The mahogany mouse mutation: further links between pigmentation, obesity and the immune system.

Completely different lines of experimentation have identified attractin, a protein that seems to have multiple roles in regulating physiological processes. It affects the balance between agonist and antagonist at receptors on melanocytes, modifies behaviour and basal metabolic rate, and mediates an interaction between activated T cells and macrophages. It may well be a target for development of drugs to treat obesity.

Effects of alpha-melanocyte-stimulating hormone on mitochondrial energy metabolism in rats of different age-groups.

Hypothalamic alpha-melanocyte-stimulating hormone (α-MSH) is a key catabolic mediator of energy homeostasis. Its anorexigenic and hypermetabolic effects show characteristic age-related alterations that may be part of the mechanism of middle-aged obesity and geriatric anorexia/cachexia seen in humans and other mammals. We aimed to investigate the role of α-MSH in mitochondrial energy metabolism during the course of aging in a rodent model. To determine the role of α-MSH in mitochondrial energy metabolism in muscle, we administered intracerebroventricular (ICV) infusions of α-MSH for 7-days to different age-groups of male Wistar rats. The activities of oxidative phosphorylation complexes I to V and citrate synthase were determined and compared to those of age-matched controls. We also quantified mitochondrial DNA (mtDNA) copy number and measured the expression of the master regulators of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPARγ). The peptide reduced weight gain in juvenile rats to one fifth of that of controls and increased the weight loss in older animals by about five fold. Mitochondrial DNA copy number inversely correlated with changes in body weight in controls, but not in α-MSH-treated animals. The strong increase in body weight in young rats was associated with a low mtDNA copy number and high PPARγ mRNA levels in controls. Expression of PGC-1α and PPARγ declined with age, whereas OXPHOS and citrate synthase enzyme activities were unchanged. In contrast, α-MSH treatment suppressed OXPHOS enzyme and citrate synthase activity. In conclusion, our results showed age-related differences in the metabolic effects of α-MSH. In addition, administration of α-MSH suppressed citrate synthase and OXPHOS activities independent of age. These findings suggest that α-MSH exposure may inhibit mitochondrial biogenesis.

alpha-MSH production, receptors, and influence on neopterin in a human monocyte/macrophage cell line.

alpha-Melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide derived from pro-opiomelanocortin, has potent antiinflammatory activity in laboratory animals. alpha-MSH inhibits nitric oxide production by murine macrophages, an influence believed to reflect activation of an autocrine circuit in these cells, one that is based on production and release of alpha-MSH and subsequent stimulation of melanocortin receptors. We found that THP-1 cells, human monocytic cells, produced alpha-MSH; this production was increased by interleukin-6, tumor necrosis factor a, or concanavalin A. These cells also expressed the gene for the human alpha-MSH receptor MC1. Unlike murine macrophages, THP-1 cells produced little nitrite in response to interferon-gamma (IFN-gamma) and lipopolysaccharide, and a-MSH inhibited this production only slightly. However, production of neopterin, a presumed primate homologue of nitric oxide in lower animals, was increased in THP-1 cells stimulated with INF-gamma plus TNF-alpha and alpha-MSH significantly inhibited this production. The evidence indicates that an autocrine regulatory circuit based on alpha-MSH occurs in human monocyte/macrophages much as in murine macrophages. alpha-MSH-induced modulation of specific inflammatory mediators/cytotoxic agents appears to differ depending on the importance of the mediators in the myelomonocytic cells of different species.

Ultraviolet B and melanocyte-stimulating hormone (MSH) stimulate mRNA production for alpha MSH receptors and proopiomelanocortin-derived peptides in mouse melanoma cells and transformed keratinocytes.

Cell lines of cutaneous origin, namely melanocytes and keratinocytes, were previously demonstrated to exhibit functional melanocyte-stimulating hormone (MSH) receptors that are up-regulated by ultraviolet (UV) radiation and by MSH itself. In this study, it is demonstrated that UVB irradiation, exposure to MSH, or exposure to N6,O2-dibutyryl cyclic adenosine monophosphate stimulates production of mRNAs for both alpha MSH receptors and proopiomelanocortin in cultured mouse Cloudman S91 melanoma cells, and that UVB stimulates production and release of MSH and adrenocorticotropin peptides in both melanoma cells and transformed PAM 212 mouse keratinocytes. The results add support to the hypothesis that the effects of UVB on cutaneous melanogenesis are mediated through a series of coordinated events in which MSH receptors and proopiomelanocortin-derived peptides play a central role.

Inhibition by melittin and fluphenazine of melanotropin receptor function and adenylate cyclase in M2R melanoma cell membranes.

Melanotropin (MSH) receptor activity in the M2R mouse melanoma cell line is tightly controlled by calcium by an unknown mechanism. The possibility that calcium regulation is mediated by calmodulin or a calmodulin-related calcium binding protein has been addressed in this report by studying the effects of two known calmodulin antagonists, fluphenazine and melittin, on MSH receptor function. Stimulation of adenylate cyclase (AC) in M2R plasma membranes by beta MSH was strongly inhibited by both antagonists. The concentrations of fluphenazine and melittin yielding half-maximal inhibition (IC50) of AC were 16 microM and 2.4 microM, respectively. Both fluphenazine and melittin also inhibit prostaglandin E1-, GTP gamma S, and forskolin-stimulated AC activity, as well as that of unstimulated enzyme, although inhibition is shown to occur at significantly higher concentrations of antagonist. We have shown that the calcium-dependent rate-limiting step in MSH stimulation of adenylate cyclase, that of hormone binding, is strongly inhibited by these antagonists at concentrations identical to, if not lower than, those required for the inhibition of AC activity (fluphenazine-IC50, 14 microM; melittin-IC50, 0.7 microM). The actions of these antagonists, furthermore, appear to be calcium insensitive, as melittin affects the stability of both the high affinity (calcium containing) and low affinity (calcium depleted) receptor-MSH complexes. The sensitivity of the MSH receptor to inhibition by calmodulin antagonists resembles that described for purified calmodulin-sensitive enzyme systems, which suggests a possible role for calmodulin in MSH receptor function. Among peptide hormone receptors, this effect by calmodulin antagonists appears to be unique for the MSH receptor.

Novel splice variants of type I pituitary adenylate cyclase-activating polypeptide receptor in frog exhibit altered adenylate cyclase stimulation and differential relative abundance.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts its various effects through activation of two types of G protein-coupled receptors, a receptor with high affinity for PACAP named PAC1-R and two receptors exhibiting similar affinity for both PACAP and vasoactive intestinal polypeptide named VPAC1-R and VPAC2-R. Here, we report the characterization of PAC1-R and novel splice variants in the frog Rana ridibunda. The frog PAC1-R has 78% homology with human PAC1-R and is highly expressed in the central nervous system. Two splice variants of the frog receptor that display additional amino acid cassettes in the third intracellular loop were characterized. PAC1-R25 carries a 25-amino acid insertion that matches the hop cassette of the mammalian receptor, whereas PAC1-R41 carries a cassette with no homology to any mammalian PAC1-R variant. A third splice variant of PAC1-R, exhibiting a completely different intracellular C-terminal domain, named PAC1-Rmc has also been identified. Determination of cAMP formation in cells transfected with the cloned receptors showed that PACAP activated PAC1-R, PAC1-R25, and PAC1-R41 with similar potency. In contrast, PACAP failed to stimulate adenylate cyclase in cells transfected with PAC1-Rmc. Fusion of PAC1-R or PAC1-Rmc with the green fluorescent protein revealed that both receptors are expressed and targeted to the plasma membrane in transfected cells. The different PAC1-R variants are highly expressed in the frog brain and spinal cord and to a lesser extent in peripheral tissues, where only certain isoforms could be detected. The present data indicate that in frog, PACAP may act through different PAC1-R splice variants that differ in their G(s) protein coupling and their abundance in various tissues.

Prostaglandin E2 is a positive regulator of adrenocorticotropin receptors, 3 beta-hydroxysteroid dehydrogenase, and 17 alpha-hydroxylase expression in bovine adrenocortical cells.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of cAMP. Herein we examine the effects of prostaglandin E2 (PGE2) on the differentiated functions of bovine adrenocortical (BAC) cells in primary culture. PGE2 (10 microM) treatment for 3 h stimulated steroidogenesis and cAMP production by over 100-fold. In addition, the cAMP antagonist Rp-cAMP (1 mM) inhibited PGE2 stimulation of steroidogenesis by 60%. This observation suggests that the cAMP second messenger system is responsible for much of the PGE2-activated steroid hormone synthesis. Chronic treatment of BAC cells with PGE2 caused induction of 3 beta-hydroxysteroid dehydrogenase and steroid 17 alpha-hydroxylase cytochrome P-450 expression as determined by the examination of enzyme activity, enzyme levels by immunoblotting, and specific messenger RNA (mRNA) levels by Northern analysis. The positive effects of PGE2 on expression of 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase cytochrome P-450 were similar to the effects seen after ACTH treatment of BAC cells. In addition, treatment of BAC cells with PGE2 for 3 days caused a 3-fold induction of ACTH receptors as determined by increased cell binding of [125I]ACTH. Finally, we determined that BAC cells produced PGE2 and that the level of synthesis increased 10-fold after treatment with the hormone angiotensin II. Taken together these data indicate that PGE2 is a positive regulator of BAC cell differentiation acting on ACTH receptors, steroid metabolizing enzymes, and steroidogenesis. The ability of BAC cells to produce PGE2 leaves open the possibility for paracrine and autocrine regulation within the adrenal.

Stimulation of the receptor for melanocyte-stimulating hormone by retinoic acid.

Treatment of Cloudman S91 melanoma cells with retinoic acid (RA) inhibits MSH-induced tyrosinase activity and melanin formation [(1990) J. Invest. Dermatol. 94, 461-464]. We report here, however, that in spite of inhibiting MSH-induced pigmentation, RA treatment caused a marked increase in MSH binding capacity for both cell surface and internal MSH binding sites. The stimulation was dose- and time-dependent and reversible, with half-maximal effects seen at 2 microM RA. Stimulation of MSH binding was seen as early as 3 h after exposure of cells to RA. Cell surface and internal binding activity increased in concert. Scatchard analysis indicated that increased MSH binding resulted from a 3-4-fold increase in the number of sites with no significant difference in their affinity for MSH. It appears that in suppressing MSH-induced melanogenesis, RA elicited a compensatory up-regulation of the MSH receptor system.

Dexamethasone-dependent expression of beta 1-24 corticotropin stimulated adenylate cyclase during adipose conversion of 3T3-F442A cells.

When 3T3-F442A preadipocytes were grown in culture media supplemented with corticosteroid poor fetal calf serum and insulin they differentiated into adipocytes. Glycerophosphate dehydrogenase, a marker of terminal differentiation, developed a 600-fold increase of activity whereas the adenylate cyclase system remained unresponsive to the synthetic ACTH(1-24) analog. In contrast, 3T3-F442A adipocytes, differentiated in the presence of dexamethasone, exhibited an adenylate cyclase activity which was stimulated 4-fold by ACTH(1-24). The stimulation of the adenylate cyclase activity by GTP gamma S remained unchanged (about 20-25-fold) suggesting that the G regulatory coupling protein was not functionally modified by dexamethasone. Binding studies with 125I-ACTH revealed that specific cellular binding could be evidenced in dexamethasone-treated cells while control adipocytes did not exhibit any specific binding of 125I-ACTH. These findings lend support to the hypothesis that the setting off of this ACTH responsiveness in 3T3-F442A cells is regulated by dexamethasone after cells are committed to adipose differentiation.

Human immune cells express ppMCH mRNA and functional MCHR1 receptor.

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT-PCR analysis revealed a low expression of prepro-MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3-stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions.

Localization and characterization of PACAP receptors in the rat cerebellum during development: evidence for a stimulatory effect of PACAP on immature cerebellar granule cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are abundant in the brain and particularly in the cerebellum of adult rats. In contrast, the occurrence of PACAP binding sites has not been investigated during ontogenesis. The aim of the present study was to determine the distribution and biochemical characteristics of PACAP binding sites in the rat cerebellum during postnatal development, and to examine the effect of PACAP on immature cerebellar granule cells. Autoradiographic studies revealed that PACAP binding sites are transiently expressed in a germinative matrix of the cerebellar cortex, the external granule cell layer, and in the medulla, from postnatal days 8 to 25. A population of PACAP binding sites persisted in the internal granule cell layer in the mature cerebellum. Emulsion-coated cytoautoradiography, performed on cultured immature granule cells from eight-day-old rat cerebellum, demonstrated that transient PACAP binding sites are expressed by cerebellar immature granule cells. Biochemical characterization of binding revealed the occurrence of two classes of PACAP recognition sites exhibiting, respectively, high (Kd = 0.39 +/- 0.08 nM) and low (Kd = 21.2 +/- 9.4 nM) affinity for PACAP27. The two naturally occurring forms PACAP38 and PACAP27 were equipotent in competing for [125I]PACAP27 binding. In contrast, the [Des-His1]PACAP38 analog was eight times less efficient and vasoactive intestinal polypeptide only induced weak displacement of the binding. Exposure of cultured immature granule cells to PACAP27 resulted in a dose-dependent stimulation of the production of cAMP, indicating that PACAP binding sites represent authentic receptors positively coupled to adenylate cyclase. These results show that PACAP receptors are actively expressed in the cerebellum of rats during postnatal development. The presence of functional PACAP receptors in the external granule cell layer suggests that PACAP may play a role in the control of proliferation and/or differentiation of granule cells.

Facilitation of noradrenaline release from sympathetic nerves through activation of ACTH receptors, beta-adrenoceptors and angiotensin II receptors.

1. In rabbit pulmonary artery and left atrial strips previously incubated with [3H]-noradrenaline, the active fragment of adrenocorticotropic hormone (ACTH 1-24, 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity when a cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM) was present, but not in the absence of these drugs. In rabbit pulmonary artery a facilitatory effect of ACTH 1-24 (0.1 microM) was also observed when only cocaine (30 microM) was present. 2. ACTH 1-24 (0.1 microM) did not affect the S-I outflow of radioactivity from rat atria, rat pulmonary artery or guinea-pig pulmonary artery, either in the presence or in the absence of the cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM). These results suggest that the presence of facilitatory prejunctional ACTH receptors may be restricted to rabbit sympathetic nerve endings. 3. Angiotensin II (0.01 microM), but not isoprenaline (0.1 microM) or ACTH 1-24 (0.1 microM), significantly enhanced the S-I outflow of radioactivity from rabbit pulmonary artery. In the presence of phentolamine (1 microM) to block inhibitory alpha 2-adrenoceptors, the facilitatory effect of angiotensin II (0.01 microM) was significantly enhanced, and a significant facilitatory effect of isoprenaline (0.1 microM) and of ACTH 1-24 (0.1 microM) was then revealed. These results suggest that feedback inhibition of noradrenaline release, mediated through the prejunctional alpha 2-adrenoceptor mechanism, buffers increases in noradrenaline release during activation of facilitatory prejunctional receptors. 4. In rabbit pulmonary artery, two concentrations of 8-Br-cyclic AMP, (270 or 540 microM), enhanced the S-I outflow of radioactivity in the presence of phentolamine (1 microM) to a similar extent. In the presence of 8-Br-cyclic AMP (270 microM) and phentolamine, the facilitatory effects of isoprenaline (0.1 microM) and of ACTH,24 (0.1 microM) were blocked, whereas that of angiotensin II (0.01 microM) was not changed. These results suggest that both prejunctional beta-adrenoceptors and ACTH receptors enhance noradrenaline release by generating cyclic AMP. The mechanism by which angiotensin II facilitates noradrenaline release is probably independent of the cyclic AMP second messenger pathway.

Involvement of PACAP receptor in primary afferent fibre-evoked responses of ventral roots in the neonatal rat spinal cord.

The role of PACAP receptor in nociceptive transmission was investigated in vitro using maxadilan, a PACAP receptor selective agonist and max.d.4, a PACAP receptor selective antagonist. Potentials, from a ventral root (L3 - L5) of an isolated spinal cord preparation or a spinal cord - saphenous nerve - skin preparation from 0 - 3-day-old rats, were recorded extracellularly. In the isolated spinal cord preparation, single shock stimulation of a dorsal root at C-fibre strength induced a slow depolarizing response lasting about 30 s (slow ventral root potential; slow VRP) in the ipsilateral ventral root of the same segment. Bath-application of max. d.4 (0.01 - 3 microM) inhibited the slow VRP in a concentration-dependent manner. In the spinal cord - saphenous nerve - skin preparation, application of capsaicin (0.1 microM) to the skin evoked a depolarization of the ventral root. This response was also depressed by max.d.4 (1 microM). Application of maxadilan evoked a long-lasting depolarization in a concentration-dependent manner in the spinal cord preparation. In the presence of max.d.4 (0.3 microM), the concentration response curve of maxadilan was shifted to the right. Reverse transcription-polymerase chain reaction (RT - PCR) experiments demonstrated the existence of PACAP receptor and VPAC(2) receptor in the neonatal rat spinal cord and [(125)I]-PACAP27 binding was displaced almost completely by maxadilan and max.d.4, but not by vasoactive intestinal peptide (VIP). These data indicate that PACAP receptor is dominantly distributed in the neonatal rat spinal cord. The present study suggests that PACAP receptor may play an excitatory role in nociceptive transmission in the neonatal rat spinal cord.

The human melanocyte stimulating hormone receptor has evolved to become "super-sensitive" to melanocortin peptides.

Melanocyte-stimulating hormone (MSH) stimulates pigmentation in mammals by activating specific cell surface MSH receptors (MC1-Rs) on melanocytes. MC1-Rs on normal human melanocytes have been difficult to detect and characterise. The pharmacological characterisation of a cloned human MC1-R (hMC1-R) is reported here, and directly compared with that of a cloned mouse MC1-R (mMC1-R). The human and mouse MC1-Rs are equally sensitive (EC50 = 1-2 pM) to the super potent analogue of alpha-MSH, NDP-MSH. In contrast with the mMC1-R, the hMC1-R is also very sensitive to alpha-MSH (EC50 = 2 pM), ACTH (EC50 = 8 pM), and Lys gamma 3-MSH (EC50 < 10(-10) M). This suggests that in man, in contrast with rodents, both ACTH and Lys gamma 3-MSH may have physiological roles in pigmentation.

Studies on the mechanism of corticotrophin-mediated desensitization of corticosterone secretion by rat adrenocortical cells.

Long-term exposure of the adrenocortical cells in vivo or in vitro to high concentrations of ACTH results in a diminution of the responsiveness of these cells to a subsequent stimulation of corticosterone release by ACTH. Conflicting studies have been published on the mechanism of this 'desensitization' phenomenon. Dispersed adrenocortical cells prepared from the hypertrophic/hyperplastic adrenal glands of rats bearing the ACTH/PRL-secreting rat pituitary tumour 7315a showed an increased basal release of corticosterone, but had lost their ability to respond further to ACTH. However, corticosterone release in response to dibutyryl cyclic AMP (dbcAMP), cholera toxin and forskolin remained intact. Pretreatment of normal rats for 3, 9 and 21 days with 50 micrograms/rat/day of a long-acting ACTH depot preparation induced a dose-dependent increase in basal corticosterone release by the adrenocortical cells prepared from these animals and a dose-dependent decrease in the sensitivity to ACTH. However, the responsiveness of the adrenocortical cells prepared from the adrenal glands of control and ACTH pretreatment rats to dbcAMP, cholera toxin and forskolin was similar. In addition, pretreatment with ACTH in vivo did not affect the sensitivity of the adrenocortical cells in vitro to calmodulin inhibition by haloperidol and 11 beta-hydroxylase inhibition by etomidate. It is concluded that long-term exposure of the adrenal gland to high concentrations of ACTH in vitro results in an excessive activation of corticosterone release by the adrenocortical cells in vitro, which is accompanied by a loss of sensitivity to ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

(N-stearyl, norleucine17)VIPhybrid is a broad spectrum vasoactive intestinal peptide receptor antagonist.

The effects of a (N-stearyl, Norleucine17) vasoactive intestinal peptide hybrid ((SN)VIPhybrid) on cells stably transfected with VPAC,, VPAC2, or PAC1 receptors were investigated. (SN)VIPhybrid inhibited specific 125I-VIP binding to membranes derived from CHO cells transfected with VPAC, or VPAC2 receptors with high affinity (IC50 = 30 and 50 nM). (SN)VIPhyb inhibited specific 125I-PACAP-27 binding to membranes derived from NIH/3T3 cells transfected with PAC1 receptors with high affinity (IC50 = 65 nM). PACAP-27 caused cAMP elevation in NIH/3T3 cells transfected with PAC1 receptors and the increase cAMP caused by pituitary adenylated cyclase (PACAP) was inhibited by (SN)VIPhyb. Also, the increase in cAMP caused by VIP using CHO cells transfected with VPAC1 or VPAC2 receptors was antagonized by (SN)VIPhyb. These results indicate that (SN)VIPhyb is an antagonist for VPAC1, VPAC2, and PAC1 receptors.

Characterization of vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptors in chick cerebral cortex.

In this study receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) were characterized in chick cerebral cortex by an in vitro binding technique, using 125I-labeled VIP ([125I]-VIP) as a ligand. The specific binding of [125I]-VIP to chick cerebral cortical membranes was found to be rapid, stable, saturable, reversible, and of high affinity. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding sites with high affinity (Kd = 0.21 nM) and low capacity (Bmax = 19.5 fmol/mg protein). The relative rank order of potency of the tested peptides to inhibit [125I]-VIP binding to chick cerebrum was VIP (chicken) > or = VIP (mammalian) > or = PACAP27 > or = PACAP38 >> VIP6-28 (mammalian) > PHI (porcine) >> neurotensin6-11-chicken VIP7-28 > neurotensin6-11-mammalian VIP7-28 >>> VIP16-28 (chicken; inactive) approximately secretin (inactive). About 60% of [125I]-VIP-binding sites in chick cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. It has been concluded that the cerebral cortex of chick, in addition to PAC1 receptors, contains a population of VPAC-type receptors.

Facilitatory effects of pituitary adenylate cyclase activating polypeptide (PACAP) on neurons in the magnocellular portion of the rat hypothalamic paraventricular nucleus (PVN) in vitro.

To establish the role of pituitary adenylate cyclase activating polypeptide (PACAP), a member of vasoactive intestinal polypeptide (VIP) family, as a neurotransmitter/neuromodulator in the central nervous system, the effects of PACAP38, PACAP27 and VIP on the single neuron activity in the magnocellular portion of the hypothalamic paraventricular nucleus (mg.PVN) were examined in rat brain slice preparations. Extracellular recordings were made from 111 neurons in the mg.PVN, which fired spontaneously at an average rate of 1.85 +/- 0.2 spikes/s (mean +/- SEM). PACAP38 and PACAP27 were applied to 78 and 33 of the 111 neurons, respectively. Perfusion with PACAP38 in doses between 10 nM and 1 microM increased the firing rate of 56 (71.8%) of the 78 neurons in a dose-dependent manner. The threshold dose of PACAP38 to excite the neurons seemed to lie below 10 nM. The application of PACAP27 (1 microM) also increased the firing rate of 19 (57.6%) of the 33 neurons tested. Eleven (52.4%) of 21 neurons which were excited by PACAP38 also showed excitation following perfusion with VIP (1 microM). The responses to PACAP38 in 12 of 20 neurons and those to VIP in 6 of 9 neurons tested were still observed in a low Ca2+ and high Mg2+ medium. Although there was no difference in the mean latency between the responses to PACAP38 (1 microM) and VIP (1 microM) (2.1 +/- 0.1 min and 2.4 +/- 0.4 min, respectively), the duration of the PACAP38-induced excitation (59.0 +/- 5.0 min) was much longer than that of the VIP-induced one (18.8 +/- 3.1 min). The PACAP38 (30 nM)-induced excitation was reversibly blocked by a concurrent application of PACAP5-38 (300 nM), a PACAP receptor antagonist. While a selective VIP receptor antagonist, [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP (1 microM), did not affect the excitatory responses to PACAP38 (300 nM), it completely blocked the VIP (1 microM)-induced excitation. These findings suggest that PACAP may therefore modulate the secretion of the pituitary hormones at least partly by its action on the neurons in the mg.PVN through the activation of specific receptors for PACAP.

Receptors and transcriptional factors involved in the anti-inflammatory activity of VIP and PACAP.

VIP and PACAP modulate the function of inflammatory cells through specific receptors. VIP/PACAP inhibit the production of TNF alpha, IL-6, IL-12, and nitric oxide (NO), and stimulate IL-10 in peritoneal macrophages and Raw 264.7 cells. Here we report on the specific VIP/PACAP receptors, transduction pathways, and transcriptional factors involved in the regulation of these macrophage factors by VIP and PACAP. Both neuropeptides inhibit IL-6 production mainly through PAC1 binding, PKC activation, and the subsequent shedding of the LPS receptor CD14 in macrophages. However, the effects on TNF alpha, IL-10, IL-12, and NO are mostly mediated through the constitutively expressed VPAC1 receptor, although the inducible expressed VPAC2 may also participate. VIP/PACAP binding to VPAC1 induces both a cAMP-dependent and a cAMP-independent pathways that regulate cytokine and NO production at the transcriptional level. VIP/PACAP inhibit TNF alpha through reduction in NFkB binding and changes in the composition of CRE-binding complexes; they inhibit IL-12 through reduction in NFkB binding and changes in the composition of the ets-2 complexes. VIP/PACAP inhibit iNOS expression through reduction in NFkB and IRF-1 binding, and augment IL-10 by increasing CREB-binding. Whereas the inhibition of IRF-1 and CRE-binding complexes seems to be mediated through the cAMP-dependent pathway, VIP/PACAP inhibition of NFkB nuclear translocation is mediated through a reduction in IkB alpha degradation mediated by the cAMP-independent pathway. This study provides new evidence for the understanding of the molecular mechanism by means of which VIP and PACAP attenuate the inflammatory response.

The long-acting vasoactive intestinal polypeptide agonist RO 25-1553 is highly selective of the VIP2 receptor subclass.

RO 25-1553 is a synthetic VIP analogue that induced a long-lasting relaxation of tracheal and bronchial smooth muscles as well as a reduction of edema and eosinophilic mobilization during pulmonary anaphylaxis. In the present study, we tested in vitro the capacity of RO 25-1553 to occupy the different VIP/PACAP receptor subclasses and to stimulate adenylate cyclase activity. The cellular models tested expressed one single receptor subtype: Chinese hamster ovary (CHO) cells transfected with the rat recombinant PACAP I, rat VIP1, and human VIP2 receptors; SUP T1 cells expressing the human VIP2 and HCT 15 and LoVo cells expressing the human VIP1 receptor. RO 25-1553 was threefold more potent than VIP on the human VIP2 receptor, 100- and 600-fold less potent than VIP on the rat and human VIP1 receptors, respectively, and 10-fold less potent than VIP and 3000-fold less potent than PACAP on the PACAP I receptor. RO 25-1553 was a full agonist on the VIP2, the PACAP I, and the rat recombinant VIP1 receptor but a partial agonist only on the human VIP1 receptor. Thus, RO 25-1553 is a highly selective agonist ligand for the VIP2 receptor subclass.