h-89 and 9-(tetrahydro-2-furyl)-adenine

Recent studies have shown the 5-HT

Topics: Adenine; Animals; Behavior, Animal; Gene Expression Regulation; Genes, fos; Indoles; Isoquinolines; Male; Methylamines; Nociception; Pain; Pain Measurement; Piperazines; Prefrontal Cortex; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; Serotonin Receptor Agonists; Spinal Cord; Sulfonamides

Calcitonin gene-related peptides (CGRP), an endogenous neuropeptide, play an important role in the development of neuroinflammation by acting upon its receptor. The CGRP receptor immunoreactivity was identified on Schwann cells. However the effects of CGRP on Schwann cells are unknown and the exact signaling mechanisms associated with CGRP receptor activation related to Schwann cells inflammatory responses are not well understood. We investigated the effect of CGRP on CGRP receptor activation mediates a proinflammatory signaling response in Schwann cells.. CGRP-induced ERK-MAPK phosphorylation and proinflammatory cytokines, interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) expressions were measured by immune blotting. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including CGRP (8-37), SQ 22536 and H-89.. Treatment with CGRP demonstrated a significant generation of IL-1β and IL-6 but not in the level of TNF-α. In addition, there was a temporal increase in the activated form of ERK caused by CGRP that was prevented after pretreatment with CGRP (8-37), SQ 22536 and H-89. Furthermore, use of the CGRP (8-37), ERK inhibitor PD 98059, SQ 22536 or H-89 abolished the CGRP mediated increase in IL-1β.. This investigation provides evidence for a novel CGRP activation on Schwann cells that mediates inflammatory response by increasing of IL-1β and IL-6 expression. CGRP activates the cAMP-PKA-ERK signaling cascade leading to IL-1β production. These results support the notion that CGRP may play a direct role to initiate inflammatory processes in the peripheral nervous system.

Topics: Adenine; Calcitonin Gene-Related Peptide; Cell Line; Cyclic AMP; Cytokines; Dose-Response Relationship, Drug; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Isoquinolines; MAP Kinase Signaling System; Peptides; Phosphorylation; Receptors, Calcitonin Gene-Related Peptide; Schwann Cells; Sulfonamides; Tumor Necrosis Factor-alpha

Liver cancer is a common human cancer with a high mortality rate and currently there is no effective chemoprevention or systematic treatment. Recent evidence suggests that prostaglandin E(2) (PGE(2)) plays an important role in the occurrence and development of liver cancer. However, the mechanisms through which PGE(2) promotes liver cancer cell growth are not yet fully understood. It has been reported that the increased expression of FUSE-binding protein 1 (FBP1) significantly induces the proliferation of liver cancer cells. In this study, we report that PGE(2) promotes liver cancer cell growth by the upregulation of FBP1 protein expression. Treatment with PGE2 and the E prostanoid 3 (EP3) receptor agonist, sulprostone, resulted in the time-dependent increase in FBP1 protein expression; sulprostone increased the viability of the liver cancer cells. The protein kinase A (PKA) inhibitor, H89, and the adenylate cyclase (AC) inhibitor, SQ22536, inhibited the cell viability accelerated by sulprostone. By contrast, the Gi subunit inhibitor, pertussis toxin (PTX), exhibited no significant effect. Treatment with PGE(2) and sulprostone caused a decrease in JTV1 protein expression, blocked the binding of JTV1 with FBP1, which served as a mechanism for FBP1 degradation, leading to the decreased ubiquitination of FBP1 and the increase in FBP1 protein expression. Furthermore, H89 and SQ22536 prevented the above effects of JTV1 and FBP1 induced by PGE(2) and sulprostone. These findings indicate that the EP3 receptor activated by PGE(2) may couple to Gs protein and activate cyclic AMP (cAMP)-PKA, downregulating the levels of JTV1 protein, consequently inhibiting the ubiquitination of FBP1 and increasing FBP1 protein expression, thus promoting liver cancer cell growth. These observations provide new insights into the mechanisms through which PGE(2) promotes cancer cell growth.

Topics: Abortifacient Agents, Nonsteroidal; Adenine; Adenylyl Cyclase Inhibitors; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colforsin; Cyclic AMP; Dinoprostone; DNA Helicases; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Humans; Isoquinolines; Liver Neoplasms; Nuclear Proteins; Pertussis Toxin; Phosphorylation; Protein Kinase Inhibitors; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Prostaglandin E, EP4 Subtype; RNA Interference; RNA-Binding Proteins; RNA, Small Interfering; Smad2 Protein; Sulfonamides; Ubiquitination

Accumulating evidence has implicated that GLP-1 may have a beneficial effect on cardiovascular and renal diseases but the mechanism is not fully understood. Here we show that GLP-1 analog, liraglutide, inhibits oxidative stress and albuminuria in streptozotocin (STZ)-induced type 1 diabetes mellitus rats, via a protein kinase A (PKA)-mediated inhibition of renal NAD(P)H oxidases. Diabetic rats were randomly treated with subcutaneous injections of liraglutide (0.3 mg/kg/12 h) for 4 weeks. Oxidative stress markers (urinary 8-hydroxy-2'-deoxyguanosine and renal dihydroethidium staining), expression of renal NAD(P)H oxidase components, transforming growth factor-β (TGF-β), fibronectin and urinary albumin excretion were measured. In vitro effect of liraglutide was evaluated using cultured renal mesangial cells. Administration of liraglutide did not affect plasma glucose levels or body weights in STZ diabetic rats, but normalized oxidative stress markers, expression of NAD(P)H oxidase components, TGF-β, fibronectin in renal tissues and urinary albumin excretion, all of which were significantly increased in diabetic rats. In addition, in cultured renal mesangial cells, incubation with liraglutide for 48 h inhibited NAD(P)H-dependent superoxide production evaluated by lucigenin chemiluminescence in a dose-dependent manner. This effect was reversed by both PKA inhibitor H89 and adenylate cyclase inhibitor SQ22536, but not by Epac2 inhibition via its small interfering RNA. Liraglutide may have a direct beneficial effect on oxidative stress and diabetic nephropathy via a PKA-mediated inhibition of renal NAD(P)H oxidase, independently of a glucose-lowering effect.

Topics: Adenine; Albuminuria; Animals; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glucagon-Like Peptide 1; Humans; Isoquinolines; Kidney; Liraglutide; Male; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxidative Stress; Rats; Rats, Wistar; Streptozocin; Sulfonamides

In cancer, VEGF-induced increase in vascular permeability results in increased interstitial pressure, reducing perfusion and increasing hypoxia, which reduce delivery of chemotherapeutic agents and increase resistance to ionizing radiation. Here, we show that both TIMP-2 and Ala + TIMP-2, a TIMP-2 mutant without matrix metalloproteinase inhibitory activity, antagonize the VEGF-A-induced increase in vascular permeability, both in vitro and in vivo. Like other agents known to preserve endothelial barrier function, TIMP-2 elevates cytosolic levels of cAMP and increases cytoskeletal-associated vascular endothelial cadherin in human microvascular endothelial cells. All of these effects are completely ablated by selective knockdown of integrin α3β1 expression, expression of a dominant negative protein tyrosine phosphatase Shp-1 mutant, administration of the protein tyrosine phosphatase inhibitor orthovanadate, or the adenylate cyclase inhibitor SQ22536. This TIMP-2-mediated inhibition of vascular permeability involves an integrin α3β1-Shp-1-cAMP/protein kinase A-dependent vascular endothelial cadherin cytoskeletal association, as evidenced by using siRNAs to integrin α3β1 and Shp-1, or treatment with Shp-1 inhibitor NSC87877 and protein kinase A inhibitor H89. Our results demonstrate the potential utility for TIMP-2 in cancer therapy through "normalization" of vascular permeability in addition to previously described antiangiogenic effects.

Topics: Adenine; Animals; Antigens, CD; Blotting, Western; Cadherins; Capillary Permeability; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Drug Antagonism; Endothelial Cells; Humans; Integrin alpha3beta1; Isoquinolines; Mice; Mice, Knockout; Microscopy, Fluorescence; Mutation; Protein Binding; Protein Tyrosine Phosphatase, Non-Receptor Type 6; RNA Interference; Signal Transduction; Sulfonamides; Tissue Inhibitor of Metalloproteinase-2; Vanadates; Vascular Endothelial Growth Factor A

The receptor-coupled intracellular signaling mechanism of endogenous opioid peptide β-endorphin (β-end) is explored for the first time in ectothermic vertebrates using wall lizard as a model. β-End inhibited the percentage phagocytosis and phagocytic index of lizard splenic phagocytes in a dose-dependent manner. The inhibitory effect of β-end on phagocytosis was completely antagonized by non-selective opioid receptor antagonist naltrexone and also by selective μ-receptor antagonist CTAP. However, selective antagonists for other opioid receptors like NTI for δ-receptor and NorBNI for κ-receptor did not alter the effect of β-end on phagocytosis. This suggests that β-end mediated its inhibitory effect on phagocytic activity of splenic phagocytes exclusively through μ opioid receptors. The μ opioid receptor-coupled downstream signaling cascade was subsequently explored using inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). Both SQ 22536 and H-89 abolished the inhibitory effect of β-end on phagocytosis in a concentration-related manner. Implication of cAMP as second messenger was corroborated by cAMP assay where an increase in intracellular cAMP level was observed in response to β-end treatment. It can be concluded that β-end downregulated the phagocytic activity of lizard splenic phagocytes through μ opioid receptor-coupled adenylate cyclase-cAMP-protein kinase A pathway.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; beta-Endorphin; Cyclic AMP-Dependent Protein Kinases; Isoquinolines; Lizards; Naltrexone; Peptides; Phagocytes; Phagocytosis; Receptors, Opioid, mu; Signal Transduction; Spleen; Sulfonamides

Present in vitro study in the wall lizard Hemidactylus flaviviridis, for the first time in ectothermic vertebrates, demonstrated the immunoregulatory role of neuropeptide Y (NPY) and its receptor-coupled downstream signaling cascade. NPY inhibited the percentage phagocytosis and phagocytic index of splenic phagocytes. The inhibitory effect of NPY on phagocytosis was completely antagonized by Y(2) and Y(5) receptor antagonists. This suggests that NPY mediated its effect on phagocytosis through Y(2) and Y(5) receptors. Further, NPY receptor-coupled downstream signaling cascade for NPY effect on phagocytosis was explored using the inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). The SQ 22536/H-89 in a concentration-related manner decreased the inhibitory effect of NPY on phagocytosis. Further, an increase in intracellular cAMP level was observed in response to NPY. Taken together, it can be concluded that NPY via Y(2) and Y(5) receptor-coupled AC-cAMP-PKA pathway downregulated the phagocytic activity of lizard splenic phagocytes.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Appetite Stimulants; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Isoquinolines; Lizards; Neuropeptide Y; Phagocytes; Phagocytosis; Receptors, Neuropeptide Y; Signal Transduction; Spleen; Sulfonamides

Present in vitro investigation for the first time in ectotherms demonstrated the immunomodulatory role of substance P in the wall lizard Hemidactylus flaviviridis. Substance P inhibited the percentage phagocytosis and phagocytic index of lizard splenic phagocytes. Inhibitory effect of substance P was completely blocked by NK-1 receptor antagonist spantide I, indicating the NK-1 receptor mediated action. Further, NK-1 receptor-coupled downstream signaling cascade involved in controlling phagocytosis was explored using inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). Both the inhibitors, in a concentration-related manner decreased the suppressive effect of substance P on phagocytosis. In addition, substance P treatment caused an increase in intracellular cAMP level in splenic phagocytes. Taken together, it can be suggested that substance P via NK-1 receptor-coupled AC-cAMP-PKA pathway modulated the phagocytic activity of splenic phagocytes in wall lizards.

Topics: Adenine; Animals; Cells, Cultured; Cyclic AMP; Enzyme Inhibitors; Female; Immunologic Factors; Isoquinolines; Lizards; Neurokinin-1 Receptor Antagonists; Phagocytes; Phagocytosis; Receptors, Neurokinin-1; Signal Transduction; Spleen; Substance P; Sulfonamides

The aim of the present study was to understand the rhythmic changes in innate immune response in freshwater fish Channa punctatus. Furthermore, the putative role of melatonin as the zeitgeber was explored. The phagocytic activity of splenic phagocytes assessed at 6-h intervals showed higher phagocytic activity during light phase than dark phase. The increased phagocytic activity during light phase was diminished by melatonin administration at 09:00 h. Implication of melatonin in control of diurnal variation in phagocytic activity was substantiated by administering irreversible tryptophan hydroxylase inhibitor, para-chlorophenylalanine (pCPA) at 18:00 h. pCPA abrogated the decrease of phagocytosis observed during dark phase, and the same was restored after melatonin administration. The direct involvement of melatonin in modulation of phagocytosis was demonstrated following in vitro experiments. Melatonin suppressed the phagocytic activity in a concentration-dependent manner without affecting the viability of phagocytes. The existence of functional membrane-bound melatonin receptors on fish phagocytes was pharmacologically demonstrated. Luzindole, melatonin membrane receptor antagonist, completely blocked the inhibitory effect of melatonin on phagocytosis. Further receptor-coupled adenylate cyclase-protein kinase A (PKA) pathway was implicated in transducing the melatonin effect as both adenylate cyclase and PKA inhibitor completely nullified the melatonin-induced suppression. An increased intracellular cAMP level in response to melatonin ascertained the second messenger status of cAMP for downstream signaling. However, manipulation of phospholipase C/PKC failed to influence the effect of melatonin on phagocytic activity. These observations in C. punctatus evidenced the diurnal rhythmicity in phagocytic activity that is regulated by melatonin following membrane-bound receptor-coupled cAMP-PKA pathway.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Survival; Central Nervous System Depressants; Circadian Rhythm; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Estrenes; Fenclonine; Isoquinolines; Melatonin; Perciformes; Phagocytes; Phagocytosis; Pyrrolidinones; Receptors, Melatonin; Signal Transduction; Spleen; Sulfonamides; Tryptamines; Tryptophan Hydroxylase

Our work assesses the effects of mu opioid receptor activation on high-threshold Ca2+/Ba2+ currents in freshly dispersed pyramidal neurons of the medial prefrontal cortex in rats. Application of the specific mu receptor agonist (D-Ala2+, N-Me-Phe4+, Gly5+-ol)-enkephalin (DAMGO) at 1 microM decreased Ca2+ current amplitudes from 0.72 to 0.49 nA. The effect was abolished by naloxone and omega-Conotoxin GVIA. Inhibition was not abolished by strong depolarisation of the cell membrane. In addition, a macroscopic Ba2+ current recorded in cell-attached configuration was inhibited when DAMGO was applied outside the patch pipette. An adenylyl cyclase inhibitor (SQ 22536) and a protein kinase A inhibitor (H-89) decreased Ca2+ current amplitude. Moreover, the inhibitory effect of mu opioid receptors on Ca2+ currents required the activation of protein kinase A. We conclude that activation of mu opioid receptors in medial prefrontal cortex pyramidal neurons inhibits N type Ca2+ channel currents, and that protein kinase A is involved in this transduction pathway.

Topics: Adenine; Analgesics, Opioid; Animals; Animals, Newborn; Calcium Channel Blockers; Calcium Channels; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Isoquinolines; Membrane Potentials; Naloxone; Narcotic Antagonists; omega-Conotoxin GVIA; Patch-Clamp Techniques; Prefrontal Cortex; Pyramidal Cells; Rats; Rats, Wistar; Receptors, Opioid, mu; Sulfonamides

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.

Topics: Adenine; Adenosine; Adenylyl Cyclases; Animals; Calcium; Catecholamines; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Dideoxyadenosine; Enzyme Inhibitors; Imines; Isoquinolines; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Kinase Inhibitors; Signal Transduction; Sperm Motility; Sperm Tail; Sulfonamides

Although traditionally recognized for maintaining extracellular matrix integrity during morphogenesis, the function of matrix metallo-proteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), in the mature nervous system is essentially unknown. Here, we report that TIMP-2 induces pheochromocytoma PC12 cell-cycle arrest via regulation of cell-cycle regulatory proteins, resulting in differentiation and neurite outgrowth. TIMP-2 decreases cyclins B and D expression and increases p21Cip expression. Furthermore, TIMP-2 promotes cell differentiation via activation of the cAMP/Rap1/ERK (extracellular signal-regulated kinase) pathway. Expression of dominant-negative Rap1 blocks TIMP-2-mediated neurite outgrowth. Both the cell-cycle arrest and neurite outgrowth induced by TIMP-2 was independent of MMP inhibitory activity. Consistent with the PC12 cell data, primary cultures of TIMP-2 knock-out cerebral cortical neurons exhibit significantly reduced neurite length, which is rescued by TIMP-2. These in vitro results were corroborated in vivo. TIMP-2 deletion causes a delay in neuronal differentiation, as demonstrated by the persistence of nestin-positive progenitors in the neocortical ventricular zone. The interaction of TIMP-2 with alpha3beta1 integrin in the cerebral cortex suggests that TIMP-2 promotes neuronal differentiation and maintains mitotic quiescence in an MMP-independent manner through integrin activation. The identification of molecules responsible for neuronal quiescence has significant implications for the ability of the adult brain to generate new neurons in response to injury and neurological disorders, such as Alzheimer's and Parkinson's diseases.

Topics: Adenine; Animals; Animals, Newborn; Blotting, Northern; Blotting, Western; Bromodeoxyuridine; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cells, Cultured; Cerebral Cortex; Cyclic AMP; Cyclin-Dependent Kinase 5; Drug Interactions; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation; Green Fluorescent Proteins; Immunohistochemistry; Immunoprecipitation; Isoquinolines; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinase Kinases; Mitosis; Molecular Biology; Nerve Growth Factor; Neurons; Neurturin; Phosphotransferases; rap1 GTP-Binding Proteins; ras GTPase-Activating Proteins; Rats; Signal Transduction; Sulfonamides; Time Factors; Tissue Inhibitor of Metalloproteinase-2; Transfection

Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2'-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Calcium; Cricetinae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Gadolinium; Glucagon; Glucagon-Like Peptide 1; Glucose; Insulin; Insulin Secretion; Islets of Langerhans; Isoquinolines; Male; Mesocricetus; Ouabain; Peptide Fragments; Protein Precursors; Sodium; Somatostatin; Sulfonamides; Tetrodotoxin

Sepsis and posttraumatic inflammatory processes are accompanied by definite changes in microvascular permeability, particularly in the lung. These permeability changes may occur because of damaged regulatory mechanisms at the level of the capillary wall. Pericytes are adventitial cells located within the basement membrane of capillaries. These cells contain multiple cytoplasmic processes that envelope endothelial cells, and are consequently thought to stabilize capillary walls and participate in microcirculation and endothelial cell permeability. Data from this laboratory and other laboratories have confirmed that pericytes are contractile cells, adding to the evidence that pericytes may influence or help regulate capillary permeability. We have already determined that hydrogen peroxide (H2O2) causes dose-dependent relaxation in microvascular lung pericytes (MLPs) at 10 minutes and, conversely, dose-dependent contraction at 30 minutes. It is the aim of this study to determine the mechanism of this biphasic contractile response. Specifically, we will determine whether cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-dependent protein kinase intracellular pathways are responsible for the hydrogen peroxide-induced contractility of MLPs.. Rat MLPs were isolated by previously published protocol and cultured on collagen gel matrices. MLPs were pretreated with either ODQ, a soluble guanylate cyclase inhibitor (100 mumol/L), for 15 minutes; GKIP, a protein kinase G inhibitor (100 mumol/L), for 1 hour; SQ22536, an adenylate cyclase inhibitor (100 mumol/L), for 15 minutes; or H89, a protein kinase A inhibitor (10 mumol/L), for 1 hour. Hydrogen peroxide was then introduced to each MLP culture at 10 mumol/L, 100 mumol/L, and 1 mmol/L. After each of these treatments, the surface area of the collagen gels was digitally quantified at 10 and 30 minutes.. SQ22536 attenuated both relaxation at 10 minutes and the contraction seen at 30 minutes for all concentrations of H2O2. H89 caused a marked basal relaxation and prevented the cells from contracting at 30-minute exposures to all concentrations of H2O2. Both ODQ and GKIP attenuated the relaxation at 10 minutes but had no affect on the later contraction.. The cGMP-dependent protein kinase pathway is a mechanism for H2O2-induced relaxation of MLPs. Up-regulation of cAMP and cGMP is responsible for early H2O2-induced relaxation and late contraction. Protein kinase A (cAMP-dependent protein kinase pathway) may be an important intracellular signaling protein in the H2O2-induced contraction of MLPs or may be unable to down-regulate cAMP once inhibited. This evidence further supports the concept that there are separate intracellular pathways that regulate divergent cellular responses. This idea parallels the clinical concept of reversible and irreversible dysfunction of cellular processes in shock, and that the cellular dysfunction is initiated by separate intracellular pathways.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Analysis of Variance; Animals; Cell Survival; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hydrogen Peroxide; Isoquinolines; Lung; Male; Muscle Contraction; Oxadiazoles; Pericytes; Quinoxalines; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Sepsis; Sulfonamides

We have previously demonstrated that, in rat, the stimulatory effect of 5-HT on aldosterone secretion is mediated through a 5-HT7 receptor subtype. The aim of the present study was to characterize the transduction mechanisms associated with activation of native 5-HT7 receptors. 5-HT induced a dose-dependent increase in cAMP production in rat glomerulosa cells. Pretreatment of cells with the adenylyl cyclase (AC) inhibitor SQ 22536 or the protein kinase A (PKA) inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Administration of 5-HT in the vicinity of glomerulosa cells induced a robust increase in cytosolic calcium concentration ([Ca2+]i) and this effect was abrogated by the T-type calcium channel blocker mibefradil. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. H-89 attenuated both the [Ca2+]i response and the activation of T-type calcium current induced by 5-HT. Reduction of extracellular calcium concentration in the medium or administration of mibefradil caused a marked reduction of the maximum effect (Emax) of 5-HT on aldosterone secretion. These data demonstrate that activation of native 5-HT7 receptors stimulates cAMP formation, which in turn provokes calcium influx through T-type calcium channels. Both the activation of the AC/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Aldosterone; Animals; Calcium; Calcium Channel Blockers; Calcium Channels, T-Type; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; In Vitro Techniques; Isoquinolines; Mibefradil; Patch-Clamp Techniques; Rats; Receptors, Serotonin; Serotonin; Signal Transduction; Sulfonamides; Zona Glomerulosa

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

In a previous study we showed that activation of a presynaptically located metabotropic glutamate receptor (mGluR) with pharmacological properties of mGluR4a causes a facilitation of glutamate release in layer V of the rat entorhinal cortex (EC) in vitro. In the present study we have begun to investigate the intracellular coupling linking the receptor to transmitter release. We recorded spontaneous alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated excitatory postsynaptic currents (EPSCs) in the whole cell configuration of the patch-clamp technique, from visually identified neurons in layer V. Bath application of the protein kinase A (PKA) activator, forskolin, resulted in a marked facilitation of EPSC frequency, similar to that seen with the mGluR4a specific agonist, ACPT-1. Preincubation of slices with the PKA inhibitor H-89 abolished the effect of ACPT-1, as did preincubation with the adenylate cyclase inhibitor, SQ22536. Activation of protein kinase C (PKC) using phorbol 12 myristate 13-acetate (PMA) did not affect sEPSC frequency; however, it did abolish the facilitatory effect of ACPT-1 on glutamate release. A robust enhancement of EPSC frequency was seen in response to bath application of the specific PKC inhibitor, GF 109203X. Both H-89 and the group III mGluR antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) abolished the effects of GF 109203X. These data suggest that in layer V of the EC, presynaptic group III mGluRs facilitate release via a positive coupling to adenylate cyclase and subsequent activation of PKA. We have also demonstrated that the PKC system tonically depresses transmitter release onto layer V cells of the EC and that an interaction between mGluR4a, PKA, and PKC may exist at these synapses.

Topics: Adenine; Animals; Colforsin; Cyclic AMP-Dependent Protein Kinases; Cyclopentanes; Entorhinal Cortex; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Glycine; In Vitro Techniques; Indoles; Isoquinolines; Male; Maleimides; Presynaptic Terminals; Protein Kinase C; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Sulfonamides; Tricarboxylic Acids

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

We previously reported that endogenous prostaglandins (PGs) may increase cAMP facilitated angiogenesis through the induction of vascular endothelial growth factor (VEGF) in rat sponge implantation models. In the present experiment, we tested whether or not adenylate cyclase / protein kinase A (AC/PKA)-dependent VEGF induction enhanced angiogenesis in this model. Topical daily injections of 8-bromo-cAMP enhanced angiogenesis in a dose-dependent manner. Forskolin, an activator of AC, also facilitated angiogenesis as did amrinone, an inhibitor of phosphodiesterase. VEGF induction was confirmed by the increased levels in the fluids in the sponge matrix after topical injection of 8-bromo-cAMP. Immunohistochemical investigation further revealed the VEGF-expressed cells in the sponge granulation tissues to be fibroblasts, and the intensity of positive reactions was enhanced by 8-bromo-cAMP, forskolin and amrinone. Angiogenesis without topical injections of the above compounds was suppressed by SQ22,536, an inhibitor for AC, or H-89, an inhibitor for PKA, with concomitant reductions in VEGF levels. Daily topical injections of neutralizing antibody or anti-sense oligonucleotide against VEGF significantly suppressed angiogenesis. PGE2-induced angiogenesis was suppressed with SQ22,536 or H-89. These results suggested that AC/PKA-dependent induction of VEGF certainly enhanced angiogenesis and that pharmacological tools for controlling this signaling pathway may be able to facilitate the management of conditions involving angiogenesis.

Topics: Adenine; Adenylyl Cyclases; Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelial Growth Factors; Isoquinolines; Lymphokines; Male; Neovascularization, Physiologic; Prostaglandins E; Rats; Rats, Sprague-Dawley; Sulfonamides; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Long-term potentiation (LTP) has several different phases, and there is general agreement that the late phase of LTP requires the activation of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). In contrast, several studies indicate that the early LTP is not affected by interfering with the cAMP pathway. We have further tested the role of the cAMP pathway in early LTP using several types of inhibitors. Bath application of the PKA inhibitor H89 suppressed the early LTP induced by a single tetanus. Similarly, the LTP induced by a pairing protocol was decreased by postsynaptic intracellular perfusion of the peptide PKA inhibitor PKI(6-22) amide. The decrease of LTP produced by these inhibitors was evident immediately after induction. These results indicate that PKA is important in early LTP, that its locus of action is postsynaptic, and that it does not act merely by enhancing the depolarization required for LTP induction. The failure of some other inhibitors of the cAMP pathway to affect the early phase of LTP might be attributable to the saturation of some step in the cAMP pathway during a tetanus. In agreement with this hypothesis we found that application of the AC inhibitor SQ 22536 by itself did not affect the early phase of LTP, but did produce a reduction if the cAMP pathway was already attenuated by the PKA inhibitor H89. Our analysis of the results of genetic modifications of the cAMP pathway, especially the work on AC knock-outs, indicates that the genetic data are generally consistent with the pharmacological results showing the importance of this pathway in early LTP.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Carrier Proteins; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dimethyl Sulfoxide; Enzyme Inhibitors; Hippocampus; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Isoquinolines; Kinetics; Long-Term Potentiation; Peptide Fragments; Pyramidal Cells; Rats; Rats, Long-Evans; Sulfonamides; Synapses

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

One of the primary physiological roles of group II and group III metabotropic glutamate receptors (mGluRs) is to presynaptically reduce synaptic transmission at glutamatergic synapses. Interestingly, previous studies suggest that presynaptic mGluRs are tightly regulated by protein kinases. cAMP analogs and the adenylyl cyclase activator forskolin inhibit the function of presynaptic group II mGluRs in area CA3 of the hippocampus. We now report that forskolin has a similar inhibitory effect on putative mGluR2-mediated responses at the medial perforant path synapse and that this effect of forskolin is blocked by a selective inhibitor of cAMP-dependent protein kinase (PKA). A series of biochemical and molecular studies was used to determine the precise mechanism by which PKA inhibits mGluR2 function. Our studies reveal that PKA directly phosphorylates mGluR2 at a single serine residue (Ser(843)) on the C-terminal tail region of the receptor. Site-directed mutagenesis combined with biochemical measures of mGluR2 function reveal that phosphorylation of this site inhibits coupling of mGluR2 from GTP-binding proteins

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Amino Acid Sequence; Animals; Anticonvulsants; CHO Cells; Cricetinae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclopropanes; Dentate Gyrus; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Glutamic Acid; Glycine; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Isoquinolines; Molecular Sequence Data; Mutagenesis; Neurons; Perforant Pathway; Phosphorylation; Protein Binding; Rats; Receptors, Metabotropic Glutamate; Serine; Sulfonamides; Transfection

Tyrphostin-23 is commonly used as inhibitor of tyrosine kinase (TK). We found that tyrphostin-23 concentration-dependently increased basal steroid-hormone secretion from dispersed human and rat adrenocortical cells, the maximal effective concentration being 10(-5) M. Tyrphostin-23 (10(-5) M) enhanced 10(-9) M angiotensin-II- and endothelin-1-stimulated secretion of human and rat adrenocortical cells, but not the secretory response to 10(-9) M ACTH However, it increased the response to lower concentrations (10(-12) or 10(-11) M) of ACTH. The secretagogue effect of tyrphostin-23 on dispersed rat adrenocortical cells was abolished by either the adenylate cyclase inhibitor SQ-22536 (10(-4) M) or the protein kinase A (PKA) inhibitor H-89 (10(-5) M). Tyrphostin-23 (10(-5) M) raised basal cyclic-AMP release by dispersed rat adrenocortical cells, but in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 10(-3) M) it was ineffective. Both tyrphostin-23 and IBMX increased cyclic-AMP release by rat adrenocortical cells in response to 10(-10) M ACTH, and their effects were not additive. Taken together, our findings suggest that tyrphostin-23, acting as an inhibitor of phosphodiesterases in adrenocortical cells, increases the intracellular concentration of cyclic-AMP available for PKA activation thereby stimulating steroid-hormone secretion. They also stress that caution must be used in interpreting the results of studies aimed at investigating the possible cross-talk between adenylate cyclase- and TK-dependent signaling cascades.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylyl Cyclase Inhibitors; Adrenal Cortex; Adrenal Cortex Hormones; Adrenocorticotropic Hormone; Aldosterone; Angiotensin II; Animals; Corticosterone; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Enzyme Inhibitors; Humans; Hydrocortisone; Isoquinolines; Male; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Sulfonamides; Tyrphostins

The exposure of freshly isolated osteoblasts and osteoblast-like cells to high-level hypergravity caused the inhibition of cell growth, elevation of cAMP content, and the stimulation of differentiated functions such as alkaline phosphatase activity, collagen synthesis, and osteocalcin synthesis. Blockage of elevation of cAMP by SQ22536, an inhibitor of adenylate cyclase, resulted in the inhibition of the hypergravity-stimulated alkaline phosphatase activity, indicating that cAMP is the intracellular mediator of this action of hypergravity. H89, an inhibitor of cAMP-dependent protein kinase (PKA), further inhibited the cell growth that was already inhibited by the hypergravity, and further stimulated the alkaline phosphatase activity that was already stimulated by hypergravity. If cAMP acts through the PKA system, H89 should have blocked the changes in cell function effected by the exposure to hypergravity. Therefore the elevated intracellular cAMP by the exposure of hypergravity caused the changes in cell function by a PKA-independent pathway.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Alkaline Phosphatase; Animals; Cell Division; Cells, Cultured; Collagen; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hypergravity; Isoquinolines; Mice; Osteoblasts; Osteocalcin; Rats; Sulfonamides

In the majority of developing neurons, GABA can exert depolarizing actions, thereby raising neuronal Ca2+. Ca2+ elevations can have broad consequences during development, inducing gene expression, altering neurite outgrowth and growth cone turning, activating enzyme pathways, and influencing neuronal survival. We used fura-2 and fluo-3 Ca2+ digital imaging to assess the effects of inhibiting or activating the cAMP signal transduction pathway on GABA activity mediating Ca2+ rises during the early stages of in vitro hypothalamic neural development. Our experiments stemmed from the finding that stimulation of transmitter receptors shown to either activate or inhibit adenylyl cyclase activity caused a rapid decrease in Ca2+ rises mediated by synaptically released GABA. Both the adenylyl cyclase activator forskolin and the inhibitor SQ-22,536 reduced the Ca2+ rise elicited by the synaptic release of GABA. Bath application of the membrane-permeable cAMP analogs 8-bromo-cAMP (8-Br-cAMP) or 8-(4-chlorophenylthio)-cAMP (0.2-5 mM) produced a rapid, reversible, dose-dependent inhibition of Ca2+ rises triggered by synaptic GABA release. Potentiation of GABAergic activity mediating Ca2+ rises was observed in some neurons at relatively low concentrations of the membrane-permeable cAMP analogs (20-50 microM). In the presence of tetrodotoxin (TTX), postsynaptic Ca2+ rises triggered by the bath application of GABA were only moderately depressed (13%) by 8-Br-cAMP (1 mM), suggesting that the inhibitory effects of 8-Br-cAMP were largely the result of a presynaptic mechanism. The protein kinase A (PKA) inhibitors H89 and Rp-3', 5'-cyclic monophosphothioate triethylamine also caused a large reduction (>70%) in Ca2+ rises triggered by synaptic GABA release. Unlike the short-term depression elicited by activation of the cAMP signal transduction pathway, Ca2+ depression elicited by PKA inhibition persisted for an extended period (>30 min) after PKA inhibitor washout. Postsynaptic depression of GABA-evoked Ca2+ rises triggered by H89 (in the presence of TTX) recovered rapidly, suggesting that the extended depression observed during synaptic GABA release was largely through a presynaptic mechanism. Long-term Ca2+ modulation by cAMP-regulating hypothalamic peptides may be mediated through a parallel mechanism. Together, these results suggest that GABAergic activity mediating Ca2+ rises is dependent on ongoing PKA activity that is maintained within a narrow zone for GABA to elicit a

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adenylyl Cyclase Inhibitors; Animals; Calcium; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Electric Stimulation; Embryo, Mammalian; Enzyme Inhibitors; gamma-Aminobutyric Acid; Hypothalamus, Middle; Isoquinolines; Neurons; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfonamides; Synapses; Tetrodotoxin; Thionucleotides

The effects of increases in cellular adenosine 3'5'-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca2+ ([Ca2+]i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC50) produced at concentrations of 7.0 +/- 0.5 and 4.9 +/- 0.4 respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca2+ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF4--induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF4-- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca2+]i increase or inhibit both responses independently.

Topics: Adenine; Aluminum Compounds; Animals; Bucladesine; Calcium; Cholera Toxin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diterpenes; Dogs; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fluorides; GTP-Binding Proteins; Isoquinolines; Male; Muscle, Smooth; PC12 Cells; Rats; Serotonin; Serotonin Antagonists; Sulfonamides; Trachea

Rat peritoneal neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) produce an aggregation response that can be inhibited by prostaglandin E2 (PGE2) with an IC50 value of 2.6 x 10(-9) M. Although PGE2 can stimulate [3H]cAMP production in neutrophils (EC50 4.3 x 10(-8) M), the anti-aggregation response cannot be significantly attenuated by inhibitors of adenylate cyclase or protein kinase A, neither can it be potentiated by inhibition of phosphodiesterase activity. Despite these observations, it still remains possible that PGE2-mediated inhibition of rat neutrophil aggregation is a cAMP-dependent response mediated by highly localized changes in neutrophil cAMP. The inhibitory effect of PGE2 does not appear to depend on effects on intracellular calcium or K(ATP) channels. Similarities exist between PGE2 and the profile of activity of phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, suggesting that PI 3-kinase is a possible target for PGE2 action in rat neutrophils.

Topics: Adenine; Androstadienes; Animals; Ascitic Fluid; Cell Aggregation; Cells, Cultured; Chemotaxis, Leukocyte; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; Enzyme Inhibitors; Glyburide; Isoquinolines; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphatidylinositol 3-Kinases; Phosphodiesterase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Potassium Channels; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Rolipram; Sulfonamides; Wortmannin