Pituitary Adenylate Cyclase-Activating Polypeptide Prevents Cisplatin-Induced Renal Failure

Cisplatin is widely used for cancer chemotherapy, but nephrotoxicity is a major dose-limiting side effect. Our recent studies in vitro have shown that pituitary adenylate cyclase-activating polypeptide (PACAP) ameliorated cisplatin nephrotoxicity and that the renoprotection with PACAP38 was mediated by the PAC₁ receptor and through the p53-dependent and -independent suppression of apoptosis of human renal proximal tubular epithelial cells. In the present studies, PACAP38 prevented the rise in blood urea nitrogen and serum creatinine in mice treated with cisplatin. Cisplatin-exposed mice treated with PACAP38 had relatively well-preserved tubular integrity, even when the treatment started 24 h after cisplatin exposure. PACAP38 also reduced plasma and kidney levels of tumor necrosis factor-α and restored collagen IV levels. The damage to mouse kidney tubules caused by cisplatin involved p53 accumulation and was partially reversed by treatment with PACAP38. PACAP38 ameliorates cisplatin-induced acute kidney injury even when treatment started 24 h after the onset of injury and increases tubular regeneration, which further facilitates restoration of kidney function in addition to its anti-apoptotic effects.     

Characterization of the Thermoregulatory Response to Pituitary Adenylate Cyclase-Activating Polypeptide in Rodents

Administration of the long form (38 amino acids) of pituitary adenylate cyclase-activating polypeptide (PACAP38) into the central nervous system causes hyperthermia, suggesting that PACAP38 plays a role in the regulation of deep body temperature (T _b). In this study, we investigated the thermoregulatory role of PACAP38 in details. First, we infused PACAP38 intracerebroventricularly to rats and measured their T _b and autonomic thermoeffector responses. We found that central PACAP38 infusion caused dose-dependent hyperthermia, which was brought about by increased thermogenesis and tail skin vasoconstriction. Compared to intracerebroventricular administration, systemic (intravenous) infusion of the same dose of PACAP38 caused significantly smaller hyperthermia, indicating a central site of action. We then investigated the thermoregulatory phenotype of mice lacking the Pacap gene (Pacap ^?/?). Freely moving Pacap ^?/? mice had higher locomotor activity throughout the day and elevated deep T _b during the light phase. When the Pacap ^?/? mice were loosely restrained, their metabolic rate and T _b were lower compared to their wild-type littermates. We conclude that PACAP38 causes hyperthermia via activation of the autonomic cold-defense thermoeffectors through central targets. Pacap ^?/? mice express hyperkinesis, which is presumably a compensatory mechanism, because under restrained conditions, these mice are hypometabolic and hypothermic compared to controls.     

Pituitary Adenylate Cyclase-Activating Polypeptide Deficiency Enhances Oxazolone-Induced Allergic Contact Dermatitis in Mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in capsaicin-sensitive sensory neurons and inflammatory/immune cells, therefore it is suggested to play a role in neuro-immune interactions. Our aim was to investigate the role of PACAP in oxazolone-induced delayed-type hypersensitivity reaction in the skin using deficient mice (PACAP^-/-). Sensitization was induced by 2% oxazolone application on the shaved abdomen on two consecutive days; inflammation was elicited by oxazolone smearing on the ears 6 days later. Ear thickness was measured by micrometry. Histological examination, cytokine profile [IL-2, IL-4, IL-5, and monocyte chemoattractant protein-1: MCP-1, IFN-γ, tumor necrosis factor alpha (TNF-α)] and myeloperoxidase activity correlating with the number of neutrophils/macrophages were determined 24 and 48 h later. Oxazolone induced a 110–130% swelling after 24–48 h in wild-type mice, which was significantly greater in PACAP-deficient mice. Histological analysis confirmed markedly increased edema in PACAP^-/- mice, but the moderately enhanced inflammatory cell accumulation was not statistically significant compared with the wild-types. There was no difference in myeloperoxidase activity of the ear homogenates. Elevation of MCP-1, but not the levels of the other cytokines, was significantly higher in the samples of the PACAP-deficient mice. These results suggest that PACAP exerts anti-inflammatory, particularly edema-inhibiting effects in allergic contact dermatitis.     

Mechanism of Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Human Nonfunctioning Pituitary Tumors

Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca²⁺]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca²⁺]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 ± 10 to 214 ± 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 ± 5.4 to 73.2 ± 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6–27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca²⁺]i rise in cells obtained from 7 NFPA (from 110 ± 34 to 151 ± 40 nM [Ca²⁺]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 μM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca²⁺ as well as blockade of L-type Ca²⁺ channels by dihydropyridine antagonists abolished [Ca²⁺]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Proliferation of Reactive Astrocytes In Vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalamus. Recently, we have shown that the PACAP receptor (PAC1-R) is expressed in reactive astrocytes following an in vivo stub wound brain injury. However, the functional role of PACAP has not yet been clarified. In order to investigate the effect of PACAP on the proliferation of reactive astrocytes, a scratch wound paradigm was applied to astrocytic monolayers. Following injury, there was an increase in PAC1-R and glial fibrillary acidic protein (GFAP) immunoreactivity in the astrocytes surrounding the scratch line. PACAP at concentrations of 10^?15 to 10^?7?M was applied immediately after scratching, and the proliferating astrocytes were visualized by multiple immunofluorescence labeling. The percentage of cells that colabeled for Ki67 (a marker of proliferating cells) and GFAP increased in the 10^?11- and 10^?13-M PACAP-treated groups. The proliferating astrocytes induced by PACAP treatment mainly occurred in the proximal wound area where many reactive astrocytes were observed. Pretreatment with the PACAP receptor antagonist PACAP6-38 significantly suppressed the PACAP-induced effects. These results strongly suggest that PACAP plays an important role in the proliferation of reactive astrocytes following nerve injury.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Increased Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin were investigated using lung cancer cells. Addition of PACAP-27 or PACAP-38 but not vasoactive intestinal peptide to NCI-H838 or NCI-H1299 human lung cancer cells significantly increased the tyrosine phosphorylation of FAK or paxillin. The increase in FAK or paxillin tyrosine phosphorylation caused by addition of PACAP-27 to NCI-H838 cells was inhibited by PACAP(6–38), a PAC1-receptor (R) antagonist. The increase in FAK or paxillin tyrosine phosphorylation caused by 100 nM PACAP-27 was maximal 2 min after addition to NCI-H838 cells. The effects of PACAP at stimulating FAK and paxillin tyrosine phosphorylation were reversed by cytochalasin D and genistein which inhibit actin polymerization and tyrosine kinase activity, respectively. The effects of PACAP at stimulating FAK and paxillin tyrosine phosphorylation were reversed by U-73122 but not H89 which inhibit phospholipase C and protein kinase A, respectively. The results show that PAC1-R regulates FAK and paxillin tyrosine phosphorylation in lung cancer cells as a result of increased phosphatidylinositol turnover but not adenylyl cylase stimulation.     

Neuroprotective Effect of Anthocyanin on Experimental Traumatic Spinal Cord Injury

Objective

We investigated the neuroprotective effect of anthocyanin, oxygen radical scavenger extracted from raspberries, after traumatic spinal cord injury (SCI) in rats.

Methods

The animals were divided into two groups : the vehicle-treated group (control group, n=20) received an oral administration of normal saline via stomach intubation immediately after SCI, and the anthocyanin-treated group (AT group, n=20) received 400 mg/kg of cyanidin 3-O-β-glucoside (C3G) in the same way. We compared the neurological functions, superoxide expressions and lesion volumes in two groups.

Results

At 14 days after SCI, the AT group showed significant improvement of the BBB score by 16.7±3.4%, platform hang by 40.0±9.1% and hind foot bar grab by 30.8±8.4% (p<0.05 in all outcomes). The degree of superoxide expression, represented by the ratio of red fluorescence intensity, was significantly lower in the AT group (0.98±0.38) than the control group (1.34±0.24) (p<0.05). The lesion volume in lesion periphery was 32.1±2.4 µL in the control and 24.5±2.3 µL in the AT group, respectively (p<0.05), and the motor neuron cell number of the anterior horn in lesion periphery was 8.3±5.1 cells/HPF in the control and 13.4±6.3 cells/HPF in the AT group, respectively (p<0.05).

Conclusion

Anthocyanin seemed to reduce lesion volume and neuronal loss by its antioxidant effect and these resulted in improved functional recovery.     

Pituitary Adenylate Cyclase-Activating Polypeptide is Protective Against Oxidative Stress in Human Retinal Pigment Epithelial Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent neurotrophic and neuroprotective effects. We have previously shown that PACAP protects against several types of retinal injuries in vivo, including retinal ischemia, glutamate-induced excitotoxicity, UV A-induced lesion, and diabetic retinopathy. We have also shown that PACAP activates antiapoptotic pathways and inhibits proapoptotic signaling in retinal lesions in vivo. PACAP receptors have been identified on the retinal pigment epithelial cells and PACAP has been shown to inhibit interleukin secretion from pigment epithelial cells. It is not known, however, whether PACAP is protective in these cells. Human retinal pigment epithelial cells (ARPE-19 cell line) were exposed to in vitro oxidative stress by hydrogen peroxide. Cell survival was decreased in cells exposed to oxidative stress, which could be significantly and dose-dependently attenuated by 10 pM–1 μM PACAP treatment, as shown by MTT viability test. The protective effect of PACAP could be blocked by the receptor antagonist PACAP6-38. In addition, flow cytometry and JC-1 assay revealed that oxidative stress-induced apoptosis in retinal pigment epithelial cells could be decreased by PACAP treatment. In summary, these results show, for the first time, that PACAP is antiapoptotic in the retinal pigment epithelial cells.     

The Role of Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-Activating Polypeptide in the Neural Pathways Controlling the Lower Urinary Tract

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are expressed in the neural pathways regulating the lower urinary tract. VIP-immunoreactivity (IR) is present in afferent and autonomic efferent neurons innervating the bladder and urethra, whereas PACAP-IR is present primarily in afferent neurons. Exogenously applied VIP relaxes bladder and urethral smooth muscle and excites parasympathetic neurons in bladder ganglia. PACAP relaxes bladder and urethral smooth muscle in some species (pig) but excites the smooth muscle in other species (mouse). Intrathecal administration of VIP in cats with an intact spinal cord suppresses reflex bladder activity, but intrathecal administration of VIP or PACAP in rats enhances bladder activity and suppresses urethral sphincter activity. PACAP has presynaptic facilitatory effects and direct excitatory effects on lumbosacral parasympathetic preganglionic neurons. Chronic spinal cord transection produces an expansion of VIP-IR (cats) and PACAP-IR (rats) in primary afferent axons in the lumbosacral spinal cord and unmasks spinal excitatory effects of VIP on bladder reflexes in cats. Intrathecal administration of PACAP6-38, a PAC1 receptor antagonist, reduces bladder hyperactivity in chronic spinal-cord-injured rats. These observations raise the possibility that VIP or PACAP have a role in the control of normal or abnormal voiding.     

Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) onHormone Secretion from Sheep Pituitary Cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 μmol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l–1 μmol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 μmol/l; P<0.05). PACAP also stimulated FSH secretion in cells from some animals although this effect was small. The GH response to PACAP was inhibited by PACAP(6–38), a putative PACAP antagonist, but not by (N-Ac-Tyr¹, D-Arg²)-GHRH(1–29)-NH_2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1–24 h, VIP (1 μmol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l–1 μmol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l–1 μmol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 μmol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.     

Functional Characterization of Neural-Restrictive Silencer Element in Mouse Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Gene Expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) is predominantly localized in the nervous system, but the underlying mechanism in its neuron-specific expression remains unclear. In addition to two neural-restrictive silencer-like element (NRSLE1 and 2), as reported previously, we have identified the third element in ?1,601 to ?1,581 bp from the translational initiation site of mouse PACAP gene and termed it as NRSLE3, of which, the sequence and location were highly conserved among mouse, rat, and human PACAP genes. In luciferase reporter assay, the deletion or site-directed mutagenesis of NRSLE3 in the reporter gene construct, driven by heterologous SV40 promoter, cancelled the repression of luciferase activity in non-neuronal Swiss-3T3 cells. Furthermore, its promoter activity was significantly repressed in Swiss-3T3 cells, but not in neuronal-differentiated PC12 cells. The electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells demonstrated a specific complex with NRSLE3 probe that exhibited the same migration with the neural-restrictive silencer element (NRSE) probe of rat type II sodium channel gene. During neuronal differentiation of PC12 cells, the increment of PACAP mRNA exhibited the correlation with that of REST4 mRNA, which is a neuron-specific variant form of neural-restrictive silencer factor (NRSF). In undifferentiated PC12 cells, trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which indirectly inhibits NRSF-mediated gene silencing, increased PACAP mRNA level and attenuated the repression of promoter activity of 5′ flanking region of mouse PACAP gene containing NRSLEs. These suggest that the NRSE–NRSF system implicates in the regulatory mechanism of neuron-specific expression of PACAP gene.     

Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs—neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H₂O₂ for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H₂O₂ and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.     

Comparison of Expression and Proliferative Effect of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its Receptors on Human Astrocytoma Cell Lines

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide considered to be a potent regulator of astrocytes. It has been reported that PACAP also affects astrocytoma cell properties, but the proliferative effects of this peptide in previous reports were inconsistent. The purpose of this study was to search for correlations between malignant potential, PACAP/PACAP receptor expression, and the proliferative potential of four astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13). Immunohistochemical observations were performed using astrocyte lineage markers with a view to establishing malignant potential, which is inversely correlated to differentiation status in astrocytoma cells. YH-13 showed the most undifferentiated astrocyte-like status, and was immunopositive to a cancer stem cell marker, CD44. These observations suggest that YH-13 is the most malignant of the astrocytoma cell lines tested. Moreover, the strongest PAC1-R immunoreactivity was observed in YH-13 cells. Using real-time PCR analysis, no significant differences among cell lines were detected with respect to PACAP mRNA, but PAC1-R and VPAC1-R mRNA levels were significantly increased in YH-13 cells compared with the other cell lines. Furthermore, when cell lines were treated with PACAP (10^?11 M) for 3 days, the YH-13 cell line, but not of the other cell lines, exhibited a significantly increased cell number. These results suggest that PACAP receptor expression is correlated with the malignant and proliferative potential of astrocytoma cell lines.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Actions on αT3-1 Gonadotrophs Show Desensitization

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived αT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation. In acute experiments, PACAP27-stimulated cAMP accumulation (intracellular plus extracellular) was measured (in the presence of phosphodiesterase inhibitor) both in intact cells and in cell membranes. The peptide increased cAMP accumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in both cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (although the deviation from linearity was less pronounced than with PACAP27). Thus, rapid homologous desensitization to PACAP27 occurs in intact αT3-1 cells, but is not entirely receptor specific. Rapid homologous desensitization of PACAP27-stimulated cAMP accumulation also occurred in the presence of a protein kinase C activating phorbol ester, which inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to cause a spike-type elevation of cytosolic Ca²⁺ concentration (a consequence of phosphoinositidase C activation). In chronic desensitization studies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC₅₀ approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%). Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) adenylyl cyclase undergoes rapid homologous desensitization in αT3-1 cells, which is paralleled by homologous desensitization of PACAP27-stimulated phosphoinositidase C activity and involves mechanisms distinct from those underlying heterologous desensitization by phorbol esters. Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.     

Neuroanatomical and Physiological Evidence for the Involvement of Pituitary Adenylate Cyclase-Activating Polypeptide in the Regulation of the Distal Lobe of the Frog Pituitary

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino-acid peptide which belongs to the glucagon/secretin/ vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ridibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP-containing neurons and fibers was examined by the indirect immunofluores-cence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose-dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED₅₀= 2 × 10^?8 M). At the maximum dose tested (5 × 10^?6 M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production. In contrast, the truncated form [Des-His¹frog PACAP38 did not affect adenylate cyclase activity demonstrating therefore that the N-terminal histidyl residue is essential for the biological activity of the peptide. [Des-His¹]frog PACAP38 did not antagonize the stimulatory effect of frog PACAP38 or PACAP27 on cAMP production. Taken together, these data support the concept that, in amphibians as in mammals, PACAP may act as a hypophysiotropic neuropeptide.