Synergistic action of melatonin and vasoactive intestinal peptide in stimulating cyclic AMP production in human lymphocytes

In the present study we investigated the synergistic effect of melatonin and vasoactive intestinal peptide (VIP) on cyclic AMP production in human blood lymphocytes. As shown by our group previously, VIP alone behaved as a potent activator of cyclic AMP production in human lymphocytes. On the other hand, melatonin alone did not affect the intracellular levels of cyclic nucleotide at any time or dose studied. However, when cells were incubated with melatonin plus VIP, melatonin potentiated the effect of the peptide. This effect can be observed in the presence of physiological doses of both melatonin (10-100 pM) and VIP (1-100 pM). The effect is specific for VIP because with other peptides belonging to the secretin-VIP family the effect was not observed. Results suggest that melatonin, in conjunction with VIP, may directly participate in the regulation of immune function in the human.     

Interaction of vasoactive intestinal peptide with human blood mononuclear cells

The binding of vasoactive intestinal peptide (VIP) and the stimulation of adenylate cyclase were studied in mononuclear cells from human peripheral blood. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Binding studies suggested the presence of 2 classes of binding site: a class with high affinity (Kd = 2.4 X 10(-10)M) and low capacity (8 fmoles/10(6) cells), and a class with low affinity (Kd = 8.0 X 10(-8)M) and high capacity (800 fmoles/10(6) cells) at 15 degrees C. Secretin displaced [125I]VIP from the cells with a 400-fold lower affinity than VIP, but glucagon, somatostatin and insulin did not show any effect. VIP was a potent and efficient stimulator of cyclic AMP production. The stimulation was observed at a concentration as low as 3 X 10(-11)M and depended on time, temperature and pH. Maximal cyclic AMP production (4-fold above basal levels) was observed with 10(-9) M at 15 degrees. Half-maximal response was obtained at 10(-10)M VIP. Secretin was an agonist of VIP but exhibited a 7000 times lower potency. Peripheral blood mononuclear cells constitute an easily accessible and suitable system for the study of VIP action in different physiological and pathophysiological conditions.     

Non-specific activation of human eosinophil functional responses by vasoactive intestinal peptide

Eosinophils and neuropeptides are thought to play effector roles in allergic diseases, such as rhinitis; however, little is known about the biological effects of neuromediators, especially vasoactive intestinal peptide (VIP), on eosinophil functional responses. In the present study, it is shown that VIP induces eosinophil chemotaxis and eosinophil-derived neurotoxin (EDN) release in potency comparable with that induced by platelet activator factor, and in a novel synergistic manner with recombinant human interleukin-5. Contrary to chemotaxis, EDN release was sensitive to staurosporine, the protein kinase C inhibitor, as well as intracellular calcium chelation. However, eosinophil treatment with inhibitors of tyrosine kinases (herbimycin A) and phosphatases (pervanadate) resulted in a dose-dependent potentiation and blockage of VIP-induced eosinophil chemotaxis, respectively. Treatment of eosinophils with VIP receptor antagonist did not modify VIP-induced chemotaxis or EDN release. Furthermore, exploration of vasoactive intestinal peptide receptor I expression was lacking in human eosinophils, but not lymphocytes. These results demonstrate two different mechanisms in triggering eosinophil activation of functional responses by VIP, a calcium-dependent degranulation and a calcium-independent chemotaxis, and elaborate on a novel cytokine–neuropeptide interaction in eosinophilic inflammation.     

Effect of vasoactive intestinal peptide on monkey prolactin secretion and cyclic AMP in culture: interaction with estradiol and phenol red

To test the hypothesis that estrogenic compounds may decrease the sensitivity of primate lactotropes to adenylate cyclase-mediated secretagogues, the effect of VIP on prolactin secretion and cAMP levels in serum-free monkey pituitary monolayer cultures was examined in the presence and absence of estradiol (E) and phenol red. In two experimental designs, E treatment was initiated on either the day after dispersion (split plate design) or 10 days after serum-free culture (whole plate design). VIP challenges (5, 50 and 500 nM) were administered for 4 h on days 10 and 20 of culture. There was a significant decrease in the maximal percent stimulation of prolactin by VIP when cultures were treated with E or phenol red. The average percent increase in prolactin at 5, 50 and 500 nM VIP equalled 23, 83 and 156% in the absence of phenol red, but equalled 14, 43 and 112% when E was added to phenol red-free cultures. The percent stimulation by VIP in the presence of phenol red averaged 32, 62 and 97%, but addition of E with phenol red decreased the average stimulation to 26, 45 and 72%, respectively. Basal levels of cAMP were increased by E and phenol red. However, the maximal percent stimulation of cAMP by VIP was decreased in the presence of E and phenol red. In summary, E and phenol red act to decrease the maximal percent stimulation of prolactin secretion by VIP. This effect is reflected by a decrease in the maximal percent stimulation of intracellular cAMP.     

Regulation by secretin, vasoactive intestinal peptide, and somatostatin of cyclic AMP accumulation in cultured brain cells.

Secretin stimulates the accumulation of cyclic AMP (half maximally stimulating concentration: 10-20 nM) in cultured mouse brain cells mainly consisting of glioblasts. Vasoactive intestinal peptide (VIP) is much less potent in raising the level of cyclic AMP in these cultures. The effect of secretin but not that of VIP is inhibited by secretin-(5-27), a synthetic antagonist of secretin. Stimulation of the adrenergic alpha-receptors and the adenosine A1-receptors present on the cells attenuates the increase in cyclic AMP evoked by secretin and VIP. Somatostatin at low concentrations inhibits the accumulation of cyclic AMP (half-maximally inhibitory concentration: 3 nM), in the absence or presence of secretin, VIP, or isoproterenol. The results suggest that secretin might regulate the concentration of cyclic AMP in brain and provoke the question of a possible involvement of glial cells in the action of peptide hormones in the brain.     

Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in turkey cerebral cortex: characterization by [125I]-VIP binding and effects on cyclic AMP synthesis

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a Kd of 0.70 nM and a Bmax of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [125I]-VIP binding to turkey cerebrum was: PACAP38 approximately PACAP27 approximately chicken VIP approximately mammalian VIP > PHI > secretin, chicken VIP16-28 (inactive). About 65% of specific [125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [125I]-VIP.     

Interaction of alpha-melanocyte-stimulating hormone, melatonin, cyclic AMP and cyclic GMP in the control of melanogenesis in hair follicle melanocytes in vitro

In short-term (48 h) cultures of hair follicles alpha-melanocyte-stimulating hormone (alpha-MSH) and cyclic AMP stimulated melanogenesis through an increase in tyrosinase activity. In contrast cyclic GMP mimicked the effects of melatonin by inhibiting melanin production without causing a concomitant decrease in tyrosinase activity. Both cyclic GMP and melatonin blocked the stimulatory effects of cyclic AMP and alpha-MSH on melanin production but they left the increased levels of tyrosinase activity unaffected. Phosphodiesterase inhibitors (3-isobutyl-1--methylxanthine and papaverine) simultaneously stimulated tyrosinase activity and inhibited melanin production, presumably by allowing endogenous cyclic AMP and cyclic GMP to accumulate intracellularly. It is suggested that whereas MSH stimulates melanogenesis through a cyclic AMP-dependent mechanism there must also be an inhibitory cyclic GMP-dependent mechanism, perhaps activated by melatonin, which operates at some post-tyrosinase step in the melanin biosynthetic pathway.     

Sequential alterations in the hepatic content and metabolism of cyclic AMP and cyclic GMP induced by DL-ethionine: Evidence for malignant transformation of liver with a sustained increase in cyclic AMP

There is evidence than adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) may have antagonistic actions on cell growth, with cAMP inhibiting and cGMP stimulating this process. However, reductions in cAMP and increases in cGMP are not charactersitic of all neoplastic tissues. Thus, benign and malignant tissues from hepatoma-bearing rats exposed to the hepatic carcinogen DL-ethionine have elevated rather than depressed cAMP, compared to control liver, and parenteral administration of this drug increases hepatic cAMP within hours. In the present study, the effects of ethionine ingestion on the hepatic content and metabolism of both cAMP and cGMP were examined sequentially in rats at 2 and then 6 wk intervals, from the initiation of drug administration until the development of hepatomas. After 2 wk, cAMP content of quick-frozen liver from rats receiving ethionine (E) was significantly increased (826 +/- 91 pmole/g wet weight) above that of liver from pair-fed controls (C, 415 +/- 44), whether calculated by tissue wet weight, protein, or DNA content. In benign tissue from E, higher cAMP was still evident after in vitro incubations of slices with 2 mM 1-methyl-3-iso-butylxanthine (MIX) and was associated with enhanced adenylate cyclase and unchanged high or low Km cAMP-phosphodiesterase activities. These findings are compatible with accelerated cAMP generation in liver from E. Protein kinase activity ratios were significantly increased in frozen liver from E (0.52 +/- 0.04 versus 0.36 +/- 0.03 in C), and the percent glycogen synthetase in the I form was clearly reduced (19% +/- 2% in E versus 47% +/- 5% in c). incubation of hepatic slices from E or C with MIX and/or 10 muM glucagon further increased cAMP and protein kinase activity ratios, data which imply higher effective, as well as total, cellular cAMP in E. Changes in cAMP metabolism and action observed at 2 wk persisted throughout the 38-wk period of drug ingestion. Adenylate cyclase activity, cAMP content, and protein kinase activity ratios of ethionine-induced hepatomas exceeded those of both the surrounding liver from tumor-bearing rats and that of control liver, but alterations in these parameters were qualitatively similar in both tissues from E. By contrast, while cGMP in quick-frozen surrounding liver from tumor-bearing rats (36 +/- 4 pmole/g wet weight) did not differ from that of control liver (30 +/- 3), cGMP in the hepatomas was increased. This change was evident in both frozen tumor (89 +/- 10) and in tumor slices incubated in vitro with MIX (C, 90 +/- 11; surrounding liver, 85 +/- 10; hepatoma 231 +/- 29). These results indicate that malignant conversion can occur in liver with a sustained elevation of both total and effective cAMP during the premalignant phase. The increase in cGMP detected in ethionine-induced hepatomas could also be a key determinant of malignant transformation in the model, although premalignant changes in cGMP were not apparent.     

Identification of cells responding to vasoactive intestinal peptide by measuring intracellular cyclic adenosine monophosphate in human colonic mucosa

BACKGROUND: Although there is great deal of evidence suggesting that vasoactive intestinal peptide (VIP) has immunomodulating effects on human colonic lamina propria mononuclear cells (LPMC), it remains unclear which type of cell carries functional VIP receptors. In this study we investigated the presence of functional VIP receptors by measuring intracellular cyclic adenosine monophosphate (cAMP) in isolated epithelial cells, bulk LPMC, T cells, and macrophages in human colonic mucosa. METHODS: Epithelial cells and LPMC were isolated from non-pathologic segment of colonic mucosa of surgical specimens from five patients with colonic cancer. Mucosal T cells and macrophages were further isolated from LPMC. Each cell population was cultured with various concentration of VIP for 60 min at most. Then, intracellular cAMP was extracted and measured by enzyme-linked immunoassay. RESULTS: When isolated epithelial cells were examined, VIP increased intracellular cAMP in a dose-dependent fashion, as observed in HT-29 cells used as a positive control. In contrast, the concentration of cAMP was essentially stable in response to VIP when isolated LPMC were examined. This was the case even when separated T cells and macrophages were individually investigated. To evaluate the possible effects of enzyme digestion for LPMC isolation on the VIP response. HT-29 cells were precultured with collagenase and deoxyribonuclease (DNase 1), resulting in less enhancement of cAMP by VIP. CONCLUSIONS: In this study we failed to show VIP-responsive enhancement of cAMP in mucosal immune cells, suggesting that epithelial cells may be major effector cells of VIP in human colonic mucosa.     

Pleiotypic Control by Cyclic AMP: Interaction with Cyclic GMP and Possible Role of Microtubules

Previous studies have shown that exogenous dibutyryl cyclic AMP inhibits the uptake of uridine, leucine, and 2-deoxyglucose by cultured mouse fibroblasts. 3':5'-cyclic GMP is shown here to counteract these inhibitory effects as well as the inhibition of precursor transport and leucine incorporation into proteins produced by prostaglandin E(1). We conclude, therefore, that cyclic GMP antagonizes the "pleiotypic" effects of cyclic AMP in these cells.Colcemid and vinblastine, but not cytochalasin B, reverse the transport inhibition caused by cyclic AMP without affecting the intracellular concentrations of cyclic AMP. These results suggest the possibility that cyclic AMP regulates the membrane transport of certain substrates by influencing the organization of microtubules.     

Dual receptor regulation of cyclic nucleotides: alpha 1-adrenergic potentiation of vasoactive intestinal peptide stimulation of pinealocyte adenosine 3',5'-monophosphate

The purpose of this investigation was to determine if alpha 1-adrenergic receptor activation would potentiate the vasoactive intestinal peptide (VIP)-stimulated increase in cAMP accumulation in the rat pinealocyte. Treatment with VIP alone increased cAMP accumulation less than 5-fold, and treatment with phenylephrine increased cAMP accumulation less than 2-fold. However, combined treatment with VIP and phenylephrine increased cAMP accumulation more than 20-fold. This is the first report that alpha 1-adrenergic and VIP receptors can interact to regulate cAMP in a cell of neural tube origin. The demonstration of such an integrated mechanism in pinealocytes is of special interest because VIP innervation of the pineal gland is of central origin, and adrenergic innervation is of peripheral origin. The magnitude of response makes the pinealocyte an attractive model for the study of such dual receptor regulation.     

Interaction of peptide YY with rat intestinal epithelial plasma membranes: binding of the radioiodinated peptide

High affinity binding sites for peptide YY (PYY) have been identified and characterized in plasma membranes prepared from rat jejunal epithelium by studying the kinetics, stoichiometry, and chemical specificity of the interaction of 125I-labeled PYY with membranes. Binding of [125I]PYY was rapid, saturable, reversible, specific, and depended on temperature, pH, and ionic strength. In optimized steady state conditions of binding (2 h of incubation at 15 C), the degradation of both [125I] PYY and binding sites did not exceed 20%. The concentration dependence of PYY binding, determined by adding increasing concentrations of [125I]PYY, indicated that specific binding saturated at 2-3 nM peptide. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 434 +/- (SE) 56 pM and a binding capacity of 336 +/- 41 fmol/mg protein (n = 11). Identical results were obtained when increasing concentrations of unlabeled PYY were added to a fixed concentration of [125I]PYY, indicating that the radioiodinated peptide has the same apparent affinity as native PYY. Peptides structurally unrelated to PYY, such as members of the vasoactive intestinal peptide family, insulin, or cholecystokinin octapeptide, were unable to compete with [125I]PYY for binding to membranes. Rat, human, and avian pancreatic polypeptides, which display, respectively, 42%, 47%, and 53% homology with PYY, did inhibit [125I]PYY binding but with an approximate or equal to 100,000-fold lower potency than PYY, indicating the strict structural requirement for recognition by PYY binding sites. In contrast, natural or synthetic neuropeptide Y, which has 25 out of 36 amino acids in common with PYY, retained a high affinity for PYY binding sites [only 4.7 +/- 1.2 (n = 5) times lower than that of PYY]. Specific [125I]PYY binding was particularly high in the upper small intestine and could not be detected in stomach, large intestine, or liver. These findings indicate that rat small intestinal epithelium expresses specific binding sites for the candidate gut hormone PYY that also binds the neuropeptide Y with high affinity, suggesting that the two peptides may regulate the function of small intestinal epithelium, through interaction with a common receptor site.     

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside

In studies on human platelets, nitroprusside (NP) alone at 1-10 micromol/l increased platelet cyclic AMP (cAMP) by 40-70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo-oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2',5'-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen-induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP-dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Ibeta isoenzyme. An inhibitor of this enzyme (Rp)-beta-phenyl-1,N2-etheno-8-bromoguanosine 3',5'-cyclic-monophosphorothioate, diminished the inhibition of collagen-induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen-induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP-dependent protein kinases had supra-additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.     

Synergistic hypotensive effect of vasoactive intestinal polypeptide and alpha-blockade with phentolamine. Evidence for vasoactive intestinal peptide alpha-adrenoceptor coupling in the cardiovascular system of newborn dogs

Vasoactive intestinal polypeptide (VIP) is a neuropeptide with potent circulatory effects in the adult animal and human. Little is known about its effects or mechanism of action in the immature animal. These series of experiments evaluated the effects and possible mechanism of action of VIP on the developing canine cardiovascular system. In all three series, measurements of mean heart rate and blood pressure were taken in the control state, after parasympathetic denervation with bilateral cervical vagotomies, and after autonomic blockade with propranolol (1 mg/kg) and phentolamine (0.5 mg i.v.). In series 1, we characterized the role of alpha-adrenergic receptors in early newborn puppies by investigating the hemodynamic effects of phentolamine alone in five early newborn puppies. In series 2, the hemodynamic effects of intravenous VIP infusion (0.2 microgram/kg/min) were recorded and compared in six early newborn puppies and in 10 late newborn puppies. In series 3, the hemodynamic effects of phentolamine in the presence of VIP receptor binding inhibitor were studied. In early newborn puppies, VIP had essentially no effect on heart rate or blood pressure until phentolamine was given; then, blood pressure decreased by 17% (p less than 0.005). In late newborn puppies, VIP resulted in an increase in heart rate in the control state but not after parasympathetic or sympathetic denervation. In early newborn puppies, phentolamine alone resulted in a 24% decrease (p less than 0.005) in blood pressure, compared with a 54% decrease (p less than 0.005) in early newborn puppies preexposed to VIP infusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective photolabeling of high and low affinity binding sites for vasoactive intestinal peptide (VIP): evidence for two classes of covalent VIP-receptor complexes in intestinal cell membranes

The biological activities of VIP derivatives as photoaffinity labels are described. The derivatives were obtained by VIP modification with azido phenyl glyoxal at arginyl residues 12 and 14 ([Az Bz Arg12-14]VIP) or only at position 14 ([Az Bz Arg14]VIP). The first derivative exhibited pronounced hydrophobic properties. The level of cAMP produced in HT 29 cells by this derivative represented 15% of that obtained by VIP at equimolar concentration (10(-10) M). The second derivative ([Az Bz Arg14]VIP) was synthesized by a new procedure, in which amino acids of VIP buried in the active site of the receptor were protected from azido phenyl glyoxal. This analog retained a high binding affinity for receptor (Kd, 0.5 nM for [125I-Tyr,Az Bz Arg14]VIP vs. 0.1 nM for [125I] VIP) and was found to be biologically active, as judged by stimulation of adenylate cyclase activity (production of cAMP was 120 pmol/10(6) cells vs. 140 pmol for VIP at 10(-10) M). Photolysis of this analog in the presence of HT 29 cell membranes resulted in a stimulation of adenylate cyclase which persisted in spite of repeated washings. Our findings indicate that [125I-Tyr,Az Bz Arg14] VIP binds covalently to active sites to form an active peptide-receptor complex. When low affinity binding sites were specifically photolabeled using a selective protection of high affinity sites by incubating membranes with 10(-10) M VIP for 5 min, the derivative did not stimulate adenylate cyclase activity. This suggests that VIP acts through a high affinity site to produce the biological activity and that the functional relevance of low affinity sites is unclear.     

Peptide specificity for stimulation of corticotropin secretion: activation of overlapping pathways by the vasoactive intestinal peptide family and corticotropin-releasing factor

The hypothalamic peptide vasoactive intestinal peptide (VIP) stimulates ACTH and endorphin secretion by the AtT20/D16 clonal strain of mouse pituitary tumor cells. The dose dependence for VIP stimulation of hormone release is biphasic, indicating that VIP is able to activate at least two classes of receptors in D16 cells (ED50 = 1.6 and 160 nM). We show that at high concentrations (ED50 greater than or equal to 150 nM), other natural peptides with primary structures homologous to that of VIP also increased ACTH secretion by D16 cells, whereas structurally unrelated peptides did not. The stimulatory actions of GH-releasing factor (GRF) and porcine heptacosapeptide with amino-terminal histidine and carboxy-terminal isoleucine amide (PHI) were mediated by high affinity VIP receptors because their effects were not additive with that of 10 nM VIP. In addition, GRF and PHI behaved as antagonists at low affinity VIP receptors; both peptides inhibited stimulation by 1 microM VIP. In contrast, glucagon and gastric inhibitory polypeptide appeared to stimulate ACTH release via low affinity VIP receptors because their effects were additive with that of 10 nM, but not 1 microM, VIP. Since all of the VIP-like peptides increased ACTH secretion only at high concentrations, they were unlikely to represent a physiological ligand for the receptor activated by high concentrations of VIP. Therefore, we determined whether cross-reactivity occurred between VIP-like peptides and corticotropin-releasing factor (CRF), a potent stimulator of ACTH secretion both in vitro and in vivo. The dose-response curve for CRF stimulation of ACTH secretion by D16 cells extended over more than a 1000-fold range of concentrations and was biphasic (ED50 = 2.6 and greater than 300 nM), indicating that CRF interacted with multiple receptor types in D16 cells. However, since the effect of 10 nM CRF was additive with that of 1 microM VIP, the CRF receptor was not the site at which high concentrations of VIP stimulated ACTH release. In contrast, the effect of 1 microM CRF was not additive with that of 1 microM VIP or other VIP-like peptides. Therefore, high concentrations of CRF and the previously recognized VIP-like peptides stimulated ACTH secretion by overlapping pathways. Comparison of the amino acid sequence of CRF with those of the VIP-like peptides showed that 18 of the 41 amino acids in CRF match a corresponding amino acid in at least 1 member of the VIP peptide family.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of calmodulin with iodothyronines: effect of iodothyronines on the calmodulin activation of cyclic AMP phosphodiesterase

The interaction between calmodulin and iodothyronines and the effect of iodothyronines on the calmodulin activation of cyclic AMP phosphodiesterase were investigated. Binding of [L-125I]triiodothyronine to calmodulin from pig brain, studied by equilibrium dialysis, was dependent on Ca2+, was saturable and reversible, with an apparent Kd of 2.79 microM and binding capacity of 0.5 nmol/20 micrograms of calmodulin L- and D-thyroxine, D-triiodothyronine and tetrac displaced [L-125I]triiodothyronine at concentrations of 8-10 microM; triac, 3,3'-diiodothyronine and reverse-triiodothyronine were weak displacers. In the presence of the antipsychotic drug trifluoperazine, binding decreased in a dose-related manner. Ultraviolet irradiation of calmodulin in the presence of trifluoperazine reduced the binding of [L-125I]triiodothyronine to calmodulin irreversibly. Calmodulin activation of cyclic AMP phosphodiesterase decreased when iodothyronines were bound to calmodulin; the calmodulin-L-triiodothyronine complex was the most active among the stereoisomers of thyroxine and triiodothyronine. These results suggest that, when triiodothyronine was bound to Ca2+-calmodulin, the activation of cyclic AMP phosphodiesterase by the latter is suppressed.     

Adrenal steroid modulation of vasoactive intestinal peptide effect on serotonin1 binding sites in the rat brain shown by in vitro quantitative autoradiography

In the present work we demonstrate by means of quantitative in vitro autoradiography that the vasoactive intestinal peptide (VIP) is able to increase the number of serotonin1 (5-HT1) binding sites in the dorsal subiculum of the rat hippocampus and to decrease them in the suprachiasmatic nucleus (SCN). Bilateral adrenalectomy (ADX) for 6 days counteracted the stimulatory effect of VIP on 5-HT1 binding sites in the dorsal subiculum, but did not modify the inhibitory effect of the peptide in the SCN. Moreover, ADX increased 5-HT1 binding sites in response to VIP in various subfields of the hippocampus as well as in the superior colliculus and in the dorsal lateral septum, but this effect was not observed in normal or in ADX rats bearing a corticosterone implant. The present data are suggestive of a possible interaction between VIP and 5-HT in the regulation of the SCN and of a modulatory role of adrenal steroids in VIP activity in the hippocampal formation.