neurons play analogous roles in synaptic plasticity, and our results suggest that serotonin and noradrenaline are critically involved in short- and long-term modulation of GABAergic transmission in the cerebellar cortex. Exogenously applied serotonin and noradrenaline selectively induced a short-term enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells, their effect subsiding in 30 min. Successive amine applications converted this effect to long-term facilitation lasting more than 2 h. During the monoamine-induced short- and long-term facilitation, spontaneously occurring miniature inhibitory synaptic responses increased in frequency, without significant changes in their mean amplitude and amplitude distribution, as well as the GABA receptor sensitivity of Purkinje cells. The actions of the two amines on the inhibitory transmission were mimicked by forskolin and blocked by kinase inhibitors, H-7, H-89 and Rp-adenosine 3',5'-cyclic monophosphothioate. Thus, serotonin and noradrenaline are likely to activate cyclic-AMP- and protein kinase-dependent pathways in GABAergic interneurons, thereby reinforcing the inhibitory transmission on to Purkinje cells. Repetitive electrical stimulation within the molecular layer mimicked the facilitatory effect induced by exogenous monoamines: namely, neural stimulation selectively elicited long-lasting enhancement of GABAergic transmission in a manner sensitive to the monoamine receptor antagonists, methiothepin and propranolol, and an uptake inhibitor, imipramine. Synaptically released monoamines thus appear to induce cyclic-AMP- and protein kinase-dependent long-term facilitation of cerebellar GABAergic transmission, thereby providing a likely mechanism of synaptic plasticity associated with motor coordination within the mammalian cerebellar system.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cerebellum; Colforsin; Cyclic AMP; Electric Stimulation; Enzyme Inhibitors; Evoked Potentials; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; In Vitro Techniques; Isoquinolines; Long-Term Potentiation; Methiothepin; Neuronal Plasticity; Norepinephrine; Propranolol; Protein Kinase Inhibitors; Purkinje Cells; Rats; Rats, Wistar; Receptors, GABA; Serotonin; Sulfonamides; Synapses; Synaptic Transmission; Tetrodotoxin; Thionucleotides

With a model of chronically compressed dorsal root ganglion (CCD), the present study was undertaken to test how the plasticity of sympathetic-sensory coupling is and whether the coupling is mediated by intracellular messenger PKA by analysing extracellularly recorded spontaneous activity of single A-fibers originating from the CCD neurons in vitro. Eighty-five out of 95 neurons from injured DRGs during application of norepinephrine (NE) were adrenosensitive. Among the 85 neurons, 44 exhibited excitation, 21 showing excitation followed by suppression, 6 displaying alternated excitation and suppression, and 14 suppression. In addition, adrenosensitivity was observed in 15 silent injured DRGs. The excitatory effect of NE was blocked by alpha 1 and alpha 2 adrenoceptor antagonists yohimbine (10 mumol/L) or prazosin (5 mumol/L). Rp-cAMPS (50-250 mumol/L, n = 6), a specific inhibitor of PKA, and H-89 (10 mumol/L, n = 6), an inhibitor of PKA catalytic subunit, obviously suppressed the NE-evoked excitation. Furthermore, the excitatory effect of NE was attenuated by SQ 22, 536 (1 mmol/L), an adenylate cyclase inhibitor (n = 6). The above results demonstrate that injury to DRG neuron body triggered the adrenosensitivity, which was mediated by alpha 1, alpha 2 adrenoceptors and PKA.

Topics: Adrenergic Fibers; Afferent Pathways; Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Female; Ganglia, Spinal; In Vitro Techniques; Isoquinolines; Male; Neurons; Radiculopathy; Rats; Rats, Sprague-Dawley; Sulfonamides; Thionucleotides

The secretion of IL-6 after stimulation of macrophages has been found to play a central role in the regulation of defense mechanism, haematopoiesis, and acute phase reaction. It was reported that cAMP is involved in the regulation of IL-6 production. Since calcitonin gene-related peptide (CGRP) is known to increase cAMP accumulation in mouse macrophages, we examined whether CGRP would induce IL-6 release in macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5 x 10(5) cells per well and allowed to adhere for 2 h. After incubation for 48 h with two changes of PRMI-1640, the macrophages were cultured with CGRP and LPS 1 microg/ml for 12 h. The IL-6 level in medium was measured by ELISA kits. The results showed that CGRP had no direct effects on IL-6 production, but it potentiated LPS-induced IL-6 production in a concentration-dependent manner. When CGRP was at a concentration of 10(-10) M, the LPS-induced IL-6 production was increased from 5.16 +/- 0.48 to 8.88 +/- 0.48 ng/ml. The effect of CGRP 10(-10) M was reversed by hCGRP(8-37) 10(-8) M, an antagonist of CGRP1 receptor. The LPS-induced IL-6 production from macrophages was also potentiated by forskolin 5 microM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 microM or Rp-cAMPS 100 microM, the inhibitors of cAMP-dependent protein kinase, inhibited the effect of CGRP by 31% and 98%, respectively. These results demonstrate that the LPS-induced IL-6 release is potentiated by CGRP via the activation of cAMP pathway in mouse resident peritoneal macrophages.

Topics: Animals; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Drug Synergism; Enzyme Inhibitors; Interleukin-6; Isoquinolines; Lipopolysaccharides; Lymphocyte Activation; Macrophages, Peritoneal; Male; Mice; Mice, Inbred BALB C; Miotics; Peptide Fragments; Receptors, Calcitonin Gene-Related Peptide; Sulfonamides; Thionucleotides

The mechanism through which kainate receptors downregulate the release of GABA in the hippocampus is not known. We have found that the action of kainate on the hippocampal inhibitory postsynaptic current (IPSC) is mediated by a metabotropic process that is sensitive to Pertussis toxin (PTx) and independent of ion channel current. The downregulation of GABA IPSCs by kainate was also prevented in a dose-dependent manner by calphostin C, a specific inhibitor of PKC, and the inhibition of phospholipase C (PLC) drastically reduced the action of kainate. The effect of kainate was completely occluded by phorbol esters and by increasing extracellular Ca2+ but remained unaltered after inhibition or activation of protein kinase A (PKA). These results demonstrate that the activation of kainate receptors triggers a second messenger cascade, which results in the stimulation of PKC, and therefore document a metabotropic action of kainate receptors, which results in the inhibition of GABA release.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Carcinogens; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Estrenes; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; GTP-Binding Protein alpha Subunits, Gi-Go; Hippocampus; Isoquinolines; Kainic Acid; Membrane Potentials; Pertussis Toxin; Phorbol 12,13-Dibutyrate; Phosphodiesterase Inhibitors; Protein Kinase C; Pyrrolidinones; Rats; Rats, Wistar; Receptors, Kainic Acid; Receptors, Metabotropic Glutamate; Second Messenger Systems; Sodium; Staurosporine; Sulfonamides; Tetraethylammonium; Tetrodotoxin; Thionucleotides; Type C Phospholipases; Virulence Factors, Bordetella

Prostaglandin E2 (PGE2) is an abundant eicosanoid in bone that has been implicated in a number of pathological states associated with bone loss. Interleukin-6 (IL-6) is a cytokine that plays a critical role in bone remodeling and appears to act as a downstream effector of most bone-resorbing agents. In light of the evidence that PGE2 induces IL-6 in the bone environment, this study was designed to investigate whether PGE2 regulated IL-6 expression by osteoblasts. Here we demonstrate that PGE2 is a potent inducer of IL-6 production by fetal rat osteoblasts and synergizes with lipopolysaccharide to enhance IL-6. We show that PGE2 stimulates the activity of the IL-6 promoter in osteoblasts, suggesting that PGE2 controls IL-6 gene expression at least at the transcriptional level. Moreover, we show that PGE2-mediated IL-6 induction is prevented by the cAMP antagonist, Rp-cAMP, and the protein kinase A (PKA) inhibitors, KT5720 and H89. Thus, our data indicate that PGE2 involves the cAMP-PKA signaling pathway to regulate IL-6 gene expression in osteoblasts.

Topics: Animals; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; Enzyme Inhibitors; Indoles; Interleukin-6; Isoquinolines; Osteoblasts; Promoter Regions, Genetic; Pyrroles; Rats; Rats, Sprague-Dawley; Sulfonamides; Thionucleotides; Transcription, Genetic

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

Effects of YT-146 [2-(1-octynyl) adenosine], an adenosine A2 receptor agonist, on cAMP production and noradrenaline (NA) release were investigated in PC12 cells. YT-146 caused a concentration-dependent cAMP accumulation (EC50: 1.2+/-0.9 nM). In [3H]NA-prelabeled cells, YT-146 increased the basal NA release and enhanced ATP-evoked NA release in a concentration-dependent manner (EC50: 0.23+/-0.15 nM). YT-146 augmented the maximal response to ATP without affecting the EC50 value of ATP. These effects of YT-146 were inhibited by several adenosine receptor antagonists with a characteristic of adenosine A2A receptor subtype. The effects of YT-146 were mimicked by forskolin, dibutylyl cAMP and Sp-cAMPS, and inhibited by H-89, a cAMP-dependent protein kinase inhibitor. YT-146 had little effect on ATP-induced increase in intracellular Ca2+ concentration. YT-146 enhanced the NA release induced by several different stimuli including Ca2+ ionophore A23187. The present results suggest that YT-146 is a potent agonist on adenosine A2A receptors in PC12 cells and causes a cAMP-dependent enhancement of NA release by affecting the exocytosis process at a point downstream of the intracellular Ca2+ increase.

Topics: Adenosine; Adenosine Triphosphate; Alkynes; Animals; Bucladesine; Calcimycin; Calcium; Carbachol; Cholinergic Agonists; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Ionophores; Isoquinolines; Membrane Potentials; Norepinephrine; PC12 Cells; Potassium Chloride; Purinergic P1 Receptor Agonists; Rats; Receptor, Adenosine A2A; Sulfonamides; Thionucleotides; Tritium; Vasodilator Agents

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cell Line; Cricetinae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Insulin; Islets of Langerhans; Isoquinolines; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Sulfonamides; Thionucleotides

Gene expression of the matrix-degrading enzyme collagenase-1 in rabbit synoviocytes and human fibroblasts is down-regulated by prostaglandin E1 (PGE1) through a cyclic adenosine monophosphate (cAMP)-dependent pathway. In the current study, we examined the role of protein kinase A (PKA) in the PGE1-mediated effect on collagenase-1 gene expression. Collagenase-1 gene expression was rapidly induced several-fold above control both by a phorbol ester, 12-o-tetradecanoyl phorbol 13 acetate, and interleukin-1 beta (IL-1 beta) in HIG-82 synoviocytes. Treatment with PGE1 and forskolin increased PKA activity in the HIG-82 cells within 15 minutes of adding the stimulating agents. Two inhibitors of PKA, the isoquinoline-sulfonamide derivative, H-89 and a cAMP analog, RpcAMP, blocked the ability of PGE1 to down-regulate collagenase-1 gene expression. However, if PGE1 was added from 6 h to 30 minutes before the PKA inhibitor H-89, collagenase-1 gene expression was inhibited. Constitutive PKA activity was increased in HIG-82 synoviocytes stably transfected with an expression vector pCMV.C alpha that caused the HIG-82 cells to overexpress an active catalytic subunit of PKA. Cells stably transfected with an inactive, mutated C-alpha-variant showed no change in PKA activity. Collagenase-1 mRNA levels in TPA-stimulated cells were reduced to baseline levels in the pCMV.C alpha but not in the mutated C-alpha-transfected cells. These data show the importance of PKA in regulating collagenase-1 gene expression in a synoviocyte cell line.

Topics: Alprostadil; Animals; Cell Line; Colforsin; Collagenases; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Induction; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Interleukin-1; Isoenzymes; Isoquinolines; Matrix Metalloproteinase 1; Protein Kinase C; Protein Kinase C-alpha; Rabbits; RNA, Messenger; Sulfonamides; Synovial Membrane; Thionucleotides; Transfection

The well-characterized enhancement of the cardiac Ca2+ L-type current by protein kinase A (PKA) is not observed when the corresponding channel is expressed in Xenopus oocytes, possibly because it is fully phosphorylated in the basal state. However, the activity of the expressed channel is reduced by PKA inhibitors. Using this paradigm as an assay to search for PKA sites relevant to channel modulation, we have found that mutation of serine 1928 of the alpha 1C subunit to alanine abolishes the modulation of the expressed channel by PKA inhibitors. This effect was independent of the presence of the beta subunit. Phosphorylation of serine 1928 of alpha 1C may mediate the modulatory effect of PKA on the cardiac voltage-dependent ca2+ channel.

Topics: Animals; Base Sequence; Calcium Channels; Calcium Channels, L-Type; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Ion Channel Gating; Isoquinolines; Molecular Sequence Data; Muscle Proteins; Mutagenesis, Site-Directed; Myocardium; Oocytes; Patch-Clamp Techniques; Phosphorylation; Protein Processing, Post-Translational; Rabbits; Recombinant Fusion Proteins; Serine; Sulfonamides; Thionucleotides; Xenopus laevis

The effects of human growth hormone-releasing hormone (hGHRH) on Ca2+ channels were examined in human growth hormone-producing adenoma cells using the perforated whole cell clamp technique. These cells exhibited T- and L-type Ca2+ channel currents, and application of 10(-8) M hGHRH increased the amplitude of both currents. Application of 10(-5) M 8-bromoadenosine 3',5'-cyclic monophosphate also increased T- and L-type currents. Additional application of 10(-8) M hGHRH did not further increase the current amplitudes. Treatment with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (10(-5) M) or H-89 (10(-5) M) inhibited the enhancement of Ca2+ channel currents by hGHRH, as did intracellular injection of protein kinase A (PKA) inhibitor peptide [PKI-(5-24)], indicating that hGHRH increased the amplitude of Ca2+ channel currents through the activation of the adenosine 3',5'-cyclic monophosphate (cAMP)-PKA system. When intracellular Ca2+ concentration ([Ca2+]i) was chelated to < 30 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTAAM), hGHRH failed to increase the Ca2+ channel currents. In this condition, hGHRH activated nonselective cation channels, which revealed that the cAMP-PKA system operated after treatment with BAPTA-AM and that the site of low [Ca2+]i-induced inhibition of hGHRH effects on Ca2+ channels was at a step after PKA activation.

Topics: Adenoma; Barium; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Enzyme Inhibitors; Growth Hormone-Releasing Hormone; Human Growth Hormone; Humans; Isoquinolines; Membrane Potentials; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptide Fragments; Peptides; Pituitary Neoplasms; Stereoisomerism; Sulfonamides; Thionucleotides; Tumor Cells, Cultured

To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis.

Topics: Animals; Antiporters; Bicarbonates; Bile; Bucladesine; Cells, Cultured; Chloride-Bicarbonate Antiporters; Chlorides; Cholagogues and Choleretics; Colchicine; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Glucagon; Hydrogen-Ion Concentration; Intracellular Fluid; Isoquinolines; Liver; Male; Nitrobenzoates; Rats; Rats, Wistar; Sodium; Sodium-Hydrogen Exchangers; Sulfonamides; Tetradecanoylphorbol Acetate; Thionucleotides

Invasive adenylate cyclase (iAC) reversibly inhibits spontaneous maturation of cumulus-enclosed bovine oocytes by increasing the intracellular concentration of cAMP, [cAMP]i. In this study, physiological aspects of maintaining meiotic arrest in bovine oocytes by iAC were investigated. The maintenance of germinal vesicle arrest by iAC in both cumulus-enclosed and denuded bovine oocytes was concentration dependent (r2 = 0.857). Denuded bovine oocytes were more sensitive to maintenance of meiotic arrest by iAC then were cumulus-enclosed oocytes. At the highest concentration, 70% of the cumulus-enclosed and 90% of the denuded bovine oocytes were maintained in meiotic arrest. The iAC increased [cAMP]i in both intact cumulus-oocyte complexes and enclosed oocytes in a concentration-dependent manner (r2 = 0.795). Cumulus-enclosed oocytes maintained in meiotic arrest by iAC retained developmental competence when subsequently cultured in iAC-free medium and then fertilized. The [cAMP]i in bovine complexes decreased precipitously upon release from follicles and remained low for the next 125 min. However, the [cAMP]i of the enclosed oocytes did not change. Bovine oocytes commit to undergo meiosis in a progressive manner. Approximately 10% of the oocytes were already committed when aspirated. This proportion increased to 40% at 2 h and 70% at 5 h. Use of two inhibitors of cAMP-dependent protein kinase A provided further evidence that cAMP functions in mediating meiotic arrest in bovine oocytes. Bovine oocytes, therefore, are sensitive to different cAMP concentrations, and are developmentally competent after iAC-induced arrest, and complexes containing oocytes exhibit a decrease in [cAMP]i before spontaneous maturation. These results suggest that maintenance of meiotic arrest by iAC is accomplished through modulation of cellular machinery, and regulation of oocyte maturation by [cAMP]i may be physiologically relevant.

Topics: Adenylyl Cyclases; Analysis of Variance; Animals; Bordetella pertussis; Cattle; Cells, Cultured; Culture Media; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Female; Intracellular Fluid; Isoquinolines; Oocytes; Oogenesis; Prophase; Sulfonamides; Thionucleotides; Time Factors

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

Topics: Adenosine; Adenosine Triphosphate; Animals; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Glyburide; Isoquinolines; Male; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Phenethylamines; Potassium Channels; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rabbits; Receptors, Purinergic P1; Sulfonamides; Thionucleotides

The effects of cyclic AMP (cAMP) on intracellular Na+ concentration ([Na+]i), membrane depolarization and intracellular Ca2+ concentration ([Ca2+]i) and the involvement of cAMP in acetylcholine (ACh)-induced such cellular events and catecholamine (CA) release were studied in cultured bovine adrenal medullary chromaffin cells. 8-Bromo-cyclic AMP (8Br-cAMP) and forskolin caused a rise in [Na+]i, membrane depolarization and a rise in [Ca2+]i and potentiated these responses and CA release to ACh. The effects of 8Br-cAMP or forskolin on ACh-induced changes of but not on basal level of [Na+]i, membrane potential and [Ca2+]i were blocked by tetrodotoxin (TTX, 1 microM). In Na+ deprivated medium, forskolin failed to produce an increase in basal [Ca2+]i level and to potentiate ACh-induced rise. The similar results as in 8Br-cAMP and forskolin were obtained using ouabain, and 8Br-cAMP or foskolin produced no further effects in the presence of ouabain. Inhibitors of cAMP-dependent protein kinase not only blocked the effects of 8Br-cAMP and forskolin on membrane depolarization, [Ca2+]i rise and CA release, but also reduced these responses to ACh. From the similarity between the effects of cAMP and those of ouabain on the cellular events and the counteraction of the effects of cAMP by ouabain, it may be suggested that cAMP produces its effects on ion fluxes and CA release probably via an inhibition of Na+, K(+)-ATPase in intact chromaffin and cAMP may participate in the responses to ACh.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Acetylcholine; Adrenal Medulla; Animals; Calcium; Catecholamines; Cattle; Cells, Cultured; Colforsin; Cyclic AMP; Enzyme Inhibitors; Isoquinolines; Membrane Potentials; Ouabain; Protein Kinase Inhibitors; Receptors, Cholinergic; Sodium; Sodium-Potassium-Exchanging ATPase; Sulfonamides; Tetrodotoxin; Thionucleotides