Pituitary Adenylate Cyclase-activating Polypeptide Infusion Research Articles

Hippocampal PACAP signaling activation triggers a rapid antidepressant response

BackgroundThe development of ketamine-like rapid antidepressants holds promise for enhancing the therapeutic efficacy of depression, but the underlying cellular and molecular mechanisms remain unclear. Implicated in depression regulation, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is investigated here to examine its role in mediating the rapid antidepressant response.MethodsThe onset of antidepressant response was assessed through depression-related behavioral paradigms. The signaling mechanism of PACAP in the hippocampal dentate gyrus (DG) was evaluated by utilizing site-directed gene knockdown, pharmacological interventions, or optogenetic manipulations. Overall, 446 mice were used for behavioral and molecular signaling testing. Mice were divided into control or experimental groups randomly in each experiment, and the experimental manipulations included: chronic paroxetine treatments (4, 9, 14 d) or a single treatment of ketamine; social defeat or lipopolysaccharides-injection induced depression models; different doses of PACAP (0.4, 2, 4 ng/site; microinjected into the hippocampal DG); pharmacological intra-DG interventions (CALM and PACAP6-38); intra-DG viral-mediated PACAP RNAi; and opotogenetics using channelrhodopsins 2 (ChR2) or endoplasmic natronomonas halorhodopsine 3.0 (eNpHR3.0). Behavioral paradigms included novelty suppressed feeding test, tail suspension test, forced swimming test, and sucrose preference test. Western blotting, ELISA, or quantitative real-time PCR (RT-PCR) analysis were used to detect the expressions of proteins/peptides or genes in the hippocampus.ResultsChronic administration of the slow-onset antidepressant paroxetine resulted in an increase in hippocampal PACAP expression, and intra-DG blockade of PACAP attenuated the onset of the antidepressant response. The levels of hippocampal PACAP expression were reduced in both two distinct depression animal models and intra-DG knockdown of PACAP induced depression-like behaviors. Conversely, a single infusion of PACAP into the DG region produced a rapid and sustained antidepressant response in both normal and chronically stressed mice. Optogenetic intra-DG excitation of PACAP-expressing neurons instantly elicited antidepressant responses, while optogenetic inhibition induced depression-like behaviors. The longer optogenetic excitation/inhibition elicited the more sustained antidepressant/depression-like responses. Intra-DG PACAP infusion immediately facilitated the signaling for rapid antidepressant response by inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII)-eukaryotic elongation factor 2 (eEF2) and activating the mammalian target of rapamycin (mTOR). Pre-activation of CaMKII signaling within the DG blunted PACAP-induced rapid antidepressant response as well as eEF2-mTOR-brain-derived neurotrophic factor (BDNF) signaling. Finally, acute ketamine treatment upregulated hippocampal PACAP expression, whereas intra-DG blockade of PACAP signaling attenuated ketamine’s rapid antidepressant response.ConclusionsActivation of hippocampal PACAP signaling induces a rapid antidepressant response through the regulation of CaMKII inhibition-governed eEF2-mTOR-BDNF signaling.

PACAP-PAC1 receptor inhibition is effective in opioid induced hyperalgesia and medication overuse headache models

Opioids prescribed for pain and migraine can produce opioid-induced hyperalgesia (OIH) or medication overuse headache (MOH). We previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) is upregulated in OIH and chronic migraine models. Here we determined if PACAP acts as a bridge between opioids and pain chronification. We tested PACAP-PAC1 receptor inhibition in novel models of opioid-exacerbated trigeminovascular pain. The PAC1 antagonist, M65, reversed chronic allodynia in a model which combines morphine with the migraine trigger, nitroglycerin. Chronic opioids also exacerbated cortical spreading depression, a correlate of migraine aura; and M65 inhibited this augmentation. In situ hybridization showed MOR and PACAP co-expression in trigeminal ganglia, and near complete overlap between MOR and PAC1 in the trigeminal nucleus caudalis and periaqueductal gray. PACAPergic mechanisms appear to facilitate the transition to chronic headache following opioid use, and strategies targeting this system may be particularly beneficial for OIH and MOH.

PACAP Induces Light Aversion in Mice by an Inheritable Mechanism Independent of CGRP.

The neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) have emerged as mediators of migraine, yet the potential overlap of their mechanisms remains unknown. Infusion of PACAP, like CGRP, can cause migraine in people, and both peptides share similar vasodilatory and nociceptive functions. In this study, we have used light aversion in mice as a surrogate for migraine-like photophobia to compare CGRP and PACAP and ask whether CGRP or PACAP actions were dependent on each other. Similar to CGRP, PACAP induced light aversion in outbred CD-1 mice. The light aversion was accompanied by increased resting in the dark, but not anxiety in a light-independent open field assay. Unexpectedly, about one-third of the CD-1 mice did not respond to PACAP, which was not seen with CGRP. The responder and nonresponder phenotypes were stable, inheritable, and not sex linked, although there was a trend for greater responses among male mice. RNA-sequencing analysis of trigeminal ganglia yielded hierarchical clustering of responder and nonresponder mice and revealed a number of candidate genes, including greater expression of the Trpc5 and Kcnk12 ion channels and glycoprotein hormones and receptors in a subset of male responder mice. Importantly, an anti-PACAP monoclonal antibody could block PACAP-induced light aversion but not CGRP-induced light aversion. Conversely, an anti-CGRP antibody could not block PACAP-induced light aversion. Thus, we propose that CGRP and PACAP act by independent convergent pathways that cause a migraine-like symptom in mice.SIGNIFICANCE STATEMENT The relationship between the neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) in migraine is relevant given that both peptides can induce migraine in people, yet to date only drugs that target CGRP are available. Using an outbred strain of mice, we were able to show that most, but not all, mice respond to PACAP in a preclinical photophobia assay. Our finding that CGRP and PACAP monoclonal antibodies do not cross-inhibit the other peptide indicates that CGRP and PACAP actions are independent and suggests that PACAP-targeted drugs may be effective in patients who do not respond to CGRP-based therapeutics.

Prefrontal cortex PACAP signaling: organization and role in stress regulation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuromodulatory peptide strongly implicated in nervous stress processing. Human polymorphism of the primary PACAP receptor (PAC1) is linked to psychiatric disorders, including posttraumatic stress disorder (PTSD). Prefrontal cortex PACAP signaling is associated with processing of traumatic stress and fear learning, suggesting a potential role in PTSD-related deficits. We used RNAscope to define the cellular location of PACAP and PAC1 in the infralimbic cortex (IL). Subsequent experiments used a pharmacological approach to assess the impact of IL PACAP infusion on behavioral and physiological stress response and fear memory. Adult male Sprague-Dawley rats were bilaterally microinjected with PACAP (1 ug) or vehicle into the IL, 30 minutes prior to forced swim test (FST). Blood was sampled at 15, 30, 60, and 120 minutes for analysis of hypothalamic pituitary adrenal (HPA) axis reactivity. Five days after, animals were tested in a 3-day passive avoidance paradigm with subsequent testing of fear retention two weeks later. We observed that PACAP is highly expressed in putative pyramidal neurons (identified by VGlut1 expression), while PAC1 is enriched in interneurons (identified by GAD). Pretreatment with PACAP increased active coping style in the FST, despite higher levels of ACTH and corticosterone. The treatment was also sufficient to cause an increase in anxiety-like behavior in a dark/light crossover test and enhanced retention of passive avoidance. Our data suggest that IL PACAP plays a role in driving stress responses and in processing of fear memories, likely mediated by inhibition of cortical drive.

Role of the PACAP system of the extended amygdala in the acoustic startle response in rats

Anxiety-related disorders are the most prevalent mental disorders in the world and they are characterized by abnormal responses to stressors. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide highly expressed in the extended amygdala, a brain macrostructure involved in the response to threat that includes the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). The aim of this series of experiments was to systematically elucidate the role of the PACAP system of the CeA and BNST under both control, unstressed conditions and after the presentation of a stressor in rats. For this purpose, we used the acoustic startle response (ASR), an unconscious response to sudden acoustic stimuli sensitive to changes in stress which can be used as an operationalization of the hypervigilance present in anxiety- and trauma-related disorders. We found that infusion of PACAP, but not the related peptide vasoactive intestinal peptide (VIP), into either the CeA or the BNST causes a dose-dependent increase in ASR. In addition, while infusion of the antagonist PACAP(6–38) into either the CeA or the BNST does not affect ASR in non-stressed conditions, it prevents the sensitization of ASR induced by an acute footshock stress. Finally, we found that footshock stress induces a significant increase in PACAP, but not VIP, levels in both of these brain areas. Altogether, these data show that the PACAP system of the extended amygdala contributes to stress-induced hyperarousal and suggest it as a potential novel target for the treatment of stress-related disorders.

The Effects of Prior Stress on Anxiety-Like Responding to Intra-BNST Pituitary Adenylate Cyclase Activating Polypeptide in Male and Female Rats.

Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in anxiety-related brain structures. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In male rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. Here, we examined whether CVS would sensitize the behavioral and/or endocrine response to a subthreshold BNST PACAP infusion. Male and cycling female rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, rats were bilaterally infused into the BNST with a normally subthreshold dose of PACAP. We found an increase in startle amplitude and plasma corticosterone levels 30 min following intra-BNST PACAP infusion in male rats that had been previously exposed to CVS. CVS did not enhance the startle response in cycling females. Equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels even in previously stressed male rats. These results suggest that repeated exposure to stressors may differentially alter the neural circuits underlying the responses to intra-BNST PACAP, and may result in different anxiety-like responses in males and females.

Central Amygdala PACAP in the Behavioral Stress Response

Anxiety‐related disorders are the most common forms of mental disorders; characterized by feelings of excessive worry in the absence of specific external stimuli, they are accompanied by physical, affective and behavioral symptoms.Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a 38‐amino acid peptide located in the hypothalamus and in various extra‐hypothalamic regions including the central nucleus of the amygdala (CeA). PACAP and PAC1 receptor (PAC1R) have been proposed to play a key role in mediating the behavioral and endocrine responses to stress; however, few studies have examined the involvement of the extrahypothalamic PACAP system in the modulation of stress. Our aim was to elucidate the role of the PACAP/PAC1 system of the CeA in the context of anxiety.We first assessed the effects of PACAP microinfused into the CeA of male rats in the elevated plus maze (EPM) and the acoustic startle response (ASR), behavioral tests sensitive to states of anxiety and fear. PACAP was also given into the basolateral amygdala (BlA) as a control brain region. We then used restraint stress to determine whether the endogenous PACAP‐PAC1 system is recruited by acute stressors.Infusion of PACAP into the CeA significantly reduced percent open arm time in the EPM and increased plasma corticosterone levels, while BlA microinfusion had no effect. Infusion of PACAP into the CeA also increased acoustic startle reactivity. Finally, we observed that an acute, 1h exposure to restraint produced a rapid elevation of PACAP and PAC1R expression in the CeA, but not the BlA.These data prove an anxiogenic role for the PACAP/PAC1 system of the CeA and suggest that hyperactivity of this system may underlie the anxiogenic effects of stress.

Pituitary adenylate cyclase-activating polypeptide (PACAP) in the bed nucleus of the stria terminalis (BNST) increases corticosterone in male and female rats.

Single nucleotide polymorphisms (SNP) in the genes for pituitary adenylate cyclase-activating polypeptide (PACAP) and the PAC1 receptor have been associated with several psychiatric disorders whose etiology has been associated with stressor exposure and/or dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. In rats, exposure to repeated variate stress has been shown to increase PACAP and its cognate PAC1 receptor expression in the bed nucleus of the stria terminalis (BNST), a brain region implicated in anxiety and depression-related behaviors as well as the regulation of HPA axis activity. We have argued that changes in BNST PACAP signaling may mediate the changes in emotional behavior and dysregulation of the HPA axis associated with anxiety and mood disorders. The current set of studies was designed to determine whether BNST PACAP infusion leads to activation of the HPA axis as determined by increases in plasma corticosterone. We observed an increase in plasma corticosterone levels 30min following BNST PACAP38 infusion in male and female rats, which was independent of estradiol (E2) treatment in females, and we found that plasma corticosterone levels were increased at both 30min and 60min, but returned to baseline levels 4h following the highest dose. PACAP38 infusion into the lateral ventricles immediately above the BNST did not alter plasma corticosterone level, and the increased plasma corticosterone following BNST PACAP was not blocked by BNST corticotropin releasing hormone (CRH) receptor antagonism. These results support others suggesting that BNST PACAP plays a key role in regulating stress responses.

PACAP causes long‐term increases in sympathetic nerve activity and is necessary for the sympathetic response to acute intermittent hypoxia

Overall objectiveTo determine if the sustained elevation of splanchnic sympathetic nerve activity (sSNA), termed long‐term facilitation (LTF), following acute intermittent hypoxia (AIH) is due to intermittent, release of sympathoexcitatory peptides, such as pituitary adenylate cyclase activating polypeptide (PACAP).MethodsIn urethane‐anaesthetized, artificially ventilated, male rats (n=18 Sprague–Dawley), we investigated the effect of 10, 45s, episodes of 10%O2–90%N2 every 5 min (AIH) on sSNA after intrathecal infusion of 10μl of vehicle, PACAP (300μM) or the PAC1/VPAC2 receptor antagonist, PACAP(6–38) (1mM). In a separate group of rats (n=6) without AIH, 10 doses of PACAP (10μL of 10μM) were given intrathecally every 5 min.ResultsVehicle‐treated rats showed a 31.0±8.1% increase in sSNA 60min after AIH, which doubled to 67.7±14.1% in PACAP treated rats. Infusing the PACAP antagonist PACAP(6–38) prior to AIH abolished sympathetic LTF (0.1±2.7%). The responses were significantly different (P<0.0001; 2‐way ANOVA). Intermittent, sub‐threshold, infusion of PACAP (10×10μM) was as effective as AIH in causing LTF (sSNA increase of 38.±8.0%).ConclusionPACAP, acting via spinal PAC1/VPAC2 receptors, is sufficient to cause sympathetic LTF in the absence of AIH and necessary for the elaboration of sympathetic LTF following AIH. Support provided by Macquarie University, ARC and NHMRC.

Effects of pituitary adenylate cyclase activating polypeptide on CD4(+)/CD8(+) T cell levels after traumatic brain injury in a rat model.

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) during traumatic brain injury (TBI) and whether it can modulate secondary injury has not been reported previously. The present study evaluated the potential protective effects of ventricular infusion of PACAP in a rat model of TBI. Male Sprague Dawley rats were randomly divided into 3 treatment groups (n=6, each): sham-operated, vehicle (normal saline)+TBI, and PACAP+TBI. Normal saline or PACAP (1 μg/5 μL) was administered intracerebroventricularly 20 minutes before TBI. Right parietal cortical contusion was produced via a weight-dropping method. Brains were extracted 24 hours after trauma. Histological changes in brains were examined by HE staining. The numbers of CD4(+) and CD8(+) T cells in blood and the spleen were detected via flow cytometry. In injured brain regions, edema, hemorrhage, inflammatory cell infiltration, and swollen and degenerated neurons were observed under a light microscope, and the neurons were disorderly arrayed in the hippocampi. Compared to the sham group, average CD4(+) CD8(-) lymphocyte counts in blood and the spleen were significantly decreased in rats that received TBI+vehicle, and CD4(-) CD8(+) were increased. In rats administered PACAP prior to TBI, damage was attenuated as evidenced by significantly increased CD4(+), and decreased CD8(+), T lymphocytes in blood and the spleen. Pretreatment with PACAP may protect against TBI by influencing periphery T cellular immune function.

Pituitary adenylate cyclase-activating polypeptide plays a key role in nitroglycerol-induced trigeminovascular activation in mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (PAC1, VPAC) are present in sensory neurons and vascular smooth muscle. PACAP infusion was found to trigger migraine-like headache in humans and we showed its central pro-nociceptive function in several mouse pain models. Nitroglycerol (NTG)-induced pathophysiological changes were investigated in this study in PACAP gene-deleted (PACAP−/−) and wildtype (PACAP+/+) mice. Chemical activation of the trigeminovascular system was induced by 10mg/kg i.p. NTG. Light-aversive behavior was determined in a light–dark box, meningeal microcirculation by laser Doppler blood perfusion scanning and the early neuronal activation marker c-Fos with immunohistochemistry. NTG-induced photophobia both in the early (0–30min) and late phases (90–120min) due to direct vasodilation and trigeminal sensitization, respectively, was significantly reduced in PACAP−/− mice. Meningeal blood flow increased by 30–35% during 4h in PACAP+/+ mice, but only a 5–10% elevation occurred from the second hour in PACAP−/− ones. The number of c-Fos expressing cells referring to neuronal activation in the trigeminal ganglia and nucleus caudalis significantly increased 4h after NTG in PACAP+/+, but not in PACAP−/− animals. Similar PAC1 receptor immunostaining was detected in both groups, which did not change 4h after NTG treatment. PACAP-38 (300μg/kg, i.p.) produced photophobia similarly to NTG and 30% meningeal vasodilatation for 30min in PACAP+/+, but not in PACAP−/− mice. It significantly increased neural activation 4h later in the trigeminal ganglia of both groups, but in the nucleus caudalis of only the PACAP+/+ mice.We provide the first experimental results that PACAP is a pivotal mediator of trigeminovascular activation/sensitization and meningeal vasodilation related to migraine.

Light Stimulates MSK1 Activation in the Suprachiasmatic Nucleus via a PACAP-ERK/MAP Kinase-Dependent Mechanism

Signaling via the p42/44 mitogen-activated protein kinase (MAPK) pathway has been shown to be a key intracellular signaling event that couples light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Because many of the physiological effects of the MAPK pathway are mediated by extracellular signal-regulated kinase (ERK)-regulated kinases, it was of interest to identify kinase targets of ERK in the SCN. In this study, we examined whether mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of ERK in the SCN and whether it couples to clock gene expression. Here we show that photic stimulation during the subjective night stimulates MSK1 phosphorylation at serine 360, an event required for robust kinase activation. Activated ERK and MSK1 were colocalized in SCN cell nuclei after photic stimulation. The in vivo administration of the MAP kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene] attenuated MSK1 phosphorylation. MSK1 phosphorylation was more responsive to late-night than early-night photic stimulation, indicating that MSK1 may differentially contribute to light-induced phase advancing and phase delaying of the clock. The potential connection between pituitary adenylate cyclase-activating polypeptide (PACAP) (a regulator of clock entrainment) and MSK1 phosphorylation was examined. PACAP infusion stimulated MSK1 phosphorylation, whereas PACAP receptor antagonist infusion attenuated light-induced MSK1 phosphorylation in the SCN. In reporter gene assays, MSK1 was shown to couple to mPeriod1 via a cAMP response element-binding protein-dependent mechanism. Together, these data identify MSK1 as both a downstream target of the MAPK cascade within the SCN and a regulator of clock gene expression.

Role of endogenous PACAP in catecholamine secretion from the rat adrenal gland.

We elucidated the contribution of endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) to neurally evoked catecholamine secretion from the isolated perfused rat adrenal gland. Infusion of PACAP (100 nM) increased adrenal epinephrine and norepinephrine output. The PACAP-induced catecholamine output responses were inhibited by the PACAP type I receptor antagonist PACAP- (6-38) (30-3,000 nM) but were resistant to the PACAP type II receptor antagonist [Lys1,Pro2,5,Ara3,4,Tyr6]-vasoactive intestinal peptide (LPAT-VIP; 30-3,000 nM). Transmural electrical stimulation (ES; 1-10 Hz) or infusion of ACh (6-200 nM) increased adrenal epinephrine and norepinephrine output. PACAP-(6-38) (3,000 nM), but not LPAT-VIP, also inhibited the ES-induced catecholamine output responses. However, PACAP-(6-38) did not affect the ACh-induced catecholamine output responses. PACAP at low concentrations (0.3-3 nM), which had no influence on catecholamine output, enhanced the ACh-induced catecholamine output responses, but not the ES-induced catecholamine output responses. These results suggest that PACAP is released from the nerve endings to facilitate the neurally evoked catecholamine secretion through PACAP type I receptors in the rat adrenal gland.

PACAP protects hippocampal neurons against apoptosis: involvement of JNK/SAPK signaling pathway.

We have demonstrated that the ischemia-induced apoptosis of neurons in the CA1 region of the rat hippocampus was prevented by either intracerebroventricular or intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP). However, the molecular mechanisms underlying the anti-apoptotic effect of PACAP remain to be determined. Within 3-6 h after ischemia, the activities of members of the mitogen-activated protein (MAP) kinase family, including extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38 were increased in the hippocampus. The ischemic stress had a potent influence on the MAP kinase family, especially on JNK/SAPK. PACAP inhibited the activation of JNK/SAPK after ischemic stress. Secretion of interleukin-6 (IL-6) into the cerebrospinal fluid was intensely stimulated after PACAP infusion. IL-6 inhibited the activation of JNK/SAPK, while it activated ERK. These observations suggest that PACAP and IL-6 act to inhibit the JNK/SAPK signaling pathway, thereby protecting neurons against apoptosis.

Effects of intravenously infused pituitary adenylate cyclase-activating polypeptide on arginine vasopressin and oxytocin secretion in man.

In order to establish possible stimulatory effects of increasing plasma concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) on arginine vasopressin (AVP) and/or oxytocin (OT) secretion, successively increasing doses of PACAP-38 (2, 4 and 8 pmol kg min-1; each dose for 20 min) were infused in seven normal men. Plasma AVP and OT levels were measured before infusion and every 20 min, just before increasing the infusion dose of PACAP. During tests, PACAP infusion did not produce untoward side effects or changes in blood osmolality and/or pressure. Plasma OT levels did not change during PACAP infusion. Plasma AVP concentrations were not modified by infusion of the lowest dose of PACAP, whereas they were significantly increased in a dose-response fashion when higher amounts of PACAP were given. These findings demonstrate for the first time in humans that the systemic administration of PACAP exerts stimulatory effects on AVP, but not OT secretion.

Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats

Pituitary adenylate cyclase activating polypeptide (PACAP) is produced by hypothalamic neurons which terminate within the median eminence suggesting that it may be a hypophysiotropic hormone. However, little endocrine activity has been ascribed to the peptide. Therefore we studied the effects of PACAP on prolactin (Prl) release from dispersed cultivated rat pituitary cells in vitro using conventional cultures as well as the reverse hemolytic plaque assay (RHPA). Furthermore the effects of the peptide on in vitro GH release were assessed. In addition, the activity of the peptide on in vivo release of Prl and GH was studied in hypothalamus-lesioned animals. PACAP dose dependently inhibited Prl release form dispersed pituitary cells in both, monolayer cell cultures and the RHPA, whereas GH secretion was not affected. In hypothalamus-lesioned rats which have high Prl levels due to the absence of hypothalamic dopamine, PACAP further stimulated Prl release. Serum GH increased more than 20 fold in response to the intravenous PACAP infusion. Thus in vitro (inhibition of Prl release, no effect on GH release) and in vivo (stimulation of both hormones) experiments yielded contradicting effects of PACAP on pituitary hormone release. We suggest that PACAP may stimulate the release of a paracrine, yet unknown factor which in the intact pituitary overrides the direct inhibitory action of PACAP on the lactotropes. The same or another paracrine factor may also enhance in vivo GH release. In cell culture the paracrine factor is diluted by the medium. Therefore the peptide never reaches effective concentrations which are present within the intact pituitary tissue.