Effects of hypothalamic-releasing hormones on LH, FSH, and prolactin in pituitary monolayer cultures.

Four-day-old pituitary monolayer cultures were incubated with various hypothalamic releasing hormones. Rat hypothalamic extract stimulated the release of LH, FSH, and PRL by these cultures in a dose-related fashion. Synthetic LH-RH stimulated the release of LH and FSH but not of PRL. Synthetic TRH increased the release of PRL but had no effect on LH or FSH. At 10(-8) M, somatostatin did not affect any of the three adenohypophyseal hormones. Incubation with DBcAMP or theophylline also stimulated PRL release without any detectable effect on LH and FSH release. These data suggest the involvement of cyclic AMP--adenylate cyclase system in the mechanism of PRL release, but their involvement in gonadotropin release requires further studies.     

Effect of dexamethasone on the isozyme pattern of adult rat liver parenchymal cells in primary cultures

Summary The influence of dexamethasone on the isozyme patterns of ATP-hexose phosphotransferases, aldolase and pyruvate kinase of adult rat hepatocytes maintained in primary cultures has been studied.A progressive loss of the typical adult liver isozymes glucokinase, pyruvate kinase L and aldolase B, with a simultaneous increase of both pyruvate kinase A and hexokinase activities, was observed in hepatocytes cultured in the absence of added glucocorticoid.When the culture medium was supplemented with 10^–7 M dexamethasone, the adult liver patterns of pyruvate kinase and aldolase were preserved for at least seven days of culture, the initial level of glucokinase was maintained for three days, and the rise of hexokinase activity was delayed and partially blocked.These results are discussed in relation to the known beneficial effect of glucocorticoids on the survival of cultured hepatocytes.     

Major metabolic pathways and hormone production in unstimulated monolayer cultures of the rat anterior pituitary

Summary In cell cultures derived from anterior pituitary glands of rats, enzyme activities of cell homogenates and hormone (GH, PRL, LH, and FSH) content of the culture media were measured. Sex differences in enzyme activities representing major metabolic pathways (citrate cycles, pentose cycles, and glycolysis) were demonstrated both in freshly dispersed cells and in 8-day-old cultures; in cultures of both sexes enzyme activities increased during cultivation. In cultures derived from female rats, cell protein doubled by the 12th day and remained constant for up to the 24th day in culture, whereas enzyme activities showed changes suggesting that cell metabolism shifted to anaerobic glycolysis during cultivation. In the culture media the presence of four pituitary hormones was demonstrated for as long as 3 weeks of cultivation with variable secretion dynamics; the release of gonadotropic hormones diminished gradually whereas that of GH remained constant and PRL levels increased with time. These results indicate that under strictly defined culture conditions pituitary cells may function in spite of profound metabolic changes.     

Major metabolic pathways and hormone production in unstimulated monolayer cultures of the rat anterior pituitary

In cell cultures derived from anterior pituitary glands of rats, enzyme activities of cell homogenates and hormone (GH, PRL, LH, and FSH) content of the culture media were measured. Sex differences in enzyme activities representing major metabolic pathways (citrate cycles, pentose cycles, and glycolysis) were demonstrated both in freshly dispersed cells and in 8-day-old cultures; in cultures of both sexes enzyme activities increased during cultivation. In cultures derived from female rats, cell protein doubled by the 12th day and remained constant for up to the 24th day in culture, whereas enzyme activities showed changes suggesting that cell metabolism shifted to anaerobic glycolysis during cultivation. In the culture media the presence of four pituitary hormones was demonstrated for as long as 3 weeks of cultivation with variable secretion dynamics; the release of gonadotropic hormones diminished gradually whereas that of GH remained constant and PRL levels increased with time. These results indicate that under strictly defined culture conditions pituitary cells may function in spite of profound metabolic changes.     

Mechanism of LH release in cultured rat pituitary cells

To investigate the mechanisms of the synthesis and the release of gonadotropin, rat anterior pituitary cells were stimulated in vitro with luteinizing hormone releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin) and 12-0-tetradecanoyl phorbol-13-acetate (TPA), and then the LH and LH-beta subunit released into the medium were determined by radioimmunoassay. Buserelin showed its biological activity at a much lower concentration than LH-RH, but both of them caused the release of LH and LH-beta subunit in a dose-dependent manner. Furthermore, intracellular LH synthesis from LH-beta subunit by stimulation with LH-RH or Buserelin was also found. After inducing various degrees of desensitization by stimulation with LH-RH or Buserelin in a dose-dependent manner (the first stimulation), pituitary cells were stimulated with a fixed dose of TPA (the second stimulation) and the released LH was assayed. LH was released almost constantly by the second stimulation, regardless of the dose used for the first stimulation. These results suggest that the C-kinase pathway was unaffected by the desensitization induced with LH-RH or Buserelin.     

Effects of Neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat

Neuropeptide Y (NPY) has been recently localized in several hypothalamic nuclei in the mammalian brain. In order to investigate the possible role of NPY on neuroendocrine function, we have investigated the effects of the peptide on the release of anterior pituitary hormones in the rat. Both intravenous (300 μg) or intraventricular (2 to 15 μg) injection of NPY produced in gonadectomized male rats a significant and long-lasting decrease of plasma LH levels. A short duration stimulating effect on prolactin plasma levels was also observed after the intravenous but not after the intraventricular injection of NPY. Plasma levels of the other pituitary hormones were not significantly modified after NPY injection. When incubated in vitro with anterior pituitary cells in monolayer culture, NPY produced no significant change in release of pituitary hormones. Thus NPY seems to exert a selective effect on LH release. Since this effect can be observed after both intravenous and intraventricular injection, it might be hypothesized that NPY could affect LHRH release in two areas which lack blood-brain barrier: the organum vasculosum of the lamina terminalis (OVLT) which contains LHRH cell bodies and NPY fibers and the median eminence which contains both LHRH and NPY fibers. The effect on prolactin release needs to be carefully evaluated in different experimental conditions.     

Electrotonic spread of current in monolayer cultures of neonatal rat heart cells

Summary The passive electrical properties of neonatal rat heart cells grown in monolayer cultures were determined. Hyperpolarizing current pulses were injected through one microelectrode via an active bridge circuit. Membrane voltage displacements caused by the injected current pulses were measured at various distances from the first with a second microelectrode. Using a modified least-squares method the experimental results were fitted to a Bessel function, which is the steady-state solution of the differential equation describing the relation between membrane voltage caused by current injection and interelectrode distance in a very large and very thin plane cell. Best fit was obtained with a space constant of 360 m and an internal resistivity of 500 cm. From these figures, specific membrane resistance was calculated to be 1,300 cm², assuming all current to leave through the upper surface of the monolayer.The time constant of the membrane was measured from the time course of the current-induced membrane voltage displacements. From its value of 1.7 msec a membrane capacity of 1.3 F/cm² was calculated.From these results and some literature data on nexus distribution (A. W. Spira,J. Ultrastruct. Res. 34:409, 1971) specific nexus resistance was calculated to range between 0.25 and 1.25 cm², depending on the amount of folding of the intercalated discs. The results suggest that spread of activation in monolayer cultures of heart cells by means of local circuit currents is very likely.     

Immunohistochemical localization of anterior pituitary hormones in S-100 protein-positive cells in the rat pituitary gland

In the anterior and intermediate lobes of the rat pituitary gland, non-hormone-producing cells that express S-100 protein coexist with various types of hormone-producing cells and are believed to function as phagocytes, supporting and paracrine-controlling cells of hormone-producing cells and stem cells, among other functions; however, their cytological characteristics are not yet fully understood. Using a transgenic rat that expresses green fluorescent protein under the promoter of the S100β protein gene, we immunohistochemically detected expression of the luteinizing hormone, thyroid-stimulating hormone, prolactin, growth hormone and proopiomelanocortin by S-100 protein-positive cells located between clusters of hormone-producing cells in the intermediate lobe. These findings lend support to the hypothesis that S-100 protein-positive cells are capable of differentiating into hormone-producing cells in the adult rat pituitary gland.     

Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20-24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-alpha 1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34-48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of alpha-amanitin in the culture media at 0 h. The inhibiting effect of alpha-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.     

Permissive effect of dexamethasone on glucagon induction of urea-cycle enzymes in perifused primary monolayer cultures of rat hepatocytes.

Parenchymal cells from adult rat liver, cultured in perifused monolayers, increased the levels of urea-cycle enzymes between 15% and 60% in response to glucagon within 24 h. This stimulation was drastically enhanced by the simultaneous presence of dexamethasone, especially in the case of argininosuccinate synthetase and argininosuccinate lyase, which increased nearly threefold. Dexamethasone itself produced only negligible stimulation, but exerted a similar effect on the stimulatory action of glucagon, if it was exclusively present during 6 h prior to the glucagon treatment, suggesting a permissive action of this hormone. The effect of glucagon, particularly in the presence of dexamethasone, was mimicked by dibutyryl adenosine 3':5'-monophosphate, whereas epinephrine was ineffective. All stimulations induced by hormones or dibutyryl adenosine 3':5'-monophosphate were abolished by cycloheximide, suggesting the involvement of protein synthesis in the induction process. Using the usual culture technique with a discontinuous supply of medium no significant effect of glucagon and dexamethasone could be measured. This striking difference between both culture systems indicates that perifusion is the more adequate in vitro system for studies of the regulation of enzyme levels. Possible reasons for the failure of hormonal stimulation of urea-cycle enzymes in normal monolayer culture are discussed.     

Influence of yohimbine on release of anterior pituitary hormones

We used a double-blind crossover design to study the effects of alpha 2 adrenoreceptor blockade with yohimbine on levels of anterior pituitary hormones. A dose of yohimbine was used which raised plasma norepinephrine from 379 +/- 74 (S.E.) to 730 +/- 143 pg/ml and mean arterial pressure from 83 +/- 4 to 92 +/- 5 torr (p less than 0.025). This dose (125 micrograms/kg, then 1 microgram/kg/min) also altered mood when compared to saline infusion. In spite of these changes, when prolactin, cortisol, ACTH, beta-endorphin, TSH and growth hormone were measured after 45 minutes of yohimbine infusion, no changes from baseline were noted. These data suggest that in normal man, at rest, alpha 2 adrenoreceptors in the hypothalamus, adenohypophysis or other brain areas do not tonically modulate release of these hormones into the blood.     

Muscarinic inhibition of prolactin production in cultures of rat pituitary cells

Acetylcholine inhibits prolactin production from cell cultures of rat pituitary glands with a half-maximal effect at about 0.2 μM, and from GH-cells, clonal strains of rat pituitary cells, with a half-maximal effect at about 1 μM. The inhibition ranges between 80 and 40 % of control values. Inhibition is detectable at 2 hours, and continues for days in the presence of the anticholinesterase, eserine. Muscarinic agonists mimic the cholinergic inhibition and nicotinic agonists do not. The inhibition is blocked by atropine, a muscarinic antagonist, and not by hexamethonium, a nicotinic antagonist.