Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in stimulating the secretion of prolactin from rat anterior pituitary cells in vitro

Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in regulating the secretion of prolactin (PRL) from the pituitary was investigated in the rat in vitro using two different methods: (1) short incubation of anterior pituitary cells and (2) superfusion of the pituitary cell column. PRL levels in the incubation medium were raised by addition of either VIP or PHI in concentrations of 10(-9) M to 10(-7) M in a dose-related manner. When both peptides were simultaneously added, additive stimulating effect on PRL release was obtained below the VIP concentration of 10(-7) M, in which no additive effect of PHI was revealed. PRL release from superfused rat pituitary cells was stimulated by 5-min pulses of VIP (10(-7) M), PHI (10(-7) M) and TRH (10(-8) M). Infusion of VIP for 200 min in the concentration of 0.3 x 10(-7) M resulted in an increase in basal release of PRL and blunted PRL release induced by not only VIP but also PHI stimulation, whereas PRL release induced by TRH was not affected. Infusion of PHI (0.3 x 10(-7) M, 0.7 x 10(-7) M and 10(-7) M) for 200 min also dose-relatedly suppressed PRL release induced by not only PHI but also VIP without any change in PRL release induced by TRH. These findings suggest that VIP and PHI may act through a common binding site on the pituitary lactotroph in the rat.     

Central galanin stimulates pituitary prolactin secretion in rats: possible involvement of hypothalamic vasoactive intestinal polypeptide

The effect of galanin, a newly identified neuropeptide, on pituitary prolactin (PRL) secretion was examined in the rat. Intracerebroventricular (i.c.v.) injection of all 5 doses of galanin (0.4, 1, 2, 5 and 10 micrograms/rat) raised plasma PRL levels in urethane-anesthetized rats. Galanin injection (2 micrograms/rat, i.c.v.) also increased plasma PRL levels in conscious rats. The intermediate dose of galanin (2 micrograms/rat, i.c.v.) produced a greater response in plasma PRL levels than either smaller or larger doses of galanin. Intravenous injection of galanin did not affect plasma PRL levels. Passive immunization with specific anti-vasoactive intestinal polypeptide (VIP) rabbit serum suppressed plasma PRL response to galanin (2 micrograms/rat, i.c.v.) in anesthetized rats. These findings indicate that central galanin has a stimulatory role in pituitary PRL secretion via the hypothalamus in the rat and that VIP may be involved in rat PRL release induced by galanin.     

Age-related changes in rates of basal secretion of immunoreactive vasoactive intestinal polypeptide and dopamine into pituitary stalk blood from the hypothalamus in anesthetized male rats

Age-related changes in prolactin (PRL) in systemic blood plasma, and in secretions of hypothalamic vasoactive intestinal polypeptide (VIP), an important candidate for PRL-releasing factor, and dopamine, a PRL-inhibiting factor, into the pituitary stalk blood were investigated. The experiments were performed on male urethane-chloralose-anesthetized Wistar rats of three different ages, i.e., (1) adult rats 5-8 months old, (2) middle-aged rats 12-15 months old, and (3) aged rats 24-26 months old. The concentration of immunoreactive PRL (iPRL) in systemic blood plasma of the aged rats was significantly higher than that of the adult rats (p less than 0.01). The secretion rate of hypothalamic immunoreactive VIP (iVIP) into the pituitary stalk blood was unchanged during aging, while that of dopamine was markedly increased in the aged rats in comparison with the value in both adult and middle-aged rats (p less than 0.01). These results indicate that the basal secretion of hypothalamic VIP is well maintained, while that of hypothalamic dopamine is augmented in aged rats with hyperprolactinemia. It can be assumed that the increase in the pituitary PRL secretion is a primary event during aging in rats, and that a high circulating level of PRL may facilitate the hypothalamic dopamine secretion through the activation of a negative feedback system of the hormone.     

The effect of dopamine on neurohypophysial hormone release in vivo and from the rat neural lobe and hypothalamus in vitro.

1. The rat hypothalamus (containing the supra-optic nuclei, paraventricular nuclei, median eminence and proximal pituitary stalk) has been incubated in vitro and shown to be capable of releasing the neurohypophysial hormones, oxytocin and arginine vasopressin, at a steady basal rate about one twentieth that of the rat neural lobe superfused in vitro. 2. The hypothalamus and neural lobe in vitro released both hormones in a similar arginine vasopressin/oxytocin ratio of about 1-2:1. However, when release was expressed relative to tissue hormone content, the hypothalamus was shown to release about three times as much arginine vasopressin and six times as much oxytocin as the neural lobe. 3. Dopamine in a concentration range of 10(-3)-10(-9)M caused graded increases in hormone release from the hypothalamus in vitro to a maximum fivefold increase over preceding basal levels. The demonstration that apomorphine also stimulated hormone release whereas noradrenaline was relatively ineffective suggested that a specific dopamine receptor was involved. A separate cholinergic component in the release process was indicated by the finding that acetylcholine stimulated release to a maximum fivefold increase in concentrations of 10(-3)-10(-9)M. 4. The fact that the isolated hypothalamus can be stimulated by dopamine and acetylcholine to release increased amount of oxytocin and arginine vasopressin raises the question of the origin and fate of the hormones released in this way. The possibility that they could be released into the hypophysial portal circulation from median eminence to affect the anterior lobe of the pituitary is discussed. 5. In similar doses, both dopamine and noradrenaline injected into the lateral cerebral ventricles of the brain of the anaesthetized, hydrated, lactating rat caused the release of arginine vasopressin and oxytocin. Apomorphine release both hormones but at a higher dose level and to less effect than the catecholamines. 6. The hormone release induced in vivo by dopamine could be prevented by the prior administration of haloperidol or phentolamine and these antagonists were equally effective in blocking the hormone release due to noradrenaline. The involvement of a specific dopamine receptor was more clearly implicated by the use of pimozide which completely inhibited the hormone release due to dopamine and apomorphine but not that due to noradrenaline. 7. It is suggested that the release of neurohypophysial hormones can be stimulated via a dopaminergic nervous pathway in addition to a cholinergic one. The possibility that the osmoreceptor mechanism for the release of antidiuretic hormone from the neural lobe of the pituitary may involve such a dopaminergic pathway is discussed.     

A combined cell column superfusion technique to study hypothalamus-pituitary-adrenal interactions in vitro

Rat anterior pituitaries and rat adrenals were dispersed into cell suspensions. In a cell column superfusion system the pituitary cells were used to study ACTH release and the adrenal cells served as a means to detect that hormone. Linear log-dose-response relationships were exhibited by the adrenal cells for ACTH and by the pituitary cell-adrenal cell system for corticotropin-releasing factor (CRF). A combined system, constructed of a superfused hypothalamus, leading into a column of pituitary cells which was connected to a column of adrenal cells, was used to study the characteristics of CRF release from the hypothalamic block. Basal and stimulated CRF release were both Ca²⁺-dependent.     

Calcitonin gene-related peptide enhances spontaneous acetylcholine release from the rat motor nerve terminal

The effect of synthetic rat calcitonin gene-related peptide (rCGRP) on neuromuscular transmission was examined in a superfused rat phrenic nerve-diaphragm preparation using an intracellular microelectrode technique. The superfusion with rCGRP (10(-8) to 2 x 10(-7) M) caused significant increases in the frequency of miniature endplate potentials (MEPPs). It, however, had no effect on the resting membrane potential, amplitude of MEPPs, or acetylcholine quantum size and content. Enhancement of spontaneous acetylcholine release from the motor nerve terminal by rCGRP was demonstrated.     

Galanin in the regulation of the hypothalamic-pituitary-adrenal axis (Review)

Galanin is a regulatory 30- or 29-amino acid peptide, widely distributed in the nervous system and gut, that acts via three subtypes of G protein-coupled receptors, named GAL-R1, GAL-R2 and GAL-R3. Findings have been accumulated that galanin regulates neuroendocrine hypothalamic axes, including the hypothalamic-pituitary-adrenal (HPA) one. Galanin and its receptors are expressed in the hypothalamic paraventricular and supraoptic nuclei, anterior pituitary and adrenal medulla. Adrenal cortex does not express galanin, but is provided with GAL-R1 and GAL-R2. The bulk of evidence indicates that galanin stimulates the activity of the central branch of the HPA axis (i.e. the release of corticotropin-releasing hormone and ACTH), thereby enhancing glucocorticoid secretion from the adrenal cortex. Investigations carried out in the rat show that galanin is also able to directly stimulate corticosterone (glucocorticoid) secretion from adrenocortical cells, through GAL-R1 and GAL-R2 coupled to the adenylate cyclase-protein kinase A signaling cascade, and nor-epinephrine release from adrenal medulla. There is indication that galanin may also enhance corticosterone release via an indirect paracrine mechanism involving the local release of catecholamines, which in turn activate beta-adrenoceptors located on adrenocortical cells. The physiological relevance in the rat of the glucocorticoid secretagogue action of galanin is suggested by the demonstration that the blockade of galanin system significantly lowers basal corticosterone secretion. There is also evidence that galanin plays a role in the modulation of HPA-axis response to stress, as well as in the pathogenesis of pituitary adenomas and perhaps of pheochromocytomas.     

Melatonin reduces the production and secretion of prolactin and growth hormone from rat pituitary cells in culture

A culture of clonal tumour cells from rat pituitary gland that secrete both prolactin and growth hormone were used to investigate whether the pineal hormone melatonin can act directly on the pituitary gland to control prolactin production. Melatonin inhibition of prolactin and growth hormone production was significant but mild. Concentrations of between 10(-8) M and 10(-6) M reduced both prolactin and growth hormone production and prolactin secretion by 10-50%. 17 beta-oestradiol (OE) and thyrotrophin-releasing hormone (TRH) stimulated prolactin production but had no significant effect on growth hormone production. Melatonin reduced the effects of both of these compounds. Both TRH and vasoactive intestinal peptide (VIP) stimulated secretion of prolactin, and TRH also of growth hormone. Melatonin reduced these effects significantly. TRH and VIP increased cAMP production two- and 12-fold, respectively. Melatonin had no effect on basal or stimulated cAMP production. The melatonin-induced changes in prolactin and growth hormone production and secretion seen here do not approach the magnitude of the fluctuations seen in plasma in vivo. It is concluded that while melatonin does have a direct effect on the lactotroph in the regulation of prolactin production, its main physiological target must be elsewhere.     

Sex differences in vasoactive intestinal peptide (VIP) concentrations in the anterior pituitary and hypothalamus of rats

Recent evidence suggests that vasoactive intestinal peptide (VIP), a putative prolactin (PRL)-releasing factor, is both synthesized and released by anterior pituitary cells, to act as a paracrine or autocrine factor. We have investigated the hypothesis that hypothalamic or pituitary VIP levels differ in male and female rats, since neuroendocrine control of PRL is sexually differentiated. Opposite sex differences were found in the hypothalamus and anterior pituitary. Random-cycle female rats had one-third higher VIP levels in the hypothalamus than males. In contrast, anterior pituitary VIP levels were 3 times as high in male rats as in females. Median eminence VIP levels were similarly low in both sexes. These results support a possible role of VIP in the sexually dimorphic regulatory mechanisms of PRL secretion. Moreover, demonstration that hypothalamic and pituitary VIP levels vary in opposite directions suggests that VIP is differentially regulated at the two sites.     

Cholecystokinin modulation of [3H]noradrenaline release from superfused hypothalamic slices

Interactions between cholecystokinin (CCK) and noradrenaline (NA) have been studied by investigating the effect of sulphated cholecystokinin octapeptide (CCK-8) on potassium (K+)-stimulated release of [3H]NA from superfused hypothalamic slices. NA and the alpha 2-agonist clonidine markedly inhibited [3H]NA release, while the alpha-antagonist yohimbine facilitated the release process. CCK was found to give a significant, albeit small, inhibition of [3H[NA release, which was maximal at a concentration of 10(-8) M. These results provide evidence that CCK-8 can exert an inhibitory effect on hypothalamic NA transmission and offer one possible mechanism for the interaction of CCK and NA in the hypothalamic control of food intake.     

Ion and ion channel involvement in alpha-melanocyte-stimulating hormone secretion from superfused slices of rat hypothalamus

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) from 250 micron frontal slices of rat hypothalamus superfused at 37 degrees C with oxygenated artificial cerebrospinal fluid (ACSF) was quantified in freeze-dried samples of ACSF by radioimmunoassay. Significant reproducible increases in alpha-MSH release were caused by 40-70 mM K+ in ACSF, maximum release being caused by 50 mM K+. Fifty mM K+-stimulated alpha-MSH release was abolished in the absence of Ca2+ from ACSF and by the presence of 10(-6) M tetrodotoxin. Tetrapentylammonium ions, 10(-4) M and 10(-3) M, stimulated dose-dependent increases in alpha-MSH release. The data support a putative neurotransmitter/neuromodulator role for alpha-MSH in the CNS.     

On the functional relationship between 45Ca2+ release and prolactin secretion in cultured rat pituitary tumour (GH3) cells

We have evaluated the role of cellular Ca2+ transport associated with stimulus-secretion coupling in prolactin (PRL) producing rat pituitary adenoma cells (GH3 cells). The action of different substances, known to modify PRL secretion, on release of 45Ca2+ from preloaded cells were examined. Surface-bound 45Ca2+ was removed by pretreatment with trypsin in EDTA buffer. During the first 6 min, basal efflux of 45Ca2+ occurred at a constant rate (0.24 min-1) at 37 degrees C. Addition of TRH (5 X 10(-7) M) resulted in an immediate enhancement of 45Ca2+ release representing about 20% of the remaining cellular 45Ca2+. In the same experiments PRL secretion increased by 45%. The EDTA in the external medium reduced the basal rate of 45Ca2+ release by 60%, but did not apparently affect the TRH-stimulated release. Somatostatin (10(-6) M) and verapamil (5 X 10(-5) M) inhibited both basal and TRH-stimulated PRL secretion, whereas high extracellular concentration of K+ (5 X 10(-2) M) had a stimulatory effect. However, neither of these treatments changed cellular 45Ca2+ release. Interference with energy-dependent Ca2+ transport by using metabolic inhibitors (iodoacetate, 6 X 10(-3) M; and antimycin, 2 X 10(-6) M) or by replacing Na+ in the medium by choline or by lowering the incubation temperature from 37 to 25 degrees C, had no effect on TRH-stimulated 45Ca2+ release although basal and TRH-stimulated PRL secretion were reduced. Thus, TRH apparently releases 45Ca2+ from calcium binding sites in the cell membrane.     

Galanin of the homologous species inhibits insulin secretion in the rat and in the pig.

It is known that pig galanin inhibits insulin secretion in dogs, rats and mice. The present study examined whether species-specific, homologous, galanin inhibits insulin secretion. Thus, the effects of rat galanin were examined in the rat, and the effects of pig galanin were examined in the pig, both in vivo and in vitro. In conscious rats, synthetic rat galanin (2 nmol kg-1) abolished the glucose- (0.56 mmol kg-1) induced increase in plasma insulin levels. In vitro, rat galanin (10(-9) to 10(-6) mol l-1) inhibited glucose- (8.3 mmol l-1) stimulated insulin release from isolated rat islets. In anaesthetized pigs, 15 min infusion of synthetic pig galanin (207 pmol min-1) into the pancreatic artery decreased the insulin output with a subsequent recovery. In vitro, pig galanin (10(-6) mol l-1) inhibited glucose- (8.3 mmol l-1) stimulated insulin release from isolated pig islets. We conclude that homologous galanin inhibits insulin secretion in both the rat and the pig.     

Regulation of amylase release from dispersed pancreatic acinar cells.

1. A study has been made of factors influencing release of amylase from dispersed pancreatic acinar cells. 2. In the basal, unstimulated, condition cells released 2-3% of the total amylase present in 30 min. 3. The rate of amylase release was stimulated 50-70% by C-terminal octapeptide of cholecystokinin (CCK-OP, maximally effective concentration, 3 X 10(-10) M); carbamylcholine (maximally effective concentration, 10(-5 M); secretin (maximally effective concentration greater than 10(-6) M); vasoactive intestinal peptide (VIP, maximally effective concentration, 10(-8) M); and adenosine 3':5' monophosphate (cyclic AMP) and guanosine 3':5' monophosphate (cyclic GMP) as well as their dibutyryl derivatives (maximally effective concentrations, 10(-3) M). 4. The responses to CCK-OP or carbamylcholine were potentiated by secretin, VIP or dibutyryl cyclic AMP. 5. The responses to secretin or VIP were potentiated by CCK-OP, carbamylcholine, or dibutyryl cyclic GMP. 6. There appear to be two pathways for the regulation of amylase release from pancreatic acinar cells: one pathway can be stimulated by cholecystokinin or cholinergic agonists, and the response to these stimuli is mediated by cyclic GMP; the other pathway can be stimulated by secretin or VIP, and the response to these stimuli is mediated by cyclic AMP.     

In vitro effects of endogenous opiate peptides on thyrotropin function: inhibition of thyrotropin-releasing hormone release and absence of effect on thyrotropin release

Thyrotropin-releasing hormone (TRH) or thyroid-stimulating hormone (TSH) was measured by radioimmunoassay in the incubation medium of rat hypothalami or anterior pituitary halves, respectively. We studied the effect of opioid peptide addition (10(-8) to 10(-6) M) on TRH or TSH release. alpha- or beta-Endorphin decreased TRH release in a dose-dependent manner while only 10(-6) M Leu- or Met-enkephalin decreased TRH release. These inhibitory effects were prevented by addition of naloxone (10(-5) M). In the dose range used none of the opioid peptides modified TSH release. These results indicate that opioid peptides may play a role in the regulation of thyrotropin secretion via a hypothalamic action on TRH release.     

Dopamine stimulates somatostatin release from perifused hypothalamic cells

We studied the release of immunoreactive somatostatin (IR-SRIF) from hypothalamic cells that were obtained from rats and dispersed with the aid of collagenase. Twenty-four hours after dispersion, cells were placed in a column supported by a matrix of preswollen Biogel P2 and perifused. Fractions were collected on ice and subsequently assayed for SRIF. SRIF release was stimulated markedly by potassium depolarization (KCl, 56 mM), by the Na+-K+-ATPase inhibitor ouabain (10(-4) M), and by dopamine at concentrations as low as 10(-11) M. The stimulatory effects of membrane depolarization were calcium dependent and were not observed in the absence of exogenous calcium in the perifusion medium or in the presence of EDTA (0.05 M). Metoclopramide, the dopamine antagonist, abolished the stimulatory effect of dopamine. In conclusion, release of IR-SRIF by dispersed rat hypothalamic cells can be studied in a simple perifusion apparatus. Release is stimulated by membrane depolarization in a calcium-dependent manner and by dopamine at physiological concentrations.     

Neurohormones coming from the normal and tumoral human anterior pituitary. Secretion and regulation in vitro

Several neuropeptides, classically associated with the hypothalamus have been found in the anterior pituitary and their local synthesis has been hypothesized. Using normal and tumoral human pituitaries we found in the tissue itself different neuropeptides (TRH, SRIH, GHRH) and dopamine in variable quantities according to the nature of the tissue. They were all present in normal pituitaries while only stimulatory neurohormones like TRH and GHRH were found in tumoral tissue implying an imbalance between the stimulatory and inhibitory control of hypophyseal hormones (PRL and GH) in pituitary adenomas. Fragments from normal pituitaries and dispersed cells from GH, PRL and nonsecreting adenomas, were perifused for 4 hours in a Krebs-Ringer medium collected every 2 min and GH, PRL, TRH, GHRH and SRIH were measured by RIA under basal conditions and in the presence of 10(-6) mol/L DA, TRH or SRIH. Neuropeptides and DA were characterized by HPLC. Both normal and tumoral pituitaries released TRH, SRIH and GHRH in large amounts suggesting their local synthesis. There was an in situ regulation between SRIH and GH as their secretion profile was negatively correlated, GH secretion decreasing while SRIH secretion was increasing. Moreover the release of TRH was stimulated 5 to 20 folds by DA, while PRL decreased at the same time. Pulses of TRH and SRIH had differential effects on GHRH and SRIH release according to the nature of the tissue as TRH stimulated SRIH release from normal pituitary while it inhibited SRIH release from adenoma. These results indicate that anterior pituitary cells can release neuropeptides which are probably endogenously synthesized and have a local regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

gamma-Aminobutyric acid decreases levels of messenger ribonucleic acid encoding prolactin in the rat pituitary

The effect of gamma-aminobutyric acid (GABA) on levels of messenger ribonucleic acid (mRNA) encoding prolactin (PRL) was studied in cultured anterior pituitary cells, in vitro and in intact rats, in vivo. As quantitated by hybridization to a 32P-labeled rat PRL complementary deoxyribonucleic acid (cDNA) probe, levels of PRL mRNA in cultured pituitary cells were decreased by about 50% following 3 days exposure to 10(-5) M GABA. This effect was mimicked by muscimol (10(-6) M) and antagonized by bicuculline (10(-5) M). An increase of endogenous GABA levels in vivo effected by injection of GABA transaminase blockers (aminooxyacetic acid, 20 mg/kg, twice daily; vinyl GABA, 800 mg/kg) into rats resulted in a similar decrease in rat PRL mRNA levels in the adenohypophysis 3-4 days following commencement of the drug treatment. These findings suggest that GABA might inhibit PRL gene expression by a direct action on lactotrophs of the adenohypophysis.     

Calcitonin gene-related peptide inhibits exocrine secretion from the rat pancreas by a neurally mediated mechanism

The mechanism by which calcitonin gene-related peptide (CGRP) inhibits exocrine secretion from the rat pancreas was examined in the isolated, vascularly perfused pancreas and in vitro using freshly isolated pancreatic acini. CGRP (10(-10)-10(-7) M) inhibited the volume and protein output from the perfused pancreas, stimulated by a mixture of the cholecystokinin octapeptide CCK8 (10(-10) M) and secretin (10(-8) M). The inhibition by CGRP was dose related and maximal at 10(-8) M (P less than 0.05). CGRP (10(-8) M) failed to inhibit amylase secretion from isolated pancreatic acini, stimulated by graded concentrations of CCK8 (10(-13)-10(-8) M). This implies an indirect mechanism of inhibition. The mechanism of inhibition was investigated in the isolated, vascularly perfused pancreas using tetrodotoxin, atropine and hexamethonium (all 10(-7) M). Tetrodotoxin and atropine but not hexamethonium prevented the inhibition of volume and protein secretion by CGRP (10(-8) M) (P less than 0.05). Tetrodotoxin, atropine and hexamethonium were without effect on exocrine secretion stimulated by CCK8 and secretin (controls). These results indicate that CGRP inhibits pancreatic exocrine secretion by an indirect, neurally mediated mechanism involving cholinergic-muscarinic transmission.