Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide-stimulated cyclic AMP synthesis in rat cerebral cortical slices

Pituitary adenylate cyclase-activating polypeptide (PACAP; 0.001-1 microM) and vasoactive intestinal peptide (VIP; 0.01-1 microM) produced a concentration-dependent stimulation of cyclic AMP (cAMP) formation in rat cerebral cortical slices prelabeled with [3H]adenine. The effects of PACAP38 and PACAP27 were similar, and more efficacious (at 0.1 and 1 microM) than those of VIP. Adrenaline and noradrenaline (each at 100 microM) also stimulated cAMP formation, with the latter compound being more effective. Combination of PACAP38, PACAP27 (each at 0.1 microM) and VIP (1 microM) with adrenaline or noradrenaline resulted in most cases in additive effects, with some supraadditive (PACAP27 plus adrenaline) or subadditive (PACAP38 or VIP plus noradrenaline) fluctuations. In contrast, combination of each of the three peptides with 3 microM forskolin resulted in synergistic effects. These results indicate that in rat cerebral cortex there is no synergism between PACAP or VIP with noradrenaline or adrenaline; however, based on the forskolin data, it seems likely that synergistic effects may take place with VIP or PACAP and other cAMP-stimulating neuroregulators.     

Antagonism of VIP-stimulated cyclic AMP formation in chick brain

Of eight peptides tested (0.01-5 microM), only two, that is, pituitary adenylate cyclase-activating polypeptide (PACAP27) and chicken vasoactive intestinal peptide (cVIP), potently stimulated cyclic AMP (cAMP) production in cerebral cortical slices of the chick. Mammalian VIP (mVIP) showed some activity only at the highest dose tested, whereas truncated forms of PACAP or VIP, that is, PACAP6-27, cVIP6-28, and mVIP6-28, or hybrid compounds, that is, neurotensin6-11-cVIP7-28 (NT-cVIP) and neurotensin6-11-mVIP7-28 (NT-mVIP), were inactive. Thirty-minute preincubation of chick cortical slices with 5 microM PACAP6-27, NT-cVIP, or NT-mVIP competitively antagonized the cAMP effects of cVIP (0.03-1 microM), with the truncated form of PACAP being the best antagonist. Preincubation of slices with 5 microM mVIP6-28 also produced a significant inhibition of the cVIP (0.1-1 microM)-induced increase in cAMP production; however its action was independent of the concentration of cVIP. In contrast to mVIP6-28, cVIP6-28 showed no antagonistic activity against the full-length peptide. In parallel experiments, 30-min pretreatment of cortical slices with 5 microM PACAP6-27 significantly antagonized the PACAP38-evoked increase in cAMP formation, whereas mVIP6-28 or the NT-mVIP hybrid was ineffective. It has been concluded that in the chick brain, PACAP and cVIP stimulate cAMP biosynthesis via PAC1 and VPAC-type receptors, respectively, and PACAP6-27 seems to be the most potent, yet PACAP/VIP receptor-nonselective antagonist. Unlike truncated PACAP, the NT-VIP hybrid peptides tested may represent VPACtype receptor-selective blocking activity.     

Receptors for VIP and PACAP in guinea pig cerebral cortex

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of ¹²⁵I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of ¹²⁵I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a Bmax of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of ¹²⁵I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) ≥ PACP38 ∼ PACAP27 ∼ guinea pig VIP (gpVIP) ≥ mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of ¹²⁵I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific ¹²⁵I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [³H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 μM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 μM, produced net increases in cAMP production in the range of 8–9% conversion, whereas 5 μM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC 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.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in melatonin release via the specific receptor PACAP-r1, but not in the circadian oscillator,in chick pineal cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates melatonin release from pineal cells and modulates glutamatergic regulation of the suprachiasmatic circadian clock in rodents. We investigated whether PACAP is involved in melatonin release and the circadian oscillation system in chick pineal cells, and if so, whether its effects are mediated by the PACAP-specific receptor (PACAP-r1) or the vasoactive intestinal polypeptide (VIP) receptor. Chick pineal cells were maintained for 4 days under a 12-h light/dark cycle, and thereafter in constant darkness. In the dose-range 10(-10) to 10(-6) M, PACAP increased melatonin release dose-dependently during the 12-h light period on day 3 of culture, and the degree of stimulation was greater than that produced by VIP. VIP receptor antagonists only slightly inhibited PACAP-stimulated melatonin release. Simultaneous addition of VIP and PACAP produced almost additive melatonin release. Under constant dark conditions, 6-h pulses of PACAP started at zeitgeber times (ZT) 15, 21, 3 and 9 h in separate groups of pineal cells did not cause any phase shift in their melatonin rhythm. In addition, PACAP did not affect the light-induced phase advance (ZT 15 h) and delay (ZT 9 h) in melatonin rhythms. The expression of mRNA for the PACAP-r1 (including its splicing variant with a hop cassette) was observed in chick pineal cells. These results suggest that PACAP participates in melatonin release, but not in the circadian oscillator system, via the specific receptor PACAP-r1 in chick pineal cells.     

Distribution of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors (VPAC1, VPAC2, and PAC1 receptor) in the rat brain

To examine the distributions of VIP/PACAP receptors (VPAC1, VPAC2, and PAC1 receptors) in the brain and to identify the cell types that express these receptors, we performed immunohistochemistry and double immunofluorescence in the rat brain with specific antibodies. The immunohistochemistry revealed that the receptors had distinctive, complementary, and overlapping distribution patterns. High levels of the VPAC1 receptor were expressed in the cerebral cortex, hippocampal formation, deep cerebellar nuclei, thalamus, hypothalamus, and brainstem. The VPAC2 receptors were concentrated in the cerebral cortex, hippocampal formation, amygdalar regions, cerebellar cortex, deep cerebellar nuclei, hypothalamus, and brainstem. On the other hand, the PAC1 receptors had a more restricted distribution pattern in the brain, and high levels of the PAC1 receptors were confined to the cerebellar cortex, deep cerebellar nuclei, epithalamus, hypothalamus, brainstem, and white matter of many brain regions. Also, many fibers expressing the PAC1 receptors were observed in various areas, i.e., the thalamus, hypothalamus, and brainstem. The double immunofluorescence showed that the VIP/PACAP receptors were confined to the neuroglia as well as the neurons. All three types of the VIP/PACAP receptors were expressed in the astrocytes, and the PAC1 receptors were also expressed in the oligodendrocytes. These findings indicate that VIP and PACAP exert their functions through their receptors in specific locations in different combinations. We hope that this first demonstration of the distributions of the VIP/PACAP receptors provides data useful in the investigation of the mechanisms of the many functions of VIP and PACAP in the brain, which require further elucidation.     

Interaction of Forskolin and Melatonin on Cyclic AMP Generation in Pars Tuberalis Cells of Ovine Pituitary

The pineal indoleamine, melatonin, acts on specific secretory cells of the pars tuberalis of the sheep pituitary. Using pars tuberalis cells in primary culture melatonin inhibited forskolin-stimulated cyclic AMP production in both a time - and dose-dependent manner, but the nature of the melatonin response was critically dependent upon the stimulatory concentration of forskolin used. Forskolin alone stimulated dose-dependent cyclic AMP accumulation, which reached an equilibrium state after 15 min. This was maintained for up to 3 h, indicating a lack of desensitization to forskolin. Melatonin (1 μM) inhibited this response by greater than 80% at all doses. However, 100 μM forskolin reduced both the affinity and the number of the melatonin receptors, relative to untreated controls. Consistent with this, melatonin was 100 times less potent at inhibiting forskolin-stimulated cyclic AMP production, when titrated against 100 μM forskolin as compared to 1 μM forskolin. The response to 1 μM forskolin could be potentiated by 10 μM phorbol 12,13 myristate acetate, but not by calcium ionophore (A23187). This provides evidence for the interaction of the phosphatidylinositol pathway with the cyclic AMP system in these cells. Nevertheless melatonin can inhibit both the potentiated and non-potentiated response with equal effect.     

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 (K _d =0.21 n M) and low capacity (B _max=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) ≥ VIP (mammalian) ≥ PACAP27 ≥ PACAP38 ≫ VIP6-28 (mammalian) > PHI (porcine) ≫ neurotensin6-11-chicken VIP7-28 > neurotensin6-11—mammalian VIP7-28 ⋙ VIP16-28 (chicken; inactive) ≈ 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.     

Effect of opioid peptides onl-noradrenaline-stimulated cyclic AMP formation in homogenates of rat cerebral cortex and hypothalamus

Morphine and the opioid peptides leucine-enkephalin (leu-enk), methionine-enkephalin (met-enk) and beta-endorphin had no effect on basal cyclic AMP levels in rat cerebral cortex and hypothalamus, but each inhibited noradrenaline (NA)-stimulated cyclic AMP formation in both brain regions. This inhibition was reversed by naloxone. Naloxone did not reverse phentolamine- or propranolol-induced inhibition of NA-stimulated cyclic AMP formation. The increase in cyclic AMP formation induced by NaF or MnCl2 was unaffected by met-enk or morphine. These data suggest that in rat cerebral cortex and hypothalamus opiates bind to opiate receptors and that the opiate-receptor complex interferes with noradrenergic receptor activity.     

Distribution of pituitary adenylate cyclase activating polypeptide mRNA in the developing rat brain

PACAP is a member of the secretin/vasoactive intestinal peptide (VIP) family, isolated from hypothalamus. Recent studies have shown that PACAP is expressed in many parts of adult brain. We have studied the precise distribution of PACAP mRNA in developing rat brain, employing in situ hybridisation. PACAP mRNA is expressed in distinct parts of the embryonic rat brain from embryonic day 13, with a robust expression in developing cortex, hippocampus, amygdala and hypothalamus as well as in spinal cord and dorsal root ganglia. The expression in hippocampus and cortex diminishes towards adulthood, compared to new-born rat brain. In the mature brain, PACAP mRNA is located in alternating layers of cerebral cortex (layers I, III and V), in the dentate gyrus, in CA4 and CA1 regions, but not in CA2 or CA3 of the hippocampus. The presence of PACAP mRNA in different structures of developing rat brain suggests an important function for this peptide during brain development.     

Development of regulation of melatonin release in pineal cells in chick embryo

Melatonin release in a pineal cell culture from 13- and 14-day-old chick embryos increased during the dark phase and decreased during the light phase of a 12 h light:12 h dark cycle. When the light-dark cycle was reversed, the pattern of melatonin release in the culture also reversed. 8-Bromo cyclic-AMP stimulated melatonin release in both the light and dark phases. However, no rhythm of melatonin release was detected under constant dark (DD) conditions in a cell culture from 14-day-old chick embryos. In 18-day-old chick embryos, the pineal cell culture expressed a circadian rhythm of melatonin release under DD conditions. These results indicate that mechanisms regulating melatonin synthesis in the avian pineal gland are established during embryonic life.     

Cyclic AMP-generating systems: regional differences in activation by adrenergic receptors in rat brain

Catecholamine, histamine, and adenosine-mediated accumulations of radioactive cyclic AMP were assessed in adenine-labeled slices from eight rat brain regions. 2-Fluoronorepinephrine, a selective beta-adrenergic agonist, elicited an an accumulation of cyclic AMP in cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, thalamus, hypothalamus, and medulla-pons. In cerebral cortex and most other brain regions, the beta-adrenergic-mediated response appeared to involve primarily beta 1-adrenergic receptors, while in cerebellum, there was a significant involvement of beta 2-adrenergic receptors. 6-Fluoronorepinephrine, a selective alpha-adrenergic agonist, elicited accumulations of cyclic AMP in all regions except cerebellum. Combinations of the two fluoro derivatives afforded in all brain regions an accumulation of cyclic AMP identical with that elicited by norepinephrine. In hypothalamus, the alpha- and beta-adrenergic responses were significantly greater than additive. In cerebral cortex, the alpha-adrenergic receptor-mediated response appeared to involve alpha 1-adrenergic receptors and to be nearly completely dependent on adenosine, while in other brain regions, the dependence of the alpha-adrenergic response on adenosine was less or absent. Combinations of 6-fluoronorepinephrine and histamine had greater than additive effects in cortex and hippocampus. The results indicate that the interactive control of cyclic AMP-generating systems by alpha-adrenergic, beta-adrenergic, adenosine, and histamine receptors differs significantly among rat brain regions.     

Agents that affect calcium influx can change cyclic nucleotide levels in cultured chick pineal cells.

Previous experiments examined interactions among the effects of cyclic AMP and calcium-related agents on melatonin output by cultured chick pineal cells, and suggested that changes in calcium influx might act through cyclic AMP. Here, effects of calcium-related agents and manipulations on cyclic AMP (and cyclic GMP) levels are demonstrated directly. These effects support a role for cyclic AMP (but not cyclic GMP) in the effects of changes in calcium influx on melatonin production by these cells.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates melatonin synthesis from rat pineal gland

The effect of pituitary adenylate cyclase polypeptide (PACAP) on rat pineal was examined. PACAP stimulated melatonin release from cultured dissociated pinealocytes with a 10⁴ higher potency than isoproterenol (EC₅₀ were 30 pM and 250 nM, respectively). The 10⁻⁹ M PACAP stimulation was not inhibited by 5 × 10⁻⁶ M VIP antagonist whereas that of 10⁻⁹ M VIP was reduced by 54%. Kinetic analysis of melatonin release indicated that PACAP acts postsynaptically via receptor activation.     

Effects of PACAP on the Circadian Changes of Signaling Pathways in Chicken Pinealocytes

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of circadian rhythms. In mammals, the brain's biological clock is the suprachiasmatic nucleus, receiving photic information from the retina through the retinohypothalamic pathway, where PACAP is the main cotransmitter of glutamate. The primary conductor of circadian rhythms of birds is the pineal gland. The presence of PACAP has been demonstrated both in the rat and avian pineal gland, where PACAP stimulates melatonin synthesis. The signaling mechanism, by which PACAP modulates melatonin synthesis and circadian rhythmic functions of the pineal gland, is only partially known. The aim of the present study was to investigate the effects of PACAP on the changes of p38 mitogen-activated protein kinase (MAPK) and 14-3-3 protein in chick pineal cell culture both of which have been shown to participate in the regulation of rhythmic functions. Pineal cells were treated with 1, 10, or 100 nM PACAP38 every 4 h during a 24-h period. The phosphorylation of p38 MAPK showed obvious changes during the observed 24 h, while the level of 14-3-3 protein did not. We found that the lowest used dose of PACAP did not cause any phase alteration in p38 MAPK phosphorylation. Ten nM PACAP induced a 4-h-long delay and 100 nM abolished the circadian changes of p38 MAPK phosphorylation. PACAP was not effective on the level of 14-3-3 protein in the early morning hours, and only the highest tested dose (100 nM) could evoke a change in the appearance of 14-3-3 between midday and midnight hours. In summary, PACAP modulated the phosphorylation of p38 MAPK and the appearance of 14-3-3 protein in the chicken pineal cells, but these effects were dose dependent and also depended on the time of day.     

Melatonin inhibits cyclic AMP and cyclic GMP accumulation in the rat pituitary

Subnanomolar concentrations of melatonin inhibit cyclic AMP and cyclic GMP accumulation in neonatal rat anterior pituitary stimulated in vitro with luteinizing-hormone releasing-hormone. Melatonin also inhibited forskolin-stimulated cyclic AMP accumulation in pars tuberalis. Inhibition of cyclic AMP accumulation is specific for melatonin, since its analogs N-acetylserotonin and 5-methoxytryptamine are 1000 times less potent. Cyclic nucleotides may thus serve as second messengers transducing the effect of melatonin on cellular level.     

Photoendocrine transduction in cultured chick pineal cells. II. Effects of forskolin, 8-bromocyclic AMP, and 8-bromocyclic GMP on the melatonin rhythm

We have recently described a system, using dispersed chick pineal cells in static culture, which displays a persistent, photosensitive, circadian rhythm of melatonin release, and the effects of light and darkness upon it. Here, we describe the effects of forskolin (FSK), a specific activator of adenylate cyclase, 8-bromocyclic AMP (8BrcA), an analogue of cyclic AMP, and 8-bromocyclic GMP (8BrcG), an analogue of cyclic GMP, on melatonin output by these cells. FSK stimulated melatonin output; it was potent and effective. 8BrcA, but not 8BrcG, also markedly stimulated melatonin output. These results support a role for cAMP (but not cGMP) in the regulation of melatonin production. Four hour pulses of white light or darkness, in otherwise constant red light, cause, in addition to acute effects, phase-dependent phase shifts of the melatonin rhythm in subsequent cycles. Such phase shifts indicate an effect on (proximal to) the pacemaker generating the rhythm. Four or 8 hour pulses of FSK, 8BrcA, or 8BrcG, however, did not appreciably alter the phase of subsequent melatonin cycles. Neither did they interfere with phase shifts induced by light pulses. These results fail to support a prominent role for cAMP or cGMP levels in regulating the pacemaker; nor do these cyclic nucleotides appear to mediate the prominent effects of light and dark on the pacemaker. Thus, cyclic AMP regulation of melatonin appears to occur distal to the pacemaker.     

Circadian rhythm of melatonin release in pineal gland culture: arg-vasopressin inhibits melatonin release

The mammalian pineal gland is known to receive a noradrenergic sympathetic efferent signal from the suprachiasmatic nucleus (SCN) via the superior cervical ganglion. Arg-vasopressin (AVP) containing neurons in the SCN is one of the output paths of circadian information to the other brain areas. AVP release from the SCN is suppressed by melatonin. In turn, we determined the direct effect of AVP on melatonin release using pineal gland explant culture. AVP (1 μM) suppressed melatonin release. Noradrenaline stimulated melatonin release was attenuated by AVP. In turn, the expression of the melatonin synthesis enzyme arylalkylamine N-acetyltransferase mRNA in the rat SCN was reported. We measured melatonin content in the SCN in rats kept under the light–dark cycle and constant dim light. Melatonin in the SCN was higher during the dark period than that in the light. A similar tendency was also observed in the SCN of animals kept under a constant dim light. It was suggested that the reciprocal regulation of melatonin release and AVP release occurs in the SCN and pineal gland.