Differential Involvement of the Arachidonic Acid Cascade on the α1-Adrenergic Potentiation of Vasoactive Intestinal Peptide-Versus β-Adrenergic-Stimulated Cyclic AMP and Cyclic GMP Accumulation in Rat Pinealocytes

In the rat pineal gland, alpha 1-adrenergic agonists, which stimulate arachidonic acid release, also potentiate vasoactive intestinal peptide (VIP)- or beta-adrenergic-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation. In this study, the possible involvement of the arachidonic acid pathway in the potentiation mechanism was examined in dispersed rat pinealocytes using two inhibitors of the arachidonic acid cascade, indomethacin and nordihydroguaiaretic acid. These two inhibitors appeared to have differential effects on the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP responses. Whereas nordihydroguaiaretic acid was effective in suppressing both the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP responses, indomethacin inhibited selectively the VIP-mediated cAMP and cGMP responses. The role of arachidonic acid metabolites was further determined using several prostaglandins--A2, I2, E2, and F2 alpha--and leukotrienes--B4, C4, and D4. Of the seven compounds tested, prostaglandins E2 and F2 alpha stimulated basal cAMP but not cGMP accumulation. The prostaglandin E2- and F2 alpha-stimulated cAMP responses were additive to those stimulated by VIP or beta-adrenergic receptors. The other five compounds had no effects on basal or VIP- or beta-adrenergic-stimulated cAMP or cGMP accumulation. Taken together, these findings indicate that the arachidonic acid cascade is likely involved in the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP accumulation. However, the specific arachidonic acid metabolite involved in the potentiation mechanisms of VIP- versus beta-adrenergic-stimulated cyclic nucleotide responses may be different.     

Pituitary Adenylate Cyclase-Activating Polypeptide: Control of Rat Pineal Cyclic AMP and Melatonin but Not Cyclic GMP

Abstract: In this study, the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10⁻⁷ M ) increased MT production 2.5-fold. PACAP (10⁻⁷ M ) also increased cyclic AMP accumulation four- to fivefold; this effect was potentiated two- to three-fold by α₁-adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because α₁-adrenergic activation is known to translocate PKC and the PACAP-stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4β-phorbol 12-myristate 13-acetate (PMA). Phenylephrine and PMA also potentiated the PACAP-stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an α₁-adrenergic → PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.     

Interaction of VIPergic and cholinergic receptors in human thyroid cell

The thyroid tissue is innervated by cholinergic and VIPergic nerves. The present study investigated the possible interactions of cholinergic agents with VIP-induced cAMP accumulation and thyroid hormone release in vitro. Carbamylcholine (Cch), acting through the muscarinic receptor increases cellular cGMP content in cultured human thyroid cells incubated with a phosphodiesterase inhibitor. Cch (10 microM) inhibits cellular cAMP accumulation and thyroxine (T4) release induced by vasoactive intestinal peptide (VIP), with or without a phosphodiesterase inhibitor. Cch (10 microM) inhibits 8-bromo-cAMP-induced T4 release from human thyroid slices. 8-Bromo-cGMP inhibits VIP-induced T4 release from human thyroid slices, only in cells incubated without the phosphodiesterase inhibitor. The results indicate that interactions between VIPergic and cholinergic receptors may be of importance in human thyroid cell.     

Neuropeptide Y Inhibits β-Adrenergic Agonist– and Vasoactive Intestinal Peptide–Induced Cyclic AMP Accumulation in Rat Pinealocytes Through Pertussis Toxin–Sensitive G Protein

The effects of neuropeptide Y (NPY) on pineal gland cyclic AMP (cAMP) accumulation were investigated using dispersed pinealocytes from rats. NPY inhibited the intracellular cAMP accumulation stimulated by isoproterenol and norepinephrine in a dose-dependent manner during a 10-min incubation of pinealocytes. NPY (1 x 10(-7) M) also inhibited vasoactive intestinal peptide (VIP)- and cholera toxin-induced cAMP accumulation. The inhibitory effect of NPY on isoproterenol-induced cAMP accumulation was completely abolished by a 5-h pretreatment of pinealocytes with 1 microgram/ml of pertussis toxin (PT). These results suggest that NPY participates in modulation of cAMP production in the rat pineal gland through PT-sensitive G protein. Yohimbine, an alpha 2-adrenergic antagonist, blocked NPY inhibition of isoproterenol-stimulated cAMP accumulation. On the other hand, the alpha 2-adrenergic agonist clonidine by itself did not affect cAMP accumulation stimulated by isoproterenol but significantly potentiated NPY action. The present study demonstrates that NPY inhibits beta-adrenergic or VIPergic stimulation of the pineal gland cAMP accumulation. The inhibitory effect of NPY is mediated through PT-sensitive G protein. Our results also suggest that NPY exerts its action to affect alpha 2-adrenoceptor function.     

Effect of radiation on cAMP,cGMP and Ca(2+)(i) pathways and their interactions in rat distal colon

The secretory response implicated in the intestinal response to luminal attack is altered by radiation. The cAMP, cGMP and Ca(2+)(i) pathways leading to secretion as well as the interactions between the cAMP pathway and the cGMP or Ca(2+)(i) pathway were studied in the rat distal colon 4 days after a 9-Gy abdominal X irradiation, when modifications mainly occurred. The secretory response in Ussing chambers and cAMP and cGMP accumulation in single isolated crypts were measured. The muscarinic receptor characteristics were determined in mucosal membrane preparations. The secretory response by the cAMP pathway (stimulated by vasoactive intestinal peptide or forskolin) and the cAMP accumulation in crypts were decreased (P < 0.05) after irradiation. The weak secretory response induced by the cGMP pathway (stimulated by nitric oxide or guanylin) was unaltered by radiation, and the small amount of cGMP determined in isolated crypts from the control group became undetectable in the irradiated group. Inducible NOS was not involved in the hyporesponsiveness to VIP after irradiation (there was no effect of an iNOS inhibitor). The secretory response by the Ca(2+)(i) pathway (stimulated by carbachol) was unaffected despite a decreased number and increased affinity of muscarinic receptors. The non-additivity of VIP and carbachol co-stimulated responses was unmodified. In contrast, VIP and SNP co-stimulation showed that NO enhanced the radiation-induced hyporesponsiveness to VIP through a reduced accumulation of cAMP in crypts. This study provides further understanding of the effect of ionizing radiation on the intracellular signaling pathways.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Interaction Between Adrenergic and Peptide Stimulation in the Rat Pineal: Pituitary Adenylate Cyclase-Activating Peptide

Abstract: The 27 amino acid peptide, pituitary adenylate cyclase-activating polypeptide (PACAP-27), and its 38 amino acid analogue, PACAP-38, stimulate serotonin- N -acetyltransferase (NAT) activity and N -acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP-38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N-terminal histidine C-terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP-38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP-38 reduces the magnitude of subsequent stimulation with PACAP-38 or forskolin. Concurrent stimulation of α-receptors or treatment with active phorbol ester augments rat pineal response to PACAP-38 stimulation just as it increases the response to VIP, PHI, and β-receptor stimulation. Pineals from newborn rats respond to PACAP-38 with an increase in NAT activity and the increase is augmented by concomitant α₁-adrenergic stimulation. The putative PACAP inhibitor PACAP (6–38) and the putative VIP inhibitor (Ac-Tyr, d -Phe)-GRF 1–29 amide, in 100–1,000-fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.     

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.     

N-Acetylation of Serotonin Is Correlated with α2- but Not with β-Adrenergic Regulation of Cyclic AMP Levels in Cultured Chick Pineal Cells

We investigated the role of cyclic AMP (cAMP) in alpha 2- and possible beta-adrenergic regulation of arylalkylamine-N-acetyltransferase (NAT), the penultimate enzyme in the biosynthesis of melatonin. The study was performed on primary cultures of dispersed chick pineal cells. Electron microscopy indicated that approximately 70% of the dispersed cells were modified photoreceptors. A similar proportion of melatoninergic cells was detected by immunocytochemical labeling of hydroxyindole-O-methyltransferase, the final enzyme in the biosynthesis of melatonin. Adrenergic agonists caused a sustained 50% inhibition of forskolin-augmented cAMP levels and NAT activity, with an alpha 2-adrenergic potency order of UK 14,304 greater than or equal to clonidine greater than norepinephrine greater than phenylephrine. Noradrenergic inhibition of 3-isobutyl-1-methylxanthine-augmented cAMP levels and NAT activity was reversed by yohimbine (an alpha 2-adrenergic antagonist) but not by prazosin (an alpha 1-adrenergic antagonist). The alpha-adrenergic inhibition of cAMP accumulation and NAT activity was prevented by pertussis toxin. Addition of propranolol (a beta-adrenergic antagonist) was necessary to observe an inhibitory effect of norepinephrine on cAMP levels but not on NAT activity. Similarly, the beta-adrenergic agonist isoproterenol transiently increased cAMP levels but did not affect NAT activity. The data indicate that the alpha 2-adrenergic inhibition of NAT activity in chick pineal cells is strongly correlated with an inhibition of cAMP accumulation. The lack of beta-adrenergic effect on NAT suggests that beta-adrenoceptors might be on a subset of cells that do not produce melatonin or that the beta-adrenergic-induced increase in cAMP levels is too transient to affect NAT.     

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line.

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.     

Differential actions of phosphodiesterase inhibitors on secretin- and vasoactive intestinal peptide-induced increases in chief cell cAMP

The actions of three different phosphodiesterase inhibitors, theophylline, 3-isobutyl-1-methylxanthine (IBMX) and Ro 20-1724 (Ro), on cellular cAMP and pepsinogen secretion from dispersed chief cells prepared from guinea pig stomach were examined. The relative order of potency for increasing cAMP and pepsinogen secretion was Ro greater than IBMX greater than theophylline. Ro, the most efficacious agent, caused a 17-fold increase in basal cAMP and a similar augmentation of the increase in cAMP caused by secretin or vasoactive intestinal peptide (VIP). Differential actions of these agents on the dose-response curves for secretin- and VIP-induced increases in cAMP suggest that chief cell receptors for these peptides are coupled to pools of cAMP that are acted upon by heterogeneous phosphodiesterases with varying sensitivities to inhibitors. Moreover, Ro, a selective inhibitor of low Km cAMP-specific phosphodiesterases, is the most potent and efficacious agent tested in this cell system.     

Cross-Regulation of Intracellular cGMP and cAMP in Cultured Human Corpus Cavernosum Smooth Muscle Cells

The goal of this study was to assess the potential cross-regulation of cyclic nucleotides in human corpus cavernosum (HCC). Incubation of primary cultures of HCC smooth muscle cells with either the NO donor sodium nitroprusside (SNP, 10 μM) or the phosphodiesterase type 5 (PDE 5) inhibitor sildenafil (50 nM) produced little or no changes in the intracellular cGMP levels. Incubation with both SNP and sildenafil produced marked increases in cGMP. Interestingly, incubation of cells with 10 μM of forskolin or PGE₁ produced significant enhancement of cGMP accumulation. These increases were not further enhanced by the addition of SNP and sildenafil. Kinetic analyses of cGMP hydrolysis by PDE 5 showed that high concentrations of cAMP reversibly inhibited the enzyme with a K_i of 258 ± 54 μM. The increase in cGMP levels in response to cAMP generating agents is not due to assay artifact since cAMP did not cross-react with cGMP antibody. Our data suggest that cAMP up-regulates intracellular levels of cGMP, in part, by inhibition of PDE 5. We also noted that cGMP down-regulates cAMP synthesis via a mechanism requiring G-protein coupling of adenylyl cyclase. These observations may have important implications in the utility of pharmacotherapeutic agents targeting cyclic nucleotide metabolism for the treatment of erectile dysfunction.     

Vasoactive Intestinal Peptide and Forskolin Stimulate Interleukin 6 Production by Rat Cortical Astrocytes in Culture via a Cyclic AMP-Dependent, Prostaglandin-Independent Mechanism

Abstract: In this study we analyzed the involvement of the cyclic AMP (cAMP)-protein kinase A system in the regulation of interleukin 6 production by cultured cortical astrocytes. Vasoactive intestinal peptide strongly increased, in a dose-dependent manner, interleukin 6 production. This effect was reduced when protein kinase A was blocked by KT-5720; it was not affected by calphostin C, a protein kinase C inhibitor. Forskolin caused a concentration-dependent increase in interleukin 6 release that was also inhibited by KT-5720. Because prostaglandins are believed to play a role in interleukin 6 production, we tried to determine whether the stimulatory effects of vasoactive intestinal peptide and forskolin on cytokine release might be mediated by stimulation of prostaglandin production in cortical astrocytes. Vasoactive intestinal peptide did not increase the production of either prostaglandin E₂ or F_2α. Conversely, forskolin concentration-dependently stimulated the production of both prostaglandins, an effect that was blocked by indomethacin. Indomethacin did not affect either vasoactive intestinal peptide- or forskolin-stimulated interleukin 6 production. To exclude the possibility that prostaglandins participate in interleukin 6 production induced by forskolin, we tested prostaglandins E₂ and F_2α. The former was completely ineffective in eliciting the cytokine production, whereas prostaglandin F_2α slightly increased interleukin 6 production only at the highest concentrations. 8-Bromo-cAMP and dibutyryl-cAMP stimulated interleukin 6 production to a lesser extent than vasoactive intestinal peptide and forskolin. In conclusion, we provide evidence that vasoactive intestinal peptide increases interleukin 6 production by astrocytes through the stimulation of the cAMP-protein kinase A pathway, an effect that is reproduced by cAMP analogues. In addition, we point out that prostaglandins are not involved in vasoactive intestinal peptide- and forskolin-mediated induction of interleukin 6 production in cultured astrocytes.     

Adrenergic-cholinergic interactions in left atria: interaction of carbachol with alpha- and beta-adrenoceptor agonists

The purpose of the present investigation was to determine the nature of the functional interaction of muscarinic agonists with cAMP-generating and cAMP-independent agonists in left atria. Negative inotropic responses of rabbit isolated left atrial strips to the muscarinic agonist carbachol were measured in the absence and presence of equi-active inotropic doses of the beta-adrenoceptor stimulant isoproterenol (Iso), the mixed alpha- and beta-adrenoceptor stimulant phenylephrine (PE) plus 1 microM timolol to block the beta-receptor mediated component of its response, and elevated extracellular Ca2+. Carbachol produced dose-dependent negative inotropic responses in left atrial strips, which were much greater than control in the presence of either Iso, or PE plus timolol. However, carbachol responses were of a similar magnitude to the control in the presence of elevated extracellular Ca2+. In the presence of timolol, PE had no significant effect on cAMP levels in left atrial strips, and inotropic responses to carbachol alone and in combination with PE plus timolol were accompanied by significant increases in cGMP levels but no change in cAMP levels. Carbachol attenuated Iso-induced increases in cAMP levels, but decreases in left atrial tension were proportionally greater than the decreases in cAMP levels produced by carbachol in the presence of Iso. These results suggest that the antiadrenergic effects of muscarinic receptor stimulation may occur by a different mechanism in left atria than has been previously reported in ventricular muscle. While the nature of this mechanism is unknown, it may involve antagonism by muscarinic agents of both alpha- and beta-adrenoceptor mediated increases in Ca2+ influx.     

Regulation of cyclic GMP levels in nerve tissue

In rat superior cervical ganglia the regulation of cyclic GMP (cGMP) formation does not involve muscarinic or adrenergic transmitters or receptors. Marked increases in cGMP content during preganglionic axonal stimulation by electric currents, elevated K+, or drugs that cause transmitter release are unaffected by muscarinic and adrenergic receptor blockade. However, the cGMP response does require Ca2+ and intact preganglionic axonal terminals. Two possibilities exist: either cGMP accumulates in the preganglionic nerves or a noncholinergic, nonadrenergic transmitter activates guanylate cyclase in postsynaptic structures. Sodium azide and nitroprusside cause cGMP accumulation in denervated ganglia, which indicates that postsynaptic structures are capable of forming cGMP. In pineal glands elevated [K+]o releases [3H]norepinephrine and causes cGMP accumulation, which suggests a relationship between the two responses and the possibility that cGMP accumulation is involved in autoinhibition of transmitter release. The finding that phentolamine, alpha-adrenergic receptor antagonists, prevent the cGMP response to K+ is compatible with this review. However, clonidine, an alpha-receptor agonist, depresses norepinephrine release but has no effect on pineal gland cGMP. Conversely, large increases in pineal gland cGMP produced by nitroprusside do not affect K+-evoked norepinephrine release. For these reasons it is not possible to relate cGMP to the auto-inhibition of [3H]norepinephrine release that is mediated by prejunctional alpha-adrenergic receptors.     

Vasoactive intestinal peptide receptor regulation of cAMP accumulation and glycogen hydrolysis in the human Ewing's sarcoma cell line WE-68

This study describes functional characteristics of receptors for vasoactive intestinal peptide (VIP) on human Ewing's sarcoma WE-68 cells. These characteristics include 125I-VIP binding capacity, cellular cAMP generation, glycogen hydrolysis, and pharmacological specificity. Binding studies with 125I-VIP showed specific, saturable, binding sites for VIP in WE-68 cells. Scatchard analysis revealed the presence of a single class of high-affinity binding sites that exhibited a dissociation constant (Kd) of 90 pM and a maximal binding capacity (Bmax) of 24 fmol/mg of protein. VIP and VIP-related peptides competed for 125I-VIP binding in the following order of potency: human (h) VIP greater than human peptide with N-terminal histidine and C-terminal methionine (PHM) greater than chicken secretin much greater than porcine secretin. Glucagon and the C-terminal fragments VIP[10-28] and VIP[16-28] and the VIP analogue (D-Phe2)VIP did not inhibit 125I-VIP binding. Addition of hVIP to WE-68 cells provoked marked stimulation of cAMP accumulation, hVIP stimulated increases in cAMP content were rapid, concentration-dependent, and potentiated by 3-isobutyl-l-methylxanthine (IBMX). Half-maximal stimulation (EC50) occurred at 150 nM hVIP. The ability of hVIP and analogues to stimulate cAMP generation paralleled their potencies in displacing 125I-VIP binding. (D-Phe2)VIP, VIP[10-28], VIP[16-28], and (p-Cl-D-Phe6, Leu17)VIP, a putative VIP receptor antagonist, affected neither basal cAMP levels nor hVIP-induced cAMP accumulation. WE-68 cell responses to hVIP were desensitized by prior exposure to hVIP. Desensitization to hVIP did not modify the cAMP response to beta-adrenergic stimulation, and beta-adrenergic agonist desensitization did not modify responses to hVIP. hVIP also induced a time- and concentration-dependent hydrolysis of 3H-glycogen newly formed from 3H-glucose in WE-68 cultures. hVIP maximally decreased 3H-glycogen content by 36% with an EC50 value of about 8 nM. The order of potency of structurally related peptides of hVIP for stimulation of glycogenolysis correlated with their order of potency for inhibition of 125I-VIP binding. IBMX potentiated the glycogenolytic action of hVIP and PHM. The simultaneous presence of the calcium channel antagonist verapamil or the calcium ionophore A 23187 did not influence the glycogenolytic and cAMP stimulatory effects of hVIP. Collectively, these data indicate that Ewing's sarcoma (WE-68) cells are endowed with genuine VIP receptors which are coupled to the formation of cAMP that probably serves a second messenger role in stimulating glycogen hydrolysis in these cells in response to VIP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vasoactive intestinal peptide receptor activity and specificity during enterocyte-like differentiation and retrodifferentiation of the human colonic cancerous subclone HT29-18

Commitment of HT29-18 cells to enterocyte-like differentiation by glucose removal is related to a decreased capacity to generate cAMP after treatment with vasoactive intestinal peptide (VIP), forskolin or sodium fluoride. In contrast, the potency of VIP (EC50 = 1.1 - 1.3 X 10(-10) M) and the pharmacological specificity of the VIP receptor (VIP greater than rh GRF 1-43 greater than PHI greater than secretin) are unchanged during differentiation and retrodifferentiation. These results indicate that disturbances in VIP receptor-post-receptor activity, involving cell surface VIP receptors, membrane and intracellular transducers of hormonal information, occur during enterocyte-like differentiation of the HT29-18 subclone.     

Transmembrane receptor cross-talk: concurrent VIP and alpha 1-adrenergic activation rapidly elevates pinealocyte cGMP greater than 100-fold

The transmembrane regulation of cGMP accumulation, which is poorly understood, was studied using isolated rat pinealocytes. It was found for the first time that VIP stimulates cGMP accumulation several-fold. This stimulation was amplified by phenylephrine acting via alpha 1-adrenoceptors, resulting in a greater than 100-fold increase in cGMP accumulation. These results raise the possibility that cGMP accumulation in other tissues might be regulated by VIP, and that the stimulating effects of VIP might be markedly amplified by catecholamine transmitters in these tissues. It is also possible that other pairs of receptors might control large changes in cGMP in the central nervous system through parallel mechanisms.     

Neuropeptidergic control of cyclic AMP accumulation in human thyroid cell

Somatostatin (SRIF), cholecystokinin (CCK), gastrin and substance P, as single agents, do not influence baseline cellular cAMP levels in human thyroid cultures. SRIF inhibits TSH-induced cAMP accumulation in human thyroid cell, while CCK, gastrin and substance P do not modify cAMP response to TSH. Vasoactive intestinal peptide (VIP) increases cellular cAMP levels in human thyroid cultures and its effect is additive to increases produced by norepinephrine (NE) and isoproterenol (ISO). Neither SRIF nor the other tested peptides influence adrenergic and VIP-ergic cAMP stimulation.     

Angiotensin II and phorbol ester enhance isoproterenol- and vasoactive intestinal peptide (VIP)-induced cyclic AMP accumulation in vascular smooth muscle cells

The importance of Ca2+ and cAMP in the regulation of cellular functions has been well demonstrated. We studied the effect of angiotensin II (AII), a potent Ca2+-mobilizing hormone, on cAMP accumulation induced by isoproterenol (ISO) and vasoactive intestinal peptide (VIP) in cultured vascular smooth muscle cells (VSMC). Although the addition of AII alone caused little increase of cAMP, it enhanced ISO- and VIP-induced cAMP accumulations in a dose-dependent manner. This enhancement was mimicked by tumor-promoting phorbol ester but not by Ca2+ ionophore. This observation suggested that AII enhanced agonist-induced cAMP accumulation through the activation of protein kinase C in VSMC.