vasoactive-intestinal-peptide and adenosine-3--5--cyclic-phosphorothioate

Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide, which belongs to a superfamily of structurally related peptide hormones including pituitary adenylate cyclase-activating polypeptide (PACAP). Although several studies have identified the involvement of PACAP in learning and memory, little work has been done to investigate such a role for VIP. At least three receptors for VIP have been identified including the PACAP receptor (PAC1-R) and the two VIP receptors (VPAC receptors). VIP can activate the PAC1-R only if it is used at relatively high concentrations (e.g., 100 nM); however, at lower concentrations (e.g., 1 nM) it is selective for the VPAC receptors. Our lab has showed that PAC1-R activation signals through PKC/CAKbeta/Src pathway to regulate NMDA receptors; however, there is little known about the potential regulation of NMDA receptors by VPAC receptors. Our studies demonstrated that application of 1 nM VIP enhanced NMDA currents by stimulating the VPAC receptors as the effect was blocked by VPAC receptor antagonist [Ac-Tyr(1), D-Phe(2)]GRF (1-29). This enhancement of NMDA currents was blocked by both Rp-cAMPS and PKI(14-22) (they are highly specific PKA inhibitors), but not by the specific PKC inhibitor, bisindolylmaleimide I. In addition, the VIP-induced enhancement of NMDA currents was accentuated by inhibition of phosphodiesterase 4, which inhibits the degradation of cAMP. This regulation of NMDA receptors also required the scaffolding protein AKAP. In contrast, the potentiation induced by high concentration of VIP (e.g., 100 nM) was mediated by PAC1-R as well as by Src kinase. Overall, these results show that VIP can regulate NMDA receptors through different receptors and signaling pathways.

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cells, Cultured; Cyclic AMP; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Vasoactive Intestinal Peptide; Signal Transduction; Synaptic Transmission; Thionucleotides; Time Factors; Vasoactive Intestinal Peptide

The interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) was investigated in isolated circular smooth muscle cells and strips of the guinea-pig gastric fundus. VIP induced a concentration-dependent inhibition of carbachol-induced contraction in smooth muscle cells with a maximum at 10(-6) M. The relaxation by 10(-6) M VIP was inhibited for 79.1+/-5.8% (mean+/-s.e. mean) by the NO-synthase (NOS) inhibitor L-N(G)-nitroarginine (L-NOARG; 10(-4) M) in a L-arginine reversible way. Also the inducible NOS (iNOS) selective inhibitor N-(3-(acetaminomethyl)-benzyl)acetamide (1400 W; 10(-6) M) inhibited the VIP-induced relaxation, but its inhibitory effect was not reversed by L-arginine. When cells were incubated with the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 10(-6) M), the protein kinase A-inhibitor (R)-p-cyclic adenosine-3', 5'-monophosphothioate ((R)-p-cAMPS, 10(-6) M) and the glucocorticoid dexamethasone (10(-5) M), the relaxant effect of VIP was decreased by respectively 80.9+/-7.6, 77.0+/-11.6 and 87.1+/-4.5%. In circular smooth muscle strips of the guinea-pig gastric fundus, the VIP (10(-9) - 10(-7) M)-induced relaxations were not significantly influenced by 10(-4) M L-NOARG, 10(-6) M 1400 W, 10(-6) M ODQ and 10(-5) M dexamethasone. These results suggest that iNOS, possibly induced by the procedure to prepare the smooth muscle cells, is involved in the relaxant effect of VIP in isolated smooth muscle cells but not in smooth muscle strips of the guinea-pig gastric fundus. This study illustrates the importance of the experimental method when studying the influence of NOS inhibitors on the relaxation induced by VIP in gastrointestinal smooth muscle preparations.

Topics: Adenine; Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Carbachol; Colforsin; Cyclic AMP; Dexamethasone; Electric Stimulation; Enzyme Inhibitors; Gastric Fundus; Guinea Pigs; In Vitro Techniques; Isoproterenol; Molsidomine; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Nitroprusside; Pinacidil; Tetrodotoxin; Thionucleotides; Vasoactive Intestinal Peptide

We have studied modulation of the slow Ca(2+)-activated K(+) current (I(sAHP)) in CA1 hippocampal pyramidal neurons by three peptide transmitters: corticotropin releasing factor (CRF, also called corticotropin releasing hormone, CRH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). These peptides are known to be expressed in interneurons. Using whole cell voltage clamp in hippocampal slices from young rats, in the presence of tetrodotoxin (TTX, 0.5 microM) and tetraethylammonium (TEA, 5 mM), I(sAHP) was measured after a brief depolarizing voltage step eliciting inward Ca(2+) current. Each of the peptides CRF (100-250 nM), VIP (400 nM), and CGRP (1 microM) significantly reduced the amplitude of I(sAHP). Thus the I(sAHP) amplitude was reduced to 22% by 100 nM CRF, to 17% by 250 nM CRF, to 22% by 400 nM VIP, and to 40% by 1 microM CGRP. We found no consistent concomitant changes in the Ca(2+) current or in the time course of I(sAHP) for any of the three peptides, suggesting that the suppression of I(sAHP) was not secondary to a general suppression of Ca(2+) channel activity. Because each of these peptides is known to activate the cyclic AMP (cAMP) cascade in various cell types, and I(sAHP) is known to be suppressed by cAMP via the cAMP-dependent protein kinase (PKA), we tested whether the effects on I(sAHP) by CRF, VIP, and CGRP are mediated by PKA. Intracellular application of the PKA-inhibitor Rp-cAMPS significantly reduced the suppression of I(sAHP) by CRF, VIP, and CGRP. Thus with 1 mM Rp-cAMPS in the recording pipette, the average suppression of I(sAHP) was reduced from 78 to 26% for 100 nM CRF, from 83 to 32% for 250 nM CRF, from 78 to 30% for 400 nM VIP, and from 60 to 7% for 1 microM CGRP. We conclude that CRF, VIP, and CGRP suppress the slow Ca(2+)-activated K(+) current, I(sAHP), in CA1 hippocampal pyramidal neurons by activating the cAMP-dependent protein kinase, PKA. Together with the monoamine transmitters norepinephrine, serotonin, histamine, and dopamine, these peptide transmitters all converge on the cAMP cascade modulating I(sAHP).

Topics: Animals; Calcitonin Gene-Related Peptide; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hippocampus; In Vitro Techniques; Ion Channel Gating; Large-Conductance Calcium-Activated Potassium Channels; Neuropeptides; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Pyramidal Cells; Rats; Rats, Wistar; Thionucleotides; Vasoactive Intestinal Peptide

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

Vasoactive intestinal peptide (VIP) is a neuropeptide with a broad range of biological activities in various tissues. Interactions of VIP and epidermal growth factor (EGF) are of particular interest for dermatology. They may be either co-mitogenic or inhibitory. HaCaT keratinocytes cultivated under serum-free conditions in vitro have been used to investigate the interactions of VIP and EGF. EGF was found to induce cell growth, whereas preincubation with VIP inhibited EGF-induced proliferation in a dose-dependent manner. Maximum growth inhibition was 46% (p < 0.01) at a VIP concentration of 10(-7) M. EGF-induced growth is mediated by tyrosine kinase (TK). Therefore we studied the effect of VIP on TK activity. Cells were incubated with VIP (10(-13)-10(-7) M) for 48 h and stimulated with EGF at a final concentration of 500 ng/ml. SDS-PAGE and Western blot with the antibody RC20H against TK were performed. We found a dose dependent decrease of EGF receptor TK activity. At VIP concentration of 10(-7) M a residual TK activity of 65% was detected. To investigate the possibly involved signal transduction pathways, we performed inhibition experiments with wortmannin, pertussis toxin, 2'5'diacylglycerol and adenosine-3':5'-mono-phosphorothioate. However, none of the inhibitors was effective in abolishing growth inhibition by VIP. VIP was shown to be growth inhibitory for human keratinocytes. The data suggest that EGF receptor TK is involved in signal transduction of VIP. Thus TK activity is a possible common target of both EGF- and VIP-induced cellular responses.

Topics: Androstadienes; Cell Division; Cells, Cultured; Culture Media, Serum-Free; Cyclic AMP; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Keratinocytes; Mitogens; Pertussis Toxin; Phosphorylation; Thionucleotides; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella; Wortmannin

The direct effects and the intracellular pathways of rCGRP were investigated on smooth muscle cells (SMC) isolated by enzymatic digestion from the circular and longitudinal layers of guinea-pig ileum. In circular SMC, rCGRP inhibited CCK8-induced contraction in a concentration-dependent manner (Cmax = 100 microM and EC50 = 0.7 +/- 0.4 nM). Preincubation of SMC with 1 microM Rp-cAMPs, a cAMP antagonist, abolished the relaxing effect of rCGRP; moreover, preincubation of SMC with 100 microM L-NAME, an inhibitor of NOS, inhibited the relaxing effect of rCGRP, hCGRP(8-37), a selective antagonist of rCGRP receptors, inhibited the rCGRP-induced relaxation in a concentration dependent manner whereas the vasoactive intestinal polypeptide (VIP) antagonist had no significant effect. In longitudinal SMC, rCGRP-induced relaxation was abolished by Rp-cAMPs, whereas L-NAME had no effect. In conclusion, rCGRP triggers different intracellular pathways to induce relaxation of circular or longitudinal intestinal SMC; cAMP is involved in cells from both layers while nitric oxide (NO) is involved only in relaxation of circular SMC.

Topics: Animals; Arginine; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Cyclic AMP; Guinea Pigs; Ileum; In Vitro Techniques; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peptide Fragments; Recombinant Proteins; Sincalide; Thionucleotides; Vasoactive Intestinal Peptide

Smooth muscle cells isolated by enzymatic digestion were used to determine the direct effects of galanin on circular and longitudinal muscle layers from dog proximal colon and to investigate the intracellular pathways involved in these effects. Effects of galanin were compared to those observed with other contracting [cholecystokinin octapeptide (CCK8)] and relaxing [vasoactive intestinal peptide (VIP)] agents. In longitudinal cells, galanin and CCK8 induced a contraction that was maximal at 1 nM galanin and 1 nM CCK8 and was 23.9 +/- 4.5% and 23.4 +/- 3.4%, respectively, of the length of resting cells. Incubation of cells in Ca(2+)-free medium or in the presence of nifedipine caused an inhibition of galanin-induced contraction whereas it had no effect on the contraction induced by CCK8. Vasoactive intestinal peptide, forskolin, and 8 bromo cAMP inhibited CCK-induced contraction but failed to inhibit contraction induced by galanin. The contraction induced by galanin was abolished; the CCK-induced contraction was unchanged by pertussis toxin. In circular cells, CCK8 induced a contraction that was maximal at 10 nM and was 24.2 +/- 2.6%. Galanin had no effect by itself. When cells were preincubated (1 min) with galanin (10 fM-1 microM), the CCK8-induced contraction was inhibited, with a maximal effect at 10 nM galanin. Likewise, VIP inhibited the CCK8-induced contraction with a maximal effect at 1 microM. Preincubation of cells with somatostatin, N-ethylmaleimide, and (R)-p-cAMPS inhibited galanin- and VIP-induced relaxation. In conclusion, galanin induces a contraction of longitudinal smooth muscle cells that is dependent on an influx of extracellular calcium and an activation of pertussis toxin G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cholecystokinin; Colforsin; Colon; Cyclic AMP; Dogs; Ethylmaleimide; Galanin; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Neuropeptides; Peptides; Pertussis Toxin; Somatostatin; Thionucleotides; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 +/- 2 microns and, with carbachol treatment, contracted to 89 +/- 2 microns. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC50 of 3-7 nM. Galanin (1 microM) and VIP (1 microM) increased cellular cAMP from 118 +/- 10 pmol/10(6) cells in control to 212 +/- 14 and 214 +/- 12 pmol/10(6) cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 microM, completely inhibited the relaxant effect of an EC50 concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, L-NNA (NG-nitro-L-arginine), at 100 microM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, L-NNA (100 microM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbachol; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Galanin; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Neuropeptides; Nitric Oxide; Peptides; Stomach; Thionucleotides; Vasoactive Intestinal Peptide

VIP plays an integral role in both protein and fluid secretion in many exocrine glands. By employing the perforated patch-clamp whole-cell recording technique we investigated the effects of VIP on membrane potential and transmembrane currents in avian exocrine salt gland cells. Prior to application of VIP, salt gland cells had a resting membrane potential close to -45 mV. When challenged with VIP (1-100 nM) a sustained depolarization to ECl- was induced which was mimicked by the application of cell-permeable cAMP analogues or forskolin (1 microM). By employing the voltage-clamp recording configuration a sustained increase in current was observed with a reversal potential which approximated ECl-. Ionic substitution experiments confirmed that the current was a Cl- conductance which was inhibited by the Cl- channel blockers flufenamic acid and niflumic acid and by the inhibitory cAMP isomer, adenosine-3',5'-cyclic monophosphothioate, Rp-isomer. Based on this, and the fact that the kinetic properties of the Cl- current activated by VIP are similar to those activated by cAMP, we propose that VIP-receptor interaction results in the activation of a cAMP-dependent Cl- current.

Topics: Animals; Chloride Channels; Cyclic AMP; Ducks; Electric Conductivity; Flufenamic Acid; Membrane Potentials; Niflumic Acid; Salt Gland; Stimulation, Chemical; Thionucleotides; Vasoactive Intestinal Peptide

Conventional inhibitors of cyclic AMP-dependent protein kinase lack membrane-permeability or selectivity, or both. The Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, Rp-cAMPS, is a novel membrane-permeable antagonist of cyclic AMP. We have assessed the ability of this compound to distinguish between cyclic AMP-dependent and cyclic AMP-independent contractile responses elicited in ventricular cardiomyocytes isolated from the hearts of adult rats. Cardiomyocytes were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM) and adenosine deaminase (5 units/ml). Contractile shortening was expressed as maximum shortening relative to prestimulus cell length (delta L%). In the presence of a maximally-effective concentration of isoprenaline (100 nM), which acts by a cyclic AMP-dependent mechanism, Rp-cAMPS inhibited the contractile response in a concentration-dependent and time-dependent manner. Following preincubation for 30 min with Rp-cAMPS (100 microM), the contractile response to isoprenaline (100 nM) was 14% of that elicited in the absence of this inhibitor. An incubation time of 30 min was chosen for all subsequent studies. Rp-cAMPS (< or = 200 microM) inhibited the contractile response to isoprenaline (100 nM) significantly and in a concentration-dependent manner, but failed to inhibit the contractile responses elicited by phenylephrine (2 microM) and calcium ion (7 mM) which act by cyclic AMP-independent mechanisms. In the presence of Rp-cAMPs (200 microM), the contractile response to isoprenaline (100 nM) was 24% of that in the absence of inhibitor. Rp-cAMPS was used subsequently to investigate the contractile-coupling mechanisms associated with some novel inotropic agents. Rp-cAMPS (< or = 200 microM) also inhibited the contractile responses to secretin (20 nM) and VIP (20 nM) significantly. In the presence of Rp-cAMPS (200 microM), the contractile response elicited by secretin (20 nM) was 19% of that in the absence of inhibitor, while that elicited by VIP (20 nM) was abolished completely. Rp-cAMPS (< or = 200 microM) failed to inhibit the contractile response elicited by CGRP (1 nM). In summary, Rp-cAMPS is a membrane-permeable, selective antagonist of cyclic AMP in ventricular cardiomyocytes and can be used, in conjunction with the bioassay of the intracellular accumulation of cyclic AMP, to distinguish between cyclic AMP-dependent and cyclic AMP-independent contractile coupling mechanisms in these cells.(ABSTRACT TRUNCATED

Topics: Adenosine Deaminase; Animals; Calcitonin Gene-Related Peptide; Calcium; Cell Size; Cells, Cultured; Cyclic AMP; Depression, Chemical; Heart Ventricles; Isoproterenol; Male; Myocardial Contraction; Rats; Rats, Sprague-Dawley; Secretin; Thionucleotides; Vasoactive Intestinal Peptide

Many studies suggest that smooth muscle relaxation caused by beta-adrenergic agents and various neuropeptides occurs as a result of an increase in cellular adenosine 3',5'-cyclic monophosphate (cAMP). However, the evidence is indirect, and furthermore does not demonstrate that an increase in cAMP is essential for mediating relaxation. To define more clearly the role of cAMP in receptor-mediated smooth muscle relaxation, we used a specific competitive antagonist of the action of cAMP on protein kinase A, (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], and its S isomer, (S)-p-cAMPS, which functions as a cAMP agonist. In gastric smooth muscle cells from guinea pig, (S)-p-cAMPS caused a dose-related relaxation [50% inhibitory concentration (IC50) 86 +/- 59 nM]. Vasoactive intestinal peptide (VIP) produced smooth muscle cell relaxation (IC50 2.3 +/- 0.8 nM) through occupation of specific VIP receptors. (R)-p-cAMPS inhibited VIP-induced relaxation, with a rightward shift in the VIP dose-response curve, suggesting competitive antagonism. Furthermore, (R)-p-cAMPS inhibited relaxation induced by other agents that increase cellular cAMP (isoproterenol, calcitonin gene-related peptide, and glucagon) but not that induced by ATP or sodium nitroprusside. (R)-p-cAMPS had no effect on contraction stimulated by carbachol, cholecystokinin, or substance P. These data demonstrate that activation of protein kinase A is primarily responsible for mediating gastrin smooth muscle relaxation produced by adrenergic agents and various neuropeptides.

Topics: Animals; Carbachol; Cyclic AMP; Muscle Relaxation; Muscle, Smooth; Neuropeptides; Protein Kinases; Stomach; Sympathomimetics; Thionucleotides; Vasoactive Intestinal Peptide