PACAP stimulates pancreatic exocrine secretion via the vagal cholinergic nerves in sheep

The present study evaluates the possible role of the vagus nerves in mediating the stimulatory effect of PACAP-27, PACAP-38 and VIP on the exocrine pancreas, especially on enzyme secretion which is atropine sensitive in sheep. The animals were equipped with two cannulae into the common bile duct, a duodenal cannula, and a ruminal cannula under anesthesia. The bilateral cervical vagus nerves were coiled with a cooling device. In conscious animals, the peptides were infused intravenously for 10 min at 10 pmol kg(-1)min(-1) in phase II of the duodenal migrating motor complexes and the same peptide infusion was repeated in the reversible cooling blockade of the vagus nerves. Increment in fluid secretion was not significantly altered by the vagal blockade in all the peptide infusions, while increment in bicarbonate ion by only PACAP-27 was inhibited by the vagal blockade. Increments in protein and amylase output decreased significantly to 32.0+/-5.0 and 23.2+/-2.6% in PACAP27, and to 26.1+/-7.7 and 20.8+/-6.4% in PACAP-38 in the vagal blockade, but the increments by VIP did not decrease. These results demonstrate that circulating PACAP stimulates pancreatic enzyme secretion via the vagal cholinergic preganglionic neurons in sheep, suggesting the central action of PACAP.     

Pituitary adenylate cyclase activating polypeptide (PACAP) is localized in human dermal neurons and causes histamine release from skin mast cells

OBJECTIVE AND DESIGN: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide homologous with vasoactive intestinal polypeptide (VIP) which is known to induce histamine release in human skin mast cells. PACAP has not been detected in human skin. The purposes of the study were to investigate the occurrence of PACAP in human skin and to evaluate the histamine releasing activity of the two common pro-PACAP products, PACAP-27 and PACAP-38. MATERIAL: Fourteen human surgical skin samples were obtained. PACAP and VIP were visualized by immunohistochemistry. A microdialysis technique was used to measure histamine release in intact skin samples following intradermal injections of the peptides. RESULTS: PACAP and VIP were localized in dermal nerves in connection with sweat glands. Intradermal injection of 3 or 10 microm PACAP significantly released histamine. Kinetics of histamine release showed peak release 2-4 min after skin challenge. Ten microm of PACAP-27, VIP and somatostatin caused histamine release with similar efficacy, whereas PACAP-38 was less effective. Substance P was twice as efficient as PACAP-27, whereas calcitonin gene-related peptide did not release histamine. CONCLUSIONS: PACAP is found in human skin and is capable of releasing histamine from skin mast cells.     

Chemoprevention of head and neck cancer

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of exocrine and endocrine pancreas was investigated in conscious sheep. Intravenous infusions of PACAP-27 and PACAP-38 (1, 3, and 10 pmol/kg/min) for 10 min during phase II of the duodenal migrating myoelectric complex accelerated pancreatic protein and amylase outputs dose-dependently. The responses in enzyme secretion to both PACAPs at the highest doses were inhibited significantly by atropine infusion (14.4 nmol/kg/min). Vasoactive intestinal polypeptide (VIP) at 3 pmol/kg/min significantly accelerated protein but not amylase outputs, although the response to the highest dose was not significantly influenced by atropine. PACAP-27 and VIP increased pancreatic juice flow and bicarbonate output dose-dependently; however, the responses to the highest dose were not altered significantly by atropine. On the other hand, intravenous injection of PACAP-38 (100 pmol/kg) did not influence basal plasma concentration of insulin, glucagon, and glucose. Moreover, PACAP-38 (1-100 pmol/kg) altered neither pancreatic endocrine response to intravenous infusion of glucose (20 mumol/kg/min) not that to n-butyric acid (33 mumol/kg/min). These results suggest that PACAP contributes to the regulation of exocrine secretion of the ovine pancreas but not to endocrine secretion. PACAP appears to accelerate pancreatic enzyme secretion mostly via the cholinergic nerves.     

Histomorphometric, physical, and mechanical effects of spaceflight and insulin-like growth factor-I on rat long bones

Pituitary adenylate cyclase activating peptide and vasoactive intestinal peptide belong to the same neuropeptide family. Both peptides are present in nerve fibers in the gastric wall and are thought to be involved in the regulation of inflammatory processes. Experimental ulcers were induced in the rat gastric mucosa by local application of acetic acid. During the healing process we examined the PACAP and VIP innervation by means of immunohistochemistry and in situ hybridization. The ulcer area was examined from day 1 to day 15 after ulcer induction. There was a marked depletion of PACAP in nerve fibers at the ulcer margin from day 1 and onwards. On day 10 and day 15, PACAP-immunoreactive nerve fibers could again be visualized at the ulcer margin. In contrast, VIP immunoreactive nerve fibers were present at the ulcer margin at all time points studied. From day 10 following ulcer induction PACAP- and VIP- immunoreactive nerve fibers were increased in frequency in the smooth muscle beneath the ulcer. An upregulation of VIP and PACAP mRNA was also demonstrated in the myenteric ganglia adjacent to ulcer. The present results indicate that neuronal PACAP and VIP react differently to the inflammation at the ulcer margin but similarly in the smooth muscle during the ulcer healing.     

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.     

Stable expression of the recombinant human VIP1 receptor in clonal Chinese hamster ovary cells: pharmacological, functional and molecular properties

We stably transfected Chinese hamster ovary (CHO) cells with the recombinant human vasoactive intestinal peptide (VIP)1 receptor. A clone referred to as Clone 15 was isolated and studied for receptor properties. The following data were obtained: (1) one class of binding site was identified by Scatchard analysis of [125I]VIP binding to cell membranes with a Kd of 0.41 nM and a Bmax of 1.62 pmol/mg protein; (2) the constant Ki for the inhibition of [125I]VIP binding by VIP and related peptides was: VIP (0.9 nM) = pituitary adenylate cyclase-activating peptide (PACAP)-27 (1.3 nM) < PACAP-38 (6.8 nM) < helodermin (46.0 nM) < human growth hormone-releasing factor (GRF) (0.6 microM) < peptide histidine methionineamide (2.0 microM) < secretin (> 10 microM); (3) cross-linking experiments using [125I]VIP identified a single M(r) 67000 recombinant receptor; (4) VIP stimulated cAMP production in Clone 15 cells with an EC50 of 0.20 nM; (5) some previously described VIP receptor antagonists including [4-Cl-D-Phe6, Leu17]VIP, [Ac-Tyr1,D-Phe2]GRF-(1-29) amide and VIP-(10-28) inhibited [125I]VIP binding with a Ki of 0.7, 1.6 and 2.5 microM, respectively. They failed to stimulate cAMP production in Clone 15 cells and inhibited, at least partially, the VIP (0.3 nM)-evoked cAMP production; (6) exposure of Clone 15 cells to 10 nM VIP for 24 h resulted in a sharp decrease in Bmax in Clone 15 cells (0.43 vs. 1.62 pmol/mg protein in control cells) and in the potency and efficacy of VIP in stimulating cAMP. Moreover, immunofluorescence studies using confocal microscopy indicated that the receptor was internalized and sequestered in vesicular structures within the cells. It is concluded that Clone 15 cells provide a valuable tool to further characterize various functional and pharmacological aspects of the human VIP1 receptor.     

Heart rate variability: significance and clinic application

In vitro effects of two bioactive forms of pituitary adenylate cyclase activating polypeptide (PACAP): PACAP-38 and PACAP-27 were studied on rabbit vascular and non-vascular smooth muscle. Segments of the ovarian artery and muscle strips from the fallopian tube were used. Two series of experiments were performed on vessels: the dose-response relationship of PACAP-38 (10(-10)-10(-7) M) was established on noradrenaline- (NA, 10(-6) M) contracted vessels. In the other set of experiments the contractile effect of 10(-8)-10(-4) M NA added cumulatively, was studied on arterial segments incubated with PACAP-38 (10(-7) M), PACAP-27 (10(-7) M) or VIP (10(-7) M). The effect of PACAP-38, PACAP-27 and VIP (10(-10)-10(-6) M) was investigated on spontaneously contracting smooth muscle of the fallopian tube. Longitudinally as well as transversally cut specimens were investigated. PACAP-38 produced a significant dose-related relaxation on the NA-precontracted vessels. However, pre-incubation of the vessels with 10(-7) M PACAP-38, PACAP-27 and vaso active intestinal polypeptide (VIP) did not induce a general rightward shift of the NA concentration-response curves, although a tendency to inhibition in the low-dose interval was observed. The peptides caused a significant, dose-dependent inhibition of both frequency and amplitude on the fallopian tube smooth muscle activity. The effects of the three peptides on longitudinally as well as transversally cut specimens were alike.     

Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland

Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.     

Regional differences in cardiac effects of pituitary adenylate cyclase-activating polypeptide-27 in the isolated dog heart

Recent observations indicate that several neuropeptides may be involved in the regulation of cardiac function, but the effects of these peptides on the atrium are not always the same as those on the ventricle. To compare the effect of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 on the atrium with that on the ventricle, we investigated the effects of PACAP-27 on the sinus rate and atrial and ventricular contractility in isolated, blood-perfused dog heart preparations. PACAP-27 (0.01-0.3 nmol) caused transient positive followed by negative chronotropic and inotropic responses in a dose-dependent manner in the isolated right atrium, whereas it caused only a dose-dependent positive inotropic response in the left ventricle. After atropine treatment, PACAP-27 caused only positive cardiac responses in isolated atria. The order of the increase in response to PACAP-27 was atrial contractile force > sinus rate > or = ventricular contractile force. Tetrodotoxin blocked the negative chronotropic and inotropic responses to PACAP-27 in isolated atria. Propranolol did not affect the positive response. PACAP-(6-27), a type I PACAP receptor antagonist, attenuated the positive responses similarly in the atropine-treated right atrium and the left ventricle. Thus, we demonstrated that (1) PACAP-27 caused negative cardiac effects in the atrium and sinoatrial node by activation of intracardiac parasympathetic nerves, but had no negative effect on the ventricle; (2) PACAP-27 had positive effects in the atrium, sinoatrial node and ventricle mediated by type I PACAP receptors, but PACAP-27 was more effective in the atrium and sinoatrial node than in the ventricle of the dog heart.     

Overexpression of glucagon-like peptide-1 receptor in an insulin-secreting cell line enhances glucose responsiveness

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the VIP (vasoactive intestinal polypeptide) family of peptides, has been demonstrated in neurons of the sensory system. PACAP expression of these neurons is sensitive to nerve damages such as nerve crush or axotomy. In the present study, PACAP expression in the mesencephalic trigeminal nucleus of the rat was examined after transsection of the main trunk of the masseteric nerve. The primary sensory neurons of the nucleus are considered to have purely proprioceptive functions. By quantitative in situ hybridization using a PACAP [35S]cRNA probe, an increase in PACAP mRNA was observed on the side ipsilateral to transsection already after 3 h and the expression reached a peak 24 h after surgery after which the levels gradually decreased during the next 14 days. A low and constant expression of PACAP mRNA could be seen on the side contralateral to transsection. PACAP immunoreactivity was demonstrated on the ipsilateral side after 18 h, using a specific monoclonal PACAP antibody. Co-existence of PACAP with NPY and galanin was demonstrated 7 days after transsection. Analysis of the masseteric nerve by radioimmunoassay on transsected and normal nerve stumps revealed an increase of PACAP-38 immunoreactivity in the nerve proximal to the transsection compared to the normal side (15.3 vs. 6.1 pmol/g wt). The results suggest that PACAP has a role in the early phase of adaptation to nerve injury in the proprioceptive neurons.     

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP-27, but not PACAP-38) degradation by the neutral endopeptidase EC 3.4.24.11

VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase-activating polypeptide), which are potent relaxing agents in the airways, were submitted to in vitro degradation by the neutral endopeptidase EC 3.4.24.11 (NEP), one of the most active peptidase in the lung, to test their relative resistance to proteolysis. Both VIP and PACAP(1-27) were cleaved by NEP, but PACAP(1-38) was not. The main fragments produced were VIP(1-22) and VIP(1-25), and PACAP(1-22) and PACAP(1-25), respectively. The degradation of VIP(1-27), PACAP(6-27), and PACAP(13-27) was also hindered by extending their C-terminal ends with the (28-38) sequence of PACAP(1-38). The sensitivity to enzyme degradation was gradually reduced when the C-terminal extension was increased from PACAP(1-27) to PACAP(1-29), PACAP(1-32) and PACAP(1-38). The biological activities of the degradation products were evaluated on the three classes of PACAP/VIP receptors, with VIP(1-25) and PACAP(1-25) retaining an important part of their activities on the VIP1 receptor. Thus, the degradation of VIP and PACAP(1-27) by the neutral endopeptidase 24.11 might produce a VIP1 receptor-selective active metabolite, provided that very high VIP or PACAP(1-27) concentrations are achieved in the receptor vicinity.     

The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibres

1. The actions of nitric oxide (NO) have been investigated in an endotoxin-evoked ocular inflammatory model in the rabbit, with particular emphasis on the relationship between NO, sensory nerves (C-fibres) and the C-fibre neuropeptides, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP). 2. Endotoxin, injected intravitreally, evoked inflammatory responses, i.e. conjunctival hyperaemia, miosis and protein extravasation, reflected by the aqueous flare response (AFR). In control rabbits, the maximum AFR was 66.5 +/- 9.5 (arbitrary units). Pretreatment with the NO synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NAME, 200 mg kg-1) given by intravenous injection, inhibited the endotoxin-evoked responses; the AFR was 16.5 +/- 1.9 (n = 8, P < 0.001) and the conjunctival hyperaemia was abolished. 3. Endotoxin-evoked ocular inflammation is associated with the release of CGRP and PACAP from C-fibres. In the eyes challenged with endotoxin, the concentrations of PACAP-27, -38 and CGRP in the aqueous humour were 58.2 +/- 10.9, 54.4 +/- 12.4 and 5526 +/- 519 (pmoll'), respectively. L-NAME inhibited the release of PACAP-27, -38 and CGRP; the concentrations were 14.3 +/- 2.5, 13.5 +/- 2.5 and 510 +/- 67 (pmoll-1), respectively (n = 8, P < 0.01 or 0.001). 4. Intravitreal injection of 0.3 nmol CGRP induced conjunctival hyperaemia and AFR; the maximum AFR was 140.2 +/- 11.4. L-NAME suppressed the response induced by CGRP; the AFR was 23.4 +/- 5.5 (n = 8, P < 0.001). L-NAME abolished the conjunctival hyperaemia induced by PACAP-27 and -38 (0.3 nmol) and reduced the AFR. 5. The inflammatory cells that infiltrated the uvea, cornea and aqueous humour in large numbers in response to intravitreal injection of endotoxin were found to express inducible NOS. L-NAME prevented the appearance of such cells. 6. Our findings suggest that NO plays an important role in the endotoxin-evoked ocular inflammation in the rabbit: NO activates C-fibres causing release of C-fibre neuropeptides into the aqueous humour. In addition, NO mediates scme of the ocular effects of CGRP and PACAP, since L-NAME suppressed the AFR induced by these peptides.     

Combined treatment with a 5HT1A receptor agonist and a muscarinic acetylcholine receptor antagonist disrupts water maze navigation behavior

In the present study, the effects of PACAP27, PACAP38 and VIP in a concentration range from 10(-13) to 10(-6) M were studied in vitro on the spontaneous and directed mobility of lymphocytes from rat spleen and thymus. The results show that VIP and both PACAPs inhibit significantly and in a similar way the mobility of lymphocytes from thymus and spleen, and the maximal effects were observed at 10(-9) M and 10(-8) M. The three neuropeptides significantly increased cAMP concentrations. Moreover, incubation with increasing PMA concentrations showed a progressive enhancement of chemotaxis of lymphocytes, which was partially prevented by VIP, and both PACAPs. Incubation with forskolin caused decrease in the chemotaxis of thymocytes and splenocytes, and the presence of VIP or PACAP peptides was not synergistic in the inhibitory effect on lymphocyte chemotaxis, suggesting that the three neuropeptides and forskolin mediate their actions by the same intracellular pathway. This study showed the ability of the VIP receptor antagonist (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 to partially reverse the inhibitory effect of both PACAPs and VIP on chemotaxis, suggesting that PACAP receptors are identical or very similar to VIP receptors in both thymocytes and splenocytes. These data suggest that PACAP27 and PACAP38 can be included as two novel immunoregulatory peptides that can modulate cell mobility on central and peripheral lymphoid organs.     

Allogeneic bone marrow transplantation for chronic myelomonocytic leukemia in childhood: a report from the European Working Group on Myelodysplastic Syndrome in Childhood

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.     

Pituitary adenylate cyclase-activating polypeptide-27 causes a biphasic chronotropic effect and atrial fibrillation in autonomically decentralized, anesthetized dogs

We investigated the effects of a neuropeptide, pituitary adenylate cyclase-activating polypeptide- (PACAP) 27, on the sinoatrial nodal pacemaker activity and the mechanisms for the cardiac effects of PACAP-27 in the autonomically decentralized heart of the anesthetized dog. PACAP-27 (0.01-0.3 nmol) injected into the sinus node artery increased followed by decreased sinus rate. PACAP-27 (0.1 and 0.3 nmol) caused atrial fibrillation spontaneously. After atropine, PACAP-27 never decreased but only increased sinus rate as did vasoactive intestinal peptide. However, propranolol did not affect the negative and positive chronotropic effects. Tetrodotoxin but not hexamethonium abolished the negative chronotropic response to PACAP-27 in atropine nontreated dogs, and tetrodotoxin also inhibited the positive chronotropic response by 34% in atropine-treated dogs. In atropine- and propranolol-treated dogs, positive chronotropic responses to PACAP-27 were inhibited by PACAP-(6-27), a PACAP receptor antagonist but not by vasoactive intestinal peptide (10-28), a vasoactive intestinal peptide receptor antagonist. These results indicate that PACAP-27 causes the negative chronotropic effect through the postganglionic parasympathetic nerve activation and it produces the positive chronotropic effect mediated by PACAP receptors with an activation of non-adrenergic, nonvasoactive intestinal peptide-ergic nerves at least in part in the dog heart. Atropine and tetrodotoxin abolished atrial fibrillation induced by PACAP-27 but other blockers did not. These results suggest that neurally released acetylcholine induced by PACAP-27 participates in the induction of atrial fibrillation.     

Effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on cyclic AMP accumulation in sheep pituitary cells in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are known to stimulate adenylate cyclase activity in rat pituitary cells but no direct effects have been reported on sheep pituitary cells. In this study we determined whether either peptide could stimulate intracellular cAMP accumulation in dispersed sheep pituitary cells in primary culture. Time course studies with PACAP showed that tachyphylaxis developed rapidly and so a short incubation time (5 min) was used to define the dose-response relationship. PACAP dose-dependently stimulated intracellular cAMP levels with a half-maximum response at 2.9 +/- 0.2 nmol/l (n = 4). In contrast, VIP only caused a small increase in intracellular cAMP levels at the highest dose tested (1 mumol/l). The VIP antagonist [4Cl-D-Phe6,Leu17]VIP had no effect on the cAMP response to either PACAP or VIP while the peptide PACAP(6-38), a putative PACAP antagonist, blocked the cAMP response to PACAP. The desensitisation to PACAP was further investigated by pretreating cells with PACAP for 30 min. After a further 15 min in culture medium alone, these cells showed no cAMP response to subsequent treatment with PACAP but could respond to forskolin. When a longer incubation period of 240 min was used between the first and second treatment with PACAP, a partial return in responsiveness to PACAP was observed. In summary, these results show that PACAP activates adenylate cyclase in sheep pituitary cells but that there is rapid development of tachyphylaxis. Experiments with the antagonists suggest that the response to PACAP is via the PACAP type I receptor. In contrast, physiological doses of VIP do not stimulate cAMP accumulation in sheep pituitary cells.