Adrenergic and peptidergic control of the regulation of cAMP efflux and melatonin secretion from perifused rat pineal gland

Mammalian pineal gland receives peptidergic (e.g., vasoactive intestinal peptide [VIP]; peptide histidine isoleucine [PHI]; neuropeptide Y, NPY; substance P, calcitonin gene-related peptide [CGRP], arginine vasopressin [AVP] and oxytocin [OXT]) fibers in addition to sympathetic innervation. The dynamics of cAMP efflux and melatonin (MT) secretion were compared during the infusion of these peptides in our long-term perifusion system. VIP and PHI enhanced both pineal cAMP efflux and MT secretion in a dose-dependent manner (10 nM to 10 μM). However, the potency of PHI was slightly less. The peak of cAMP release always precedes that of MT production. The possible interactions between adrenergic and peptidergic compounds in the regulation of pineal cAMP efflux and MT secretion were also studied. VIP acts on specific peptidergic receptors, since its stimulatory effect could only be reduced by a VIP receptor antagonist. VIP has an additive effect at a lower (100 nM) concentration combined with norepinephrine (NE). NPY (100 nM) can completely block NE-induced MT secretion, but the decrease in cAMP efflux is less. However, NPY does not significantly influence VIP-stimulated cAMP efflux or MT secretion. These data suggest that NE, VIP, and NPY are differently involved in the cAMP and calcium signaling. The other neuropeptides are ineffective.     

Vasoactive intestinal peptide stimulates N-acetyltransferase and hydroxyindole-O-methyltransferase activities and melatonin production in cultured rat but not in Syrian hamster pineal glands

The purpose of this study was to compare the responses of the Syrian hamster and rat pineal glands in organ culture to vasoactive intestinal peptide (VIP). The endpoints in these studies were the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as pineal and medium melatonin levels. When rat pineal glands were incubated with either VIP (1 microM) or isoproterenol (1 microM), a beta-adrenergic agonist, a significant increase in NAT and HIOMT activities and melatonin levels were observed within 3 hr. Conversely, during the day, VIP (1 microM) was ineffective in stimulating these parameters in hamster pineal gland after incubation times of either 2, 4, 6, or 8 hr. In another experiment, hamster pineal glands were collected from animals killed in the late dark period (after 30 min light exposure). In these glands, isoproterenol promoted NAT activity and melatonin production; however, VIP was ineffective in stimulating either NAT or HIOMT activities; likewise, VIP had no stimulatory effect on pineal melatonin levels at night. Finally, when hamster pineal glands at night were incubated with either 0, 10 nM, 100 nM, 10 microM, or 100 microM VIP, no changes in any parameter of melatonin synthesis were measured. The results indicate that the hamster pineal gland, unlike that of the rat, may not respond to VIP with an increased melatonin production.     

The effect of PACAP on rhythmic melatonin release of avian pineals

Pituitary adenylate cyclase activating polypeptide (PACAP) is a ubiquitous member of the VIP/secretin/glucagon bioactive peptide family. The distribution, concentration of PACAP, and its role in the control of rhythmic melatonin (MT) secretion from chicken pineal gland were studied. In the chicken pineal gland (ChPG), 40ng PACAP/g tissue was measured with radioimmunoassay. No midday-midnight differences in the PACAP content of the ChPG could be detected. Immunohistochemical studies of ChPG showed PACAP immunoreactive nerve fibers in the perivascular space and around the pinealocytes. Neither PACAP-labeled perikarya nor PACAP immunopositivity in the pinealocytes could be detected. In five day perifusion experiments, carried out under darkness, both MT and cAMP showed circadian rhythmic release pattern from explanted ChPG. One hour exposure of ChPG to PACAP induced transient (3-4h) elevation of MT and cAMP release. The responses were dose-dependent in the range from 1 to 100nM PACAP concentrations. The magnitude of the response was independent on the phase of the daily cycle in which PACAP was applied. cAMP levels during normal daily cycle and also PACAP-induced cAMP responses always preceded similar changes of MT by about an hour suggesting that cAMP is an intracellular intermediate in controlling MT release from the ChPG. At the same time PACAP, similarly to VIP, did not shift the phase of the in vitro circadian MT rhythm of the pineals. Our data reveal, that PACAP is present in nerve fibers in the chicken pineals and pineal cells contain functioning PACAP-sensitive receptors. PACAP apparently exerts a modulatory effect of the rhythmic MT release from the chicken pineal but does not modify the intrinsic biological clock in the avian pineal gland. Thus, cAMP-mediated intracellular mechanisms in ChPG are not components of the pineal circadian clock, but intermediaries between the clock-mechanism and MT release and may also be components of clock-independent MT release modifiers.     

Peptide specificity for stimulation of corticotropin secretion: activation of overlapping pathways by the vasoactive intestinal peptide family and corticotropin-releasing factor

The hypothalamic peptide vasoactive intestinal peptide (VIP) stimulates ACTH and endorphin secretion by the AtT20/D16 clonal strain of mouse pituitary tumor cells. The dose dependence for VIP stimulation of hormone release is biphasic, indicating that VIP is able to activate at least two classes of receptors in D16 cells (ED50 = 1.6 and 160 nM). We show that at high concentrations (ED50 greater than or equal to 150 nM), other natural peptides with primary structures homologous to that of VIP also increased ACTH secretion by D16 cells, whereas structurally unrelated peptides did not. The stimulatory actions of GH-releasing factor (GRF) and porcine heptacosapeptide with amino-terminal histidine and carboxy-terminal isoleucine amide (PHI) were mediated by high affinity VIP receptors because their effects were not additive with that of 10 nM VIP. In addition, GRF and PHI behaved as antagonists at low affinity VIP receptors; both peptides inhibited stimulation by 1 microM VIP. In contrast, glucagon and gastric inhibitory polypeptide appeared to stimulate ACTH release via low affinity VIP receptors because their effects were additive with that of 10 nM, but not 1 microM, VIP. Since all of the VIP-like peptides increased ACTH secretion only at high concentrations, they were unlikely to represent a physiological ligand for the receptor activated by high concentrations of VIP. Therefore, we determined whether cross-reactivity occurred between VIP-like peptides and corticotropin-releasing factor (CRF), a potent stimulator of ACTH secretion both in vitro and in vivo. The dose-response curve for CRF stimulation of ACTH secretion by D16 cells extended over more than a 1000-fold range of concentrations and was biphasic (ED50 = 2.6 and greater than 300 nM), indicating that CRF interacted with multiple receptor types in D16 cells. However, since the effect of 10 nM CRF was additive with that of 1 microM VIP, the CRF receptor was not the site at which high concentrations of VIP stimulated ACTH release. In contrast, the effect of 1 microM CRF was not additive with that of 1 microM VIP or other VIP-like peptides. Therefore, high concentrations of CRF and the previously recognized VIP-like peptides stimulated ACTH secretion by overlapping pathways. Comparison of the amino acid sequence of CRF with those of the VIP-like peptides showed that 18 of the 41 amino acids in CRF match a corresponding amino acid in at least 1 member of the VIP peptide family.(ABSTRACT TRUNCATED AT 400 WORDS)     

The immature rat ovary is innervated by vasoactive intestinal peptide (VIP)-containing fibers and responds to VIP with steroid secretion

The nature and role of the peptidergic innervation of the ovary were examined by determining the location and function of vasoactive intestinal peptide (VIP)-containing nerve fibers in the immature rat ovary. Immunohistofluorescence analysis of prepubertal ovaries using a specific VIP antibody revealed sparse delicate VIP-immunoreactive fibers localized around veins and arterioles, in the interstitial tissue, and associated with the thecal layers of developing follicles. Radioimmunoassayable VIP content was found to be approximately 100 pg/ovary (3 nM). The VIP immunoreactivity coeluted with authentic VIP when subjected to Sephadex G-25 chromatography. VIP enhanced in vitro progesterone release from infantile (12 days old), juvenile (30 days old), and peripubertal ovaries and estradiol release during the two latter developmental periods. The maximal estradiol response to VIP occurred during the early and first proestrous phases of puberty. No response was observed during estrus or first diestrus. The progesterone response to VIP increased moderately between day 12 and first proestrus, and then strikingly at estrus and first diestrus. The stimulatory effect of VIP on ovarian steroid production was dose related, as determined in ovaries from PMSG-treated immature rats (ED50 = 215, 44, and 51 nM for estradiol, androgen, and progesterone, respectively). The specificity of the VIP effect was tested using five other gastrointestinal peptides (porcine peptide histidine isoleucine, gastric inhibitory polypeptide, secretin, motilin, and glucagon). Only peptide histidine isoleucine, which has the greatest sequence homology with VIP, enhanced ovarian steroid production at 50% of VIP effectiveness. VIPergic nerves thus appear to be involved in the developmental regulation of ovarian steroidogenesis.     

Beta-adrenergic and peptide N-terminal histidine and C-terminal isoleucine stimulation of N-acetyl-transferase activity and melatonin production in the cultured rat pineal gland.

The purpose of this investigation was to compare the effect of peptide N-terminal histidine and C-terminal isoleucine (PHI) with that of the beta-adrenergic agonist isoproterenol (ISO) on N-acetyltransferase (NAT) activity and melatonin production in the cultured rat pineal gland. Pineal glands were removed and placed in organ culture containing PHI, ISO, or PHI plus ISO at different dosages, and subsequent changes in NAT activity and melatonin levels were measured. PHI stimulated these parameters in both a time- and dose-dependent manner. Combined treatment with PHI (10(-6) M) and high doses of ISO (either 10(-7) or 10(-8) M) did not potentiate the effect of the peptide in terms of either NAT activity or melatonin levels in the pineal gland. However, at a lower concentration, ISO (10(-9) M) had additive effects to those of PHI in both cultured pineals and medium. The results suggest that PHI modulates melatonin synthesis in the rat pineal gland. Furthermore, stimulation of the pineal with both PHI and ISO demonstrates an additive effect rather than a synergistic action of these compounds. It is presumed that ISO and PHI stimulate pineal melatonin production via separate receptors, but they probably use the same intracellular second messenger, cAMP, to do so. This is the first study showing an effect of the peptide PHI on pineal melatonin production in any vertebrate.     

Involvement of 5-lipoxygenase pathway in norepinephrine stimulation of rat pineal melatonin synthesis

The effect of lipoxygenase inhibition, leukotriene agonists and antagonists, and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was examined in the rat pineal gland in organ culture. To study melatonin secretion pineal explants were incubated for 6 h in tissue culture medium 199 with the different drugs. Melatonin concentration in the pineal gland and the medium was measured by RIA. Exposure of explants to norepinephrine (NE) brought about a 2- to 5-fold increase in both parameters, an effect that was reduced but not abolished, by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 10(-5) M). Lilly 171883 (10(-5) M) or FPL 55712 (10(-5) M; both antagonists of leukotrienes) reduced NE-induced melatonin production. Neither NDGA nor Lilly 171883 affected melatonin production in the absence of NE. Leukotrienes C4 and D4 increased melatonin release to the media at all concentrations tested (1-1,000 nM) with a maximum effect at 1 nM (leukotriene C4) and 10 nM (leukotriene D4). Significantly higher tissue melatonin concentrations as compared to controls were observed after exposure of pineal explants to 1 and 100 nM of leukotriene C4, or 100 nM of leukotriene D4. Another 5-lipoxygenase metabolite, 5-HETE, increased pineal melatonin content at concentrations of 1, 10 and 100 nM whereas only 1,000 nM stimulated melatonin release. These results suggest that the 5-lipoxygenase pathway plays a significant role in NE-stimulated melatonin production by the rat pineal gland.     

Vasoactive intestinal peptide binding to specific receptors on rat parotid acinar cells induces amylase secretion accompanied by intracellular accumulation of cyclic adenosine 3'-5'-monophosphate

Specific binding sites for vasoactive intestinal polypeptide (VIP) were characterized in dispersed rat parotid acini. The binding of [125I]VIP was rapid, saturable, reversible, and temperature dependent. Scatchard analysis indicated two functionally independent classes of receptor sites: 41,000 high affinity-low capacity sites per cell with a dissociation constant (Kd) of 6.4 nM and 420,000 low affinity-high capacity sites per cell with a Kd of 150 nM. A peptide with N-terminal histidine and C-terminal isoleucine and secretin, which are structurally related to VIP, inhibited the tracer binding 30 and 200 times less strongly, respectively, than VIP. Epinephrine and carbachol did not inhibit [125I]VIP binding to parotid acinar cells. VIP stimulated cAMP accumulation in parotid lobules and induced amylase secretion in a dose-dependent manner. A peptide with N-terminal histidine and C-terminal isoleucine and secretin were less potent than VIP regarding cAMP accumulation (1/12 and 1/80 of VIP, respectively) and amylase secretion (1/40 and 1/500 of VIP, respectively). Substance P did not stimulate cAMP accumulation but stimulated amylase secretion more strongly than VIP. These observations clearly demonstrated the presence of VIP receptors coupled to adenylate cyclase system in the rat parotid gland, which plays an important role in the regulation of the amylase secretion. The regulation of parotid function by VIP was independent of the adrenergic or muscarinic regulatory system and of the influence of substance P.     

Adenosine effects on the rat pineal gland in vitro: Cyclic adenosine monophosphate levels, N-acetyltransferase, and thyroxine type II 5''-deiodinase activities and melatonin production

The presence of adenosine and its receptors in the pineal gland suggests that they could have a regulatory influence on pineal physiology. Rat pineal glands cultured in the presence of the adenosine analog 5'-N-ethylcarboxamido adenosine (NECA) promoted a significant increase in pineal adenosine 3'5'-monophosphate (cAMP) content, similar to that caused by the beta-adrenergic receptor agonist isoproterenol (ISO). A potentiation of the cAMP response occurred when the glands were simultaneously stimulated with both NECA and ISO. Whereas high cAMP levels induced by ISO were associated with an increased N-acetyltransferase (NAT) activity and rise in the melatonin production and release, the NECA-induced rise in cAMP concentration failed to promote an increase in the activity of either NAT or another pineal enzyme thyroxine 5'-deiodinase type II; likewise, pineal melatonin levels did not rise.     

Somatostatin inhibits basal and vasoactive intestinal peptide-stimulated hormone release by different mechanisms in GH pituitary cells

Somatostatin (SRIF) inhibits both basal and vasoactive intestinal peptide (VIP)-stimulated hormone secretion by the GH4C1 clonal strain of rat pituitary tumor cells. We have previously shown that SRIF inhibits cAMP accumulation stimulated by VIP but does not alter basal cAMP levels in this cell line. To determine the importance of changes in cAMP accumulation in the mechanism of SRIF action, we have compared the effect of SRIF on hormone release stimulated by VIP and two other secretagogues which increase effective intracellular cAMP concentrations: forskolin and 8-Bromo-cAMP (8-Br-cAMP). VIP stimulated GH and PRL secretion to the same maximal extent (220% of control) with similar ED50 values (0.37 +/- 0.03 and 0.43 +/- 0.08 nM, mean +/- SE, respectively). SRIF (100 nM) reduced maximal VIP-stimulation of both GH and PRL release from 220 to 140% of control; however, it did not significantly change the ED50 values for VIP. The effect of SRIF on VIP-stimulated hormone release parallels its action on VIP-stimulated cAMP accumulation. Furthermore, the concentrations of SRIF required to produce half-maximal inhibition of VIP-stimulated GH and PRL release (0.8 +/- 0.2 nM and 0.7 +/- 0.1 nM, respectively) were similar to its potency to inhibit VIP-stimulated cAMP accumulation (1.2 +/- 0.1 nM). These data indicate that changes in cAMP levels mediate inhibition of VIP-stimulated hormone secretion by SRIF. Forskolin increased cAMP accumulation with an ED50 value of 2.4 +/- 0.5 microM. A maximal concentration of forskolin (100 microM) stimulated cAMP accumulation to a greater extent than 100 nM VIP (34 +/- 4-fold vs. 9 +/- 1-fold). Together, forskolin (100 microM) and VIP (100 nM) stimulated cAMP accumulation by more than 50-fold. However, PRL secretion in response to maximal concentrations of VIP or forskolin individually or together were the same (approximately 200% of control). These results support the conclusion that both compounds stimulate PRL secretion by a cAMP-mediated mechanism which can be fully activated by either one alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diurnal changes in cyclic nucleotide response to pineal indoles in murine mammary glands

The aim of this study was to determine whether pineal indoles affect cyclic nucleotide levels in mammary gland slices of BALB/c adult mice. Melatonin at 0.1 nM-10 microM concentrations decreased cAMP and augmented cGMP concentration in murine mammary gland slices in the presence of a phosphodiesterase inhibitor (1 mM theophylline), an index of cyclic nucleotide synthesis. Melatonin-induced changes in cyclic nucleotide levels were significantly larger at the end of the light period (2000) than in the morning (at 1000). Indole-induced inhibition of cyclic AMP levels by mammary slices exhibited the following order of potency: 5-methoxytryptamine > melatonin > or = 6-hydroxymelatonin > serotonin, N-acetylserotonin > 5-hydroxytryptophol. The order of potency for indole-induced augmentation of cyclic GMP levels was: 5-methoxytryptamine > melatonin > 6-hydroxymelatonin > serotonin, N-acetylserotonin, 5-hydroxytryptophol. When melatonin or 5-methoxytryptamine (10 nM) were examined for their effects on cAMP and cGMP levels in mammary glands of mice killed at six different time intervals during the 24-hr cycle, the activity was maximal during night. The data demonstrate that 5-methoxytryptamine and melatonin decreased cAMP and increased cGMP levels in mammary gland slices. Methoxyindole-induced changes in cyclic nucleotide synthesis in murine mammary glands exhibit the time-dependency known to occur in several other melatonin-influenced responses.     

Stimulation of Serotonin-N-Acetyltransferase Activity in the Pineal Gland of the Mongolian Gerbil (Meriones unguiculatus) by Intracerebroventricular Injection of Vasoactive Intestinal Polypeptide

There is ever-increasing evidence that intrapineal peptides have an important role in the modulation of pineal melatonin synthesis. In the pineal gland of the Mongolian gerbil ( Meriones unguiculatus ), we have previously shown the presence of VIP-immunoreactive nerve fibers as well as pinealocytic VIP receptors. To assess the functional significance of these findings, 10 μl of a 1 μM or 1 nM solution of VIP were injected into the lateral ventricle of gerbils over a period of 10 min. Animals were killed 1.5 hr after injection, and the superficial pineal glands were excised and assayed for N-acetyltransferase (NAT) activity. Injection of the 1 μM VIP solution stimulated the NAT activity to values four times the control values. The results are compatible with an in vivo influence on the pineal gland indole metabolism of the nonsympathetic VIP-containing nerve fibers via VIP-receptors present in the gland.     

Adenosine stimulates adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission

Adenosine produces a concentration-dependent increase in pinealocyte cAMP (EC50, approximately 0.3 nM) and cGMP accumulation (EC50, approximately 0.7 nM). Maximal increases in both nucleotides are evident 10 min after treatment; 1 h later values return to pretreatment levels. Concentration-dependent effects on cAMP are also observed with N6-(L-2-phenylisopropyl)adenosine (EC50, approximately 0.75 nM), 5'-N-ethylcarboxy aminoadenosine (EC50, approximately 0.75 nM), and 2-chloroadenosine (EC50, approximately 2.0 nM); the EC50 values for stimulation of cGMP with these agents are higher by a factor of 2-10. In the case of 5'-N-ethylcarboxy amidoadenosine, the concentration-response curve is biphasic, with a significant effect evident within the range of 1-100 pM. The stimulatory nature of this response and the relative potency of the agonists tested are consistent with the involvement of an A2-like adenosine receptor. Comparison of adenosine and the selective beta-adrenergic agonist isoproterenol indicated that their maximal EC50 values were generally similar. Studies with antagonists revealed that both 8-(p-sulfophenyl)theophylline (1 microM) and the xanthine amine congener (8-[4-[[[(2-aminoethyl)carbonyl]methyl]oxy]phenyl]1,3- dipropylxanthine (1 microM) inhibited the effects of adenosine (1 nM to 1 microM), but xanthine amine congener was more potent; the latter was markedly effective at 0.1 nM, whereas 8-(p-sulfophenyl)theophylline was nearly ineffective at this concentration. It was also determined that pineal cells generate extracellular adenosine from extracellular ATP. ATP is thought to be released along with catecholamines during neurotransmission. Hence, these studies support the view that adenosine could participate in the transsynaptic regulation of pineal function.     

Commonalities in vasoactive intestinal peptide and peptide N-terminal histidine C-terminal isoleucine stimulation of N-acetyltransferase activity in the rat pineal

Abstract: Exposure of adult rat pineal glands in organ culture to the polypeptides vasoactive intestinal polypeptide (VIP), and peptide N-terminal histidine C-terminal isoleucine (PHI) increases pineal serotonin N-acetyltransferase (NAT) activity and melatonin synthesis. The following research results are taken to indicate that VIP and PHI share common components of the NAT induction system: (1) The effects of the two peptides are additive at concentrations of 10 nM VIP and 100 nM PHI but not at higher peptide concentrations. (2) Pineals from newborns also respond to PHI with a dose dependent increase in NAT activity. NAT responses are additive at the same concentrations as seen with the adult pineals. (3) Light exposure affects the sensitivity of pineals to VIP and PHI stimulation in a similar manner; pineals taken after 3 hr of light are much less sensitive to PHI or VIP than those taken after 13 hr of light. (4) Pineals exposed for 48 hr to either PHI or VIP have a reduced NAT response to either agonist, which is reversible by culture in agonist-free media. (5) Neither VIP nor PHI stimulation of NAT activity is affected by concentrations of the VIP antagonists (N-Ac-Tyr¹, D-Phe²)-GRF(l-29)-NH2 (NAcTDGRF), L-8-K, VIP-Neurotensin Hybrid (VIPNET), or (4Cl-D-Phe⁶, Leu¹⁷)-VIP (4C1PLVIP), which affect VIP binding or function in other tissues.     

Status of Dopamine in Bovine Pineal Glands and the Stimulation of N-Acetyltransferase Activity by D2-Dopaminergic Receptor Agonists in the Rat Pineal Glands in Culture

In a previous study, we identified in the bovine pineal gland two [3H]spiroperidol-binding sites with KD values of 0.18 and 2.1 nM and Bmax values of 37 and 630 fmol/mg protein, respectively. In this study, the status of dopamine in the bovine pineal glands was delineated further by measuring the relative concentrations of dopamine and norepinephrine and the relative concentrations of serotonin and melatonin. Furthermore, the presence of 4.0 +/- 0.6 micrograms/dopamine/gm tissue encouraged us to delineate the effects of select dopaminergic receptor agonists and antagonists on the synthesis of melatonin in vivo and on the activity of N-acetyltransferase in the rat pineal gland in culture. The acute administration of haloperidol (3 mg/kg intraperitoneally [ip]) or sulpiride (200 mg/kg ip) increased the concentration of melatonin in the pineal gland from 160.6 +/- 8.18 to 327.6 +/- 45.43 and 306.5 +/- 40.53 pg/gland, respectively. Dopamine exhibited dual effects on the activity of N-acetyltransferase, inhibiting the basal activity at 0.1 microM and stimulating it at 10 microM, and the later effect was blocked by propranolol. D2-dopaminergic receptor agonists such as bromocriptine (4.0 microM) or LY-171555 (10.0 microM) partially attenuated the norepinephrine-induced stimulation of N-acetyltransferase, and these attenuating effects were reversed by D2-dopaminergic antagonists such as haloperidol (10 microM) or domperidone (10 microM). The results of these studies are interpreted to indicate that for the synthesis of melatonin, the pineal D2-dopaminergic receptors may function independently from those of the beta 1-adrenergic receptor sites. Furthermore, the said D2-dopaminergic receptor are amenable to down regulation since the activity of N-acetyltransferase remained unaltered (0.0717 vs. 0.0729 nmol/gland/h) following chronic treatment (4 mg/kg ip/day for 30 days) with bromocriptine.     

Prostaglandin E2 increases adenosine 3',5'-monophosphate concentration and binding-site occupancy, and stimulates serotonin-N-acetyltransferase activity in rat pineal glands in vitro

The effects of prostaglandins (PGs) on rat pineal metabolism were examined in vitro. PGE2 (0.01-1 microM) increased the activity of serotonin-N-acetyltransferase (SNAT), the stimulation curve exhibiting a maximum at 0.1 microM. PGE1 increased SNAT activity only at the highest dose (1 microM) whereas PGF2 alpha, 15-keto-PGF2 alpha or PGI2 did not affect the enzymic activity. The stimulation of SNAT activity brought about by PGE2 in pineals from ganglionectomized rats was greater than in sham-operated controls at all the doses studied, suggesting that the observed effect is predominantly post-synaptic. Only PGE2 significantly increased pineal cAMP accumulation in vitro at doses between 0.01 and 1 microM, and depressed the unoccupied cAMP-binding sites in pineal 900 g supernatants. The total number of cAMP-binding sites remained unaltered after incubation of PGE2. The present observations together with the previously reported NE-induced release of PGs in incubated pineal glands, the occurrence of pineal PG-binding sites and the indomethacin blockade of the nocturnal rise of pineal SNAT and melatonin content, support a role for PGs in the control of melatonin synthesis.     

Interleukin-1 stimulates interleukin-6 release from rat anterior pituitary cells in vitro

We have reported previously that anterior pituitary cells released interleukin-6 (IL-6) and that this release was stimulated by lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or agents that increased intracellular cAMP concentrations. We now report that IL-1 stimulates IL-6 release from anterior pituitary cells in vitro. IL-1 alpha and IL-1 beta (0.04-25 ng/ml) significantly increased IL-6 release 3- to 4-fold in a concentration-related manner during 6-h incubations; however, there was no change in extracellular or intracellular cAMP concentrations. IL-1 alpha and IL-1 beta (10 ng/ml), vasoactive intestinal peptide (VIP, 500 nM), prostaglandin E2 (PGE2, 1 microM), and LPS (1 ng/ml) stimulated IL-6 release to a similar degree. In the presence of VIP and PGE2, IL-1 alpha and IL-1 beta increased IL-6 release without any apparent further change in extracellular or intracellular cAMP. Conversely, LPS did not increase cAMP concentrations, and IL-1 did not significantly increase IL-6 release in the presence of LPS. The preexposure of anterior pituitary cells to 1 microM PMA caused the apparent down-regulation of protein kinase C activity because 100 nM PMA was no longer effective to stimulate IL-6 release; however, the ability of IL-1 alpha, IL-1 beta, PGE2, or LPS to stimulate IL-6 release was not altered. In addition, IL-1 alpha and IL-1 beta stimulated IL-6 release in the presence of maximally stimulative concentrations of PMA. The synthetic glucocorticoid dexamethasone (10 nM) significantly inhibited IL-6 release induced by IL-1 alpha, IL-1 beta, or LPS. The separation of anterior pituitary cells on unit gravity BSA gradients generated fractions of IL-6-producing cells that were inducible by LPS and IL-1 beta and separate from the PRL-, ACTH-, GH-, or LH-producing cell fractions. These data suggest that IL-1 stimulates IL-6 release from a subpopulation of anterior pituitary cells via a glucocorticoid-sensitive and non-cAMP-mediated pathway that is different from those pathways used by VIP, PGE2, and PMA.     

Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GH4C1) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca2+

Vasoactive intestinal peptide (VIP) and peptide (P) with N-terminal histidine and C-terminal isoleucine (PHI) stimulated prolactin (PRL) secretion from GH4C1 cells equipotent with ED50 values of 30-50 nM. In a parafusion system optimized to give high time resolution both VIP and PHI increased PRL secretion with a delay of about 60 s and subsequent to the activation of the adenylate cyclase. Thyroliberin (TRH) increased PRL secretion within 4 s. The dose-response curves for VIP- and PHI-stimulated cAMP accumulation were superimposable on those for PRL secretion. At submaximal concentrations the effects of VIP and PHI on both cAMP accumulation and PRL secretion were additive, whereas the effects were not additive at concentrations giving maximal effects. VIP and PHI increased [Ca2+]i measured by quin-2 in a different way than TRH, without inducing changes in the electrophysiological membrane properties of the GH4C1 cells. We conclude that both VIP and PHI stimulate PRL secretion via a cAMP-dependent process involving an increase in [Ca2+]i.     

How important is stimulation of α‐adrenoceptors for melatonin production in rat pineal glands?

The objective of this study was to determine the role of alpha-adrenoceptors in melatonin production by rat pineal gland. Pineal glands were isolated from adult male rats and maintained in organ baths. The perfusate was sampled every 5 min, stored, and later assayed for melatonin. Exposure to norepinephrine (10 microM) or the beta-adrenoceptor agonist orciprenaline (2-10 microM) increased the glands' production of melatonin. The time courses of melatonin production in response to these agonists were unaffected by the rats' pretreatment in vivo with the alpha-adrenoceptor antagonist prazosin (2 mg/kg i.p., three times). Rats that had had their superior cervical ganglia removed were primed with either orciprenaline (2 mg/kg i.p) or both orciprenaline and phenylephrine (1 mg/kg i.p) 1 hr before decapitation. Exposure of the pineal glands from these rats to orciprenaline evoked melatonin release that was similar in each group. These results lend weight to the suggestion that the marked potentiation by alpha-adrenoceptor agonists of the stimulation of cAMP and N-acetyltransferase (NAT) by beta-adrenoceptor agonists, demonstrated most readily in cultured glands or dispersed rat pinealocytes, does not carry over into significant augmentation of melatonin production in intact pineal glands.