Endothelin-3 inhibits prolactin and stimulates LH, FSH and TSH secretion from pituitary cell culture

The influence of endothelin-3 (ET-3) on anterior pituitary hormone secretion was investigated over a wide range of concentrations (from 10(-14) to 10(-6) M) and incubation times (from 4 to 48 hours). ET-3 elicited a concentration-dependent inhibition of prolactin (PRL) secretion and stimulated the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH) from primary monolayer cultures of anterior pituitary cells derived from female rats. The responsiveness of different pituitary cells to ET-3 differs markedly in terms of onset and duration: the maximal inhibition of PRL secretion occurred after 12 hours and the stimulation of LH, FSH and TSH reached the maximum after 4, 48 and 48 hours of incubation, respectively. These data corroborate the concept that ET-3 has an important role as a neuroendocrine modulator. Moreover, the data presented suggest different intracellular mechanisms underlying ET-3 actions.     

Rat RFamide-related peptide-3 stimulates GH secretion, inhibits LH secretion, and has variable effects on sex behavior in the adult male rat

A recently described avian neuropeptide, gonadotropin inhibitory hormone (GnIH), has been shown to have seasonal regulatory effects on the hypothalamic-pituitary-gonadotropin axis (HPG) in several avian species. In the bird, GnIH expression is increased during the photorefractory period and has inhibitory effects on the HPG. A recently described mammalian neuropeptide, RF-amide-related peptide-3 (RFRP-3), may be genetically related and functionally similar to this avian neuropeptide. The purposes of this study were to first see if rat RFRP-3 is expressed in the male rat brain and second to determine if ICV injections of RFRP-3 will have effects on feeding and sex behaviors, as well as hormone release from the anterior pituitary. Results confirm other studies in that immunoreactive cell bodies and fibers are observable in areas of the male rat brain known to control the HPG and feeding and sex behaviors. RFRP-3 fibers are also observed in close proximity to GnRH immunoreactive cell bodies. Behavioral tests indicate that high but not low ICV RFRP-3 (500 vs. 100 ng, respectively) significantly (p<0.05) suppressed all facets of male sex behavior while not having any observable effects on their ability to ambulate. Sex behavior was later exhibited when those same male rats received the ICV vehicle. While suppressing sex behavior, ICV RFRP-3 significantly (p<0.05) increased food intake compared to controls. ICV RFRP-3 also significantly reduced plasma levels of luteinizing hormone but increased growth hormone regardless of the time of day; however, at no time did RFRP-3 alter plasma levels of FSH, thyroid hormone, or cortisol. These results indicate that although RFRP-3 has similar effects on LH as observed with GnIH in avian species, in the rat RFRP-3 has additional roles in regulating feeding and growth.     

Comparison of hyposmolar and hyperosmolar effects on in vitro luteinizing hormone secretion by anterior pituitary cells

It has previously been described that perifusion of acutely dispersed adenohypophyseal cells with hypotonic medium causes an immediate high-amplitude "on" burst of luteinizing hormone (LH) secretion. In the present report the converse study with hyperosmolar solutions has been made. Perifusion with hypertonic medium depressed LH secretion; return to isotonicity caused an immediate high-amplitude "off" burst of LH secretion closely resembling that induced by hypotonic perifusion. The data give further support to the theory that exocytotic secretion may involve expansion of the outer cell membrane, thus drawing secretory granules to the cell surface where their contents are extruded.     

Galanin stimulates rat pituitary growth hormone secretion in vitro

The effect of galanin on growth hormone (GH) secretion was investigated in monolayer cultures of rat anterior pituitary cells. Galanin caused a gradual increase in GH concentrations into the culture medium that was maximal at 90 minutes and sustained after 180 minutes. The ED50 for galanin-stimulated GH secretion was approximately 200 nM compared to an ED50 for rat GH-releasing factor (rGRF)-stimulated GH secretion of 10pM. Galanin and rGRF were additive in increasing GH release into the incubation medium. These data indicate that porcine-derived galanin has a direct effect on pituitary GH secretion in vitro.     

Growth hormone inhibits growth hormone secretion from the rainbow trout pituitary in vitro

Growth hormone (GH) secretion in salmonids and other fish is under the control of a number of hypothalamic factors, but negative feed-back regulation by circulating hormones can also be of importance for the regulation of GH secretion. Mammalian studies show that GH has a negative feed-back effect on its own secretion. In order to elucidate if GH levels present a direct ultra-short negative feedback loop at the pituitary level GH secretion was studied in intact pituitaries from 50 g fish in an in vitro perifusion system. Following an initial equilibrium period pituitaries were exposed to five increasing concentrations (1-1,000 ng ml(-1)) of ovine GH (oGH) in 20-min steps, before being returned to a GH-free perifusion. Ovine GH caused a significant dose-dependent inhibition of GH secretion and it is concluded that GH can exert a direct negative feedback control on GH secretion at the pituitary level.     

Long-term maintenance of growth hormone secretion in perifused rat anterior pituitary cells

We studied the effect of rat growth hormone-releasing factor-(1-43) acid, rGRF(1-43)OH, on the long-term secretion of rat growth hormone (rGH) in dispersed primary cultured cells of rat anterior pituitaries over a period of 7 days or longer. Results of the perifusion assay show that freshly dispersed cells secrete more rGH than 4-day-old redispersed cells (P less than 0.05), that a stabilization period ranging from 4 to 24 h allows a greater production of rGH per day than longer periods (P less than 0.05) and that the working concentrations of rGRF-(1-43)OH and prostaglandin E2 (PGE2) that insured the best responsiveness and longer viability are 50 pM and 10-1000 nM, respectively. Under these conditions, the cells continued secreting rGH after 42 days of perifusion, and 315 milligrams of rGH was produced over that period.     

Cadmium chronotoxicity at pituitary level: effects on plasma ACTH,GH, and TSH daily pattern

Cadmium is an endocrine disruptor that has been shown to induce chronotoxic effects. The present study was designed to evaluate the possible cadmium effects on the daily secretory pattern of adrenocorticotropin hormone (ACTH), growth hormone (GH), and thyroid-stimulating hormone (TSH) in adult male Sprague-Dawley rats. For this purpose, animals were treated with cadmium at two different doses [25 and 50 mg/l cadmium chloride (CdCl₂)] in the drinking water for 30 days. Control age-matched rats received cadmium-free water. After the treatment, rats were killed at six different time intervals throughout a 24-h cycle. Cadmium exposure modified the 24-h pattern of plasma ACTH and GH levels, as the peak of ACTH content between 12:00 and 16:00 h in controls appeared at 12:00 h in the group treated with the lowest dose used, while it appeared between 16:00 and 20:00 h in rats exposed to 50 mg/l CdCl₂. In addition, the peak of GH content found at 04:00 h in controls moved to 16:00 h in rats exposed to 25 mg/l CdCl₂, and the highest dose used abolished 24-h changes of GH secretion. The metal treatment did not modify ACTH secretory pattern. Exposure to cadmium also increased ACTH and TSH medium levels around the clock with both doses used. These results suggest that cadmium modifies ACTH and TSH medium levels around the clock, as well as disrupted ACTH and GH secretory pattern, thus confirming the metal chronotoxicity at pituitary level.     

Effects of interleukin-1 beta on secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by cultured rat anterior pituitary cells

Interleukin-1 beta (IL-1 beta) at doses of 0.15 and 1.5 nM significantly inhibited FSH secretion and stimulated LH secretion by cultured rat pituitary cells after 24-72 hr incubation whereas 15 pM of IL-1 beta was not effective. Treatment with IL-1 beta for 12-48 hr did not affect intracellular content of FSH. However, treatment with 0.15 and 1.5 nM of IL-1 beta for 72 hr significantly suppressed intracellular content of FSH whereas various doses of IL-1 beta incubated with the cells for 12-72 hr showed no effect on the intracellular content of LH. Pretreatment with IL-1 beta for 48 hr inhibited both GnRH-mediated LH and FSH secretions by the pituitary. The secretion of FSH and LH mediated by an activator of protein kinase C, phorbol 12-myristate 13-acetate, was also significantly suppressed by pretreatment with IL-1 beta for 48 hr. These results suggest that (a) IL-1 beta has opposite effects on the secretion of LH and FSH and (b) pretreatment with IL-1 beta suppresses GnRH-mediated stimulation of LH and FSH by the pituitary and this suppressive effect of IL-1 beta may involve the suppression of a protein kinase C-dependent mechanism.     

Dynamics and regulation of TSH secretion by superfused anterior pituitary cells

Superfused dispersed cells respond rapidly to 2- to 10-min pulses of TRH (10(-10) to 10(-7) M) in a dose-dependent manner. The effects of decreasing the stimulus duration can be overcome by a proportional increase in concentration of TRH. A TRH stimulus of 10 min or greater duration results in a sharp peak in TSH secretion followed by a lower plateau. Somatostatin (10(-8) M inhibits the response to TRH (t X 10(-9) M). T3 (2.0 microgram/dl) inhibits TRH-induced TSH secretion by superfused pituitary fragments, but not by dispersed cells. Corticosterone (50 microgram/dl), however, inhibits crude CRF-induced ACTH secretion by such cells.     

Muscarinic acetylcholine receptor activation causes inhibition of cyclic AMP accumulation, prolactin and growth hormone secretion in GH3 rat anterior pituitary tumour cells

Acetylcholine, oxotremorine and carbachol, compounds that exhibit muscarinic agonist activity, maximally inhibited basal prolactin secretion from GH3 cells by approx. 50% and intracellular cyclic AMP levels by approx. 20%. Both parameters were inhibited with similar potencies by each agonist. These inhibitory effects were blocked by a muscarinic but not by a nicotinic receptor antagonist. In the presence of VIP or IBMX, which raise intracellular cyclic AMP levels and stimulate hormone release, the degree of muscarinic inhibition was increased, but the potency remained unchanged. Similar changes in the secretory rate of prolactin and growth hormone occurred in these and in cell perifusion experiments. These results suggest that the inhibition of hormone secretion from GH3 cells by muscarinic agonists is mediated by a decrease in intracellular cyclic AMP levels.     

Stimulatory effect of thyrotrophin-releasing hormone (TRH) on the release of luteinizing hormone (LH) from rat anterior pituitary cells in vitro

The effect of thyrotrophin-releasing hormone (TRH, 10(-7) M) on luteinizing hormone (LH) release from rat anterior pituitary cells was examined using organ and primary cell culture. The addition of TRH to the culture medium resulted in a slightly enhanced release of LH from the cultured pituitary tissues. However, the amount of LH release stimulated by TRH was not greater than that produced by luteinizing hormone-releasing hormone (LH-RH, 10(-7) M). Actinomycin D (2 X 10(-5) M) and cycloheximide (10(-4) M) had an inhibitory effect on the action of TRH on LH release. The inability of TRH to elicit gonadotrophin release from the anterior pituitary glands in vivo may partly be due to physiological inhibition of its action by other hypothalamic factor(s).     

Somatostatin fibers and their relationship to specific cell types (GH and TSH) in the rat anterior pituitary

Fibers immunocytochemically stained for somatostatin (growth hormone-release-inhibiting hormone) were localized in the anterior pituitaries of rats. Initial studies of fiber localization involved the use of thick frozen (30 micron) sections which allowed visualization of fibers as they coursed along the periphery of the anterior lobe in the sagittal plane and along blood vessels throughout the lobe. Fibers were observed most often at the rostral, caudal, and lateral poles. In thinner (1-3 micron) paraffin sections, stained somatostatin fibers could be localized in close proximity to cells that were stained for growth hormone or thyroid stimulating hormone in a double stain with a second peroxidase substrate. These and our previous light microscopic studies show that a few neuronal processes containing neurotransmitters extent beyond the level of the median eminence (or perhaps from a peripheral source), penetrate the anterior lobe in specific regions, and lie in close proximity to cells known to be controlled by the transmitter.     

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.     

Synthesis and secretion of phosphorylated growth hormone by rat pituitary glands in vitro

Rat anterior pituitary glands were incubated in buffered medium, pH 7.4, containing 32Pi. After incubation the tissue and medium were separated and a post-mitochondrial supernate (PMS) of the tissue homogenate was prepared. Gel filtration of the PMS and medium resulted in a radioactive peak which coincided with the elution volume of authentic rat growth hormone (rGH). Polyacrylamide gel electrophoresis of the radioactive peak under denaturing condition resulted in a protein-staining band having the same mobility as authentic rGH. Autoradiography of the gels revealed radioactivity precisely at the position of growth hormone as well as elsewhere. The specific radioactivity of the PMS [32P]GH was estimated to be 5 to 10 times greater than that of tissue [32P]GH. These results indicate that phosphorylated GH is synthesized and secreted by pituitary glands in vitro.     

The effects of tunicamycin on anterior pituitary hormone producing cells in the rat

An electron microscopic study was performed to clarify the effects of tunicamycin, a glycosylation inhibitor, on rat anterior pituitary cells. Tunicamycin (10, 50, and 100 micrograms/250 g B.W.) was intraperitoneally injected into rats, which were sacrificed 24 hrs later. Protein hormone producing GH and prolactin cells, and ACTH cells which are known to have a glycosylated precursor, showed no recognizable ultrastructural changes. TSH cells and gonadotrophs, both of which secrete glycoprotein hormones consisting of alpha and beta subunits, showed remarkable dilatation of the rough endoplasmic reticulum, and decreased numbers of secretory granules. These results suggest that the role of glycosylation in TSH cells and gonadotrophs may have a different biological significance from that in ACTH cells.     

Studies of growth hormone secretion VIII. Effects of alpha,beta-methylene-ATP on prostglandin induced GH release and cyclic AMP accumlation in rat anterior pituitary glands.

Alpha, beta-methylene-ATP, a competitive inhibitor of adenylate cyclase of liver and fat cell membrane preparations, caused a dose related inhibition of PGE1 and PGE2-induced cyclic AMP accumulation in rat anterior pituitary explants. At the same time, this ATP analog potentiated PGE1 and PGE2-promoted growth hormone secretion. The possible functional role of prostaglandins and cyclic nucleotides in the regulation of growth hormone secretion remains to be defined.