Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

Effect on an antagonistic analog of gonadotropin releasing hormone upon ovarian granulosa cell function.

We have recently demonstrated that gonadotropin releasing hormone (GnRH) acts directly on ovarian granulosa cells to inhibit the follicle stimulating hormone (FSH)-induced increase in granulosa cell steroidogenesis $\text{in}$$\text{vitro}$. A GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6] GnRH (A), which is known to antagonize GnRH-stimulated gonadotropin release by cultured pituitary cells, was tested in the granulosa cell system. GnRH (10^?8M) inhibited estrogen and progesterone production by FSH-treated granulosa cells $\text{in}$$\text{vitro}$, whereas the antagonist A (10^?6M) did not affect FSH stimulation of steroidogenesis. Antagonist A, when added together with GnRH and FSH, blocked the GnRH inhibition of FSH-induced steroidogenesis. Estrogen and progesterone production by granulosa cells was increased by 50% at a molar ratio (IDR₅₀) of $\text{20}\text{1}\text{and}\text{12}\text{1}$ ([antagonist]/[GnRH]), respectively. At 10^?6M, antagonist A completely prevented the GnRH (10^?8M) inhibition. A similar effect of antagonist A was seen in FSH-induced increase of luteinizing hormone (LH) receptor content. FSH treatment for 2 days $\text{in}$$\text{vitro}$ induced an 8-fold increase in LH receptor content in cultured granulosa cells; concomitant treatment with 10^?8M GnRH completely inhibited the FSH effect. Antagonist A (10^?6M), by itself, had no effect on the FSH action. However, when added together with FSH and GnRH, antagonist A completely abolished the inhibitory effect of GnRH. These results demonstrate that the direct inhibitory effect of GnRH on granulosa cell function can be prevented by a GnRH antagonist and that the GnRH action at the ovarian level may require stringent stereospecific interactions of these peptides with putative GnRH recognition sites.     

The influence of vasoactive intestinal polypeptide and cholecystokinin of prolactin release in rat and human monolayer cultures

We have evaluated the effects of the gut-brain peptides, VIP and CCK, on pituitary PRL secretion in monolayer cultures of normal and tumor bearing rodent and human pituitary tissue. In cultures prepared with normal human pituitary tissue obtained from three patients with metastatic breast cancer, VIP at 10^?7M and 10^?9M (but not 10^?11M) significantly (p<.05) increased PRL secretion in the wells by 6 hrs. Similar concentrations of VIP also significantly (p<.05) promoted PRL release from pituitary tissue obtained by transphenoidal hypophysectomy from one of two prolactinoma patients. Dopamine (10^?5M) inhibition of PRL secretion was not affected by 10^?11 to 10^?7M VIP. In contrast to these findings VIP did not significantly influence 6 hr rat PRL release in monolayer cultures of normal or transformed cells (GH₃) with or without the addition of bacitracin (10^?5M).CCK₃₃ significantly (p<.01) increased rat PRL release in human pituitary monolayer cultures at 10^?5M. The more biologically potent CCK₈ significantly (p<.02) increased rat PRL release at a 10-fold lower concentration, 10^?6M. In contrast, CCK₈ 10^?8 to 10^?6M, did not significantly influence PRL release from normal human pituitary cultures or from tumor bearing human (prolactinoma) and rat (GH₃) cultures. We conclude that 1) the gut-brain peptides, VIP and CCK, can directly stimulate pituitary PRL release and 2) VIP may be a physiologic prolactin releasing factor in man.     

Hydrocortisone increases the number of receptors for thyrotropin-releasing hormone on pituitary cells in culture.

Hydrocortisone (cortisol) increased the binding of thyrotropin-releasing hormone (TRH) to specific membrane receptors in 4 clonal strains of rat pituitary cells. At the highest effective cortisol concentration (3–5 × 10^?6 M), the increase was observed within 6–8 hr and became maximal (140 to 160% of control binding) by 18–24 hr. Half-maximum stimulation occurred in serum-containing medium at 9 × 10^?8 M cortisol, and a significant increase in TRH binding was seen at 3 × 10^?8 M. Equilibrium binding studies showed that enhanced TRH binding was explained by an increase in receptor number with no change in affinity. Similar effects were seen with Dexamethasone, but no increase in TRH binding was noted when testosterone, methyltestosterone, progesterone, estradiol or the antiestrogen Lilly 88571 were added to the culture medium. Cortisol treatment did not cause the appearance of specific TRH binding sites in cell strains previously shown to lack receptors for the tripeptide (F₄C₁, GH₁2C₁ and R₅ cells). When added cortisol was removed from medium, receptor number decayed to control values with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 30 hr. Previous studies have shown that TRH receptors in GH-cells can be down-modulated by TRH and thyroid hormones; the present findings demonstrate that glucocorticoid hormones can increase the number of TRH receptors in GH-cells.     

Adenylyl cyclase from a prolactin producing tumor cell: The effect of phenothiazines

An adenylyl cyclase stimulated by low concentrations of chlorpromazine was observed in homogenates of a clonal pituitary tumor cell line (GH₃/C14) which releases prolactin and growth hormone. A half-maximal increase in activity of the GH₃/C14 cyclase occurred in the presence of 0.5 × 10^?6M chlorpromazine and a significant increase in activity was observed with a concentration of chlorpromazine as low as 10^?7M. Several derivatives (7-methoxychlorpromazine, 7-hydroxychlorpromazine and 8-hydroxychlorpromazine) were found to mimic the stimulatory action of chlorpromazine on adenylyl cyclase, whereas chlorpromazine-5, N-dioxide was ineffective. Under the assay conditions used, sodium fluoride caused a four-fold increase in activity. However, dopamine at concentrations up to 2 × 10^?4M was ineffective in stimulating or inhibiting the enzyme whether present alone or in combination with chlorpromazine. The ergot alkaloids, ergotamine and ergocryptine, blocked the stimulation of cyclase activity observed in the presence of chlorpromazine (10^?5M). Homogenates of normal pituitaries showed no enhancement of adenylyl cyclase activity by chlorpromazine alone. However, when chlorpromazine was tested in the presence of 5′ guanylimidophosphate [GPP(NH)P], there was a significant increase in cyclase activity in the pituitary similar to that observed in the GH₃/C14 preparation. These results suggest that hyperprolactinemia resulting as a side effect of phenothiazine treatment may be attributable to a direct action of these drugs to increase adenylyl cyclase activity in prolactin-producing cells of the anterior pituitary.     

Specificity of the stimulatory effect of prostaglandins on hormone release in rat anterior pituitary cells in culture

Specificity of the effect of prostaglandins (PGs) on hormone release by the anterior pituitary gland was studied using cells in primary culture. Growth hormone (GH) release is stimulated by all eight PGs studied, PGE₁ and E₂ being 1000-fold more potent than the corresponding PGFs. The release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) remains unchanged upon addition of PGEs. While the basal release of thyrotropin (TSH) is only slightly stimulated by concentrations of PGEs above 10⁻⁶M, an important potentiation of the stimulatory effect of thyrotropin-releasing hormone on TSH release is observed. The release of GH, TSH and LH is stimulated equally well by PGAs and PGBs at concentrations higher than 10⁻⁶M, 3 × 10⁻⁶M, and 10⁻⁵M, respectively. PGFs do not affect the release of any of the measured pituitary hormones at concentrations below 10⁻⁴M. The stimulation of GH release by PGE₂ can be inhibited by the PG antagonist 7-oxa-13-prostynoic acid, a half-maximal inhibition being found at a concentration of 4 × 10⁻⁵M of the antagonist in the presence of 10⁻⁶M PGE₂. In the presence of somatostatin (10⁻⁸M), the inhibition of GH release cannot be reversed by PGE₂ at concentrations up to 10⁻⁴M. 8-bromo-cyclic AMP-induced GH release is additive with that produced by PGE₂.The present data show that 1) of the five pituitary hormones measured, only GH release is stimulated by prostaglandins at relatively low concentrations, 2) the PGE-induced GH release can be competitively inhibited by 7-oxa-13-prostynoic acid, 3) the inhibition of GH release by somatostatin cannot be reversed by PGE₂ and 4) the PGEs increase the responsiveness of the thyrotrophs to TRH.     

Stimulatory effect of prostaglandin E1 on thyroliberin-induced α-melanotropin release from perifused neuro-intermediate lobes of frog pituitary gland

A possible direct effect of prostaglandins on α-melanotropin (α-MSH) release at the level of the intermediate lobe of the frog pituitary was investigated $\text{in vitro}$ using a perifusion system technique. The effect of prostaglandins was studied on both spontaneous and TRH-stimulated α-MSH secretion. No significant effect of PGE₁, PGE₂, PGF_1α or PGF_2α on basal release of α-MSH could be detected. Indomethacin did not alter the α-MSH release induced by TRH. Conversely a significant increase in TRH-induced α-MSH secretion was observed in the presence of 1 x 10^?6M PGE₁. This magnifying effect was directly related to the concentration of TRH for doses ranging from 1 x 10^?8M to 1 x 10^?6M.     

Thyrotropin-releasing hormone stimulates the release of melanotropin from frog neurointermediate lobes in vitro

The hypothalamus of Amphibia contains large amounts of tripeptide P-Glu-His-Pro-NH₂ (mammalian thyrotropin-releasing hormone, TRH). However, synthetic TRH is unable to stimulate thyrotropin release from frog pituitary gland. The recent discovery of TRH in the skin of the frog suggests a possible role of this peptide in skin-colour adaptation. Thus we have investigated the role of TRH upon melanotropin (α-MSH) release from perifused frog neurointermediate lobes. A dose related increase in α-MSH release was observed when TRH was added to the perifusion medium. Half-maximum stimulation occurred with the 1 × 10^?8M dose. Theophylline at a dose of 2 × 10^?3M strongly enhanced TRH-induced α-MSH release, indicating that cyclic AMP may be the second messenger. α-MSH releade was not modified by crude homogenates of rat hypothalamus but was significantly reduced when the hypothalamus extracts were preincubated with specific TRH antibodies. As far is known, these results provide the first evidence that P-Glu-His-Pro-NH₂ stimulates the release of α-MSH from frog neurointermediate lobes $\text{in vitro}$. The present findings suggest a possible feedback loop between skin TRH and pituitary MSH in Amphibia.     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of growth hormone in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of growth hormone (GH) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma GH level after 10 and 20 min. CCK-8 at concentrations of 10(-11)M to 10(-7)M also caused dose-dependent stimulation of GH release from dispersed cells of rat anterior pituitary. On the other hand, somatostatin (SRIF) inhibited GH release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-7)M to 10(-9)M. Release of GH from the cells was increased by addition of K+ at high concentration (50 mM) in a Ca++-dependent manner. Addition of 10(-3)M verapamil to the incubation medium inhibited CCK-8-induced GH release from the cells. Addition of SRIF (10(-7)M) to the incubation medium inhibited GH release from the cells induced by CCK-8 or high K+ (50 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate GH release and that calcium ion is involved in the mechanism of this effect.     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

Studies on growth hormone secretion VI. Effects of dibutyryl cyclic AMP,prostaglandin E1, and indomethacin on growth and hormone secretion by rat pituitary tumor cells in culture

The growth of rat pituitary tumor cells (GH₁ line) maintained in monolayer culture was inhibited by dibutyryl cyclic AMP in a dose-related fashion. Neither PGE₁ (2.8 × 10^?5M) nor indomethacin (2.8 × 10^?6M) had any significant effect on cell proliferation. Release of GH into the culture medium was stimulated by the cyclic AMP derivative but not by PGE₁ or indomethacin. In short term experiments (15 min.) both in intact monolayers and in trypsin-treated cells incubated in suspension, PGE₁ caused a 2–10 fold increase in cyclic AMP levels. This response, however, appeared to be of short duration reaching a maximum in 10 minutes. It is suggested that, at least in this line of pituitary tumor cells, PGE₁ does not mimic the effect of cyclic AMP, for it probably cannot sustain the elevated intracellular levels of this nucleotide which seem to be necessary for growth inhibition and enhanced GH secretion.     

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied $\text{in}$$\text{vitro}$ by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10^?7 M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [¹²⁵I]iodo-[D-Ala⁶]des-Gly¹⁰-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.     

Activation of Na+ Channels in GH3 Cells and Human Pituitary Adenoma Cells by PACAP

Koshimura, K., Y. Murakami, M. Mitsushima, T. Hori and Y. Kato. Activation of Na⁺ channels in Gh₃ cells and human pituitary adenoma cells by Pacap. Peptides 18(6) 877–883, 1997.—The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on ion channels were examined in GH₃ cells and human pituitary adenoma cells. In GH₃ cells, PACAP-38 (10-9 M) reversibly activated tetrodotoxin-sensitive Na⁺ channels but had little effect on nicardipine-sensitive Ca²⁺ channels. PACAP-induced increase in Na⁺ currents was inhibited by PACAP(6-38), a specific PACAP receptor antagonist, and Rp-cAMPs, an inhibitor for protein kinase A, and mimicked by 8-bromo-cAMP. In human pituitary adenoma cells, PACAP also activated tetrodotoxin-sensitive Na⁺ channels and growth hormone secretion. These results suggest the possibility that PACAP can activate voltage-gated Na⁺ channels via adenylate cyclase-protein kinase A pathway in the pituitary.     

In vitro stimulation of human red blood cell Ca2+-ATPase by thyroid hormone

Ca²⁺-ATPase activity in human erythrocyte ghosts previously washed to remove endogenous thyroid hormone is stimulated $\text{in}\text{vitro}$ by physiologic concentrations of thyroxine (T₄) and triiodothyronine (T₃). Two- to three-fold increases (P <0.005) in Ca²⁺-ATPase activity occurred after 60–120 minutes' exposure of membranes to iodothyronines at concentrations of T₄ and T₃ of 10^?8 M to 10^?12 M. T₄ was more active than T₃ and its activity did not depend upon prior conversion to T₃. The Ca²⁺-ATPase effect represents an extranuclear action of thyroid hormone in a human cell model.     

Synthesis and biological activity of four gamma-melanotropin peptides derived from the cryuptic region of the adrenocorticotropin/beta-lipotropin precursor.

A third melanotropin coding fragment named γ-MSH was discovered by Nakanishi et al (Nature $\text{278}$, 423–427 (1979)) in the cryptic region outside the portion coding for ACTH and β-LPH in the ACTH/β-LPH precursor mRNA isolated from the intermediate lobe of bovine pituitary. Four possible γ-MSH peptides derived from this coding fragment were synthesized by solid-phase methodology and their bioactivity determined in an $\text{in vitro}$ MSH assay as well as the anterior pituitary primary culture assay. Relative to α-MSH, the melanotropic activities of Ac-γ₁-MSH, γ₁-MSH, γ₂-MSH and γ₃-MSH are 7.3 × 10^?4, 3.3 × 10^?5, 1.4 × 10^?4 and 4.6 × 10^?7 respectively. None of these γ-MSH peptides releases LH, FSH, PRL, GH and TSH in the pituitary culture medium at a dose as high as 100 ng per dish.     

Medium flow rate modulates autocrine-paracrine feedback of GH and PRL release by perifused GH3 cells

Summary We previously documented both the spontaneous acceleration of growth hormone (GH) and prolactin (PRL) production by GH₃ cells during periffusion and the suppression of their production during plate culture. We here present the role played by medium flow itself in this differential behavior. Increasing rates of perifusion flow (pump rates of 1 to 5 ml/h, equivalent to chamber flow rates of 0.19 to 1.3 μl·min⁻¹·mm⁻² of cross-sectional area) were associated with enhanced GH and PRL secretion. Flow rate-dependent basal hromone secretion rates were established quickly and were stable for the first 10 to 14 h of perifusion. The previously documented independent, spontaneous, and continuously accelerating production of both hormones that followed during the subsequent 40 (PRL) to 60 (GH) h of perifusion was also shown to be flow-rate related. Any time the rate of medium flow was changed within an experiment, the rate of hormone secretion was modulated. However, that modulation did not interrupt ongoing flow-associated acceleration of hormone production once the latter had begun. In addition, GH₃ cell product(s) from one cell column reversibly inhibited secretion from cells in a downstream column. The inhibition did not occur when cells in the downstream column had been exposed to trypsin. Other work had suggested that neither GH, PRL, insulinlike growth factor-I, leucine, nor nutrient exhaustion were responsible for the effect. These data are consistent with autocrine-paracrine feedback regulation of GH₃ cells by a secretory product(s). Feedback would thus provide a mechanism to effect flow-rate-dependent modulation of GH and PRL release, and to explain accelerating hormone production during perifusion. This work was supported by a grant to M. E. S. from the National Institutes of Health (DK33388), Bethesda, MD, and in part, by the Medical Research Service of the Veterans Administration.     

Formation of guanosine 3′,5′-cyclic monophosphate by thyroliberlin in cultured rat pituitary cells a possible role in stimulation of prolactin synthesis

In a clonal strain of rat pituitary tumour cells (GH₄C₁ cells), thyroliberin stimulated prolactin secretion and synthesis: effects that could be demonstrated after 5 min and 4–5 h of treatment, respectively. Within 0.5–5 min after addition of thyroliberin, maximal increases (2–4 hold) in cellular cyclic GMP concentrations were observed, and this rise preceded or occurred simultaneously with that of cyclic AMP. After 60 min of treatment the concentrations of the cyclic nucleotides had returned to control values. Half maximal and maximal stimulation of cyclic GMP elevations were obtained with approx. 2·10⁹ and approx. 27·10^?9 thyroliberin, respectively. Aminophylline increased both cyclic GMP and cyclic AMP, and potentiated the stimulatory effects of thyroliberin on both cyclic nucleotides. The dibutyryl derivative of cyclic GMP (10^?4–10^?6 M) stimulated prolactin synthesis, but not hormone release. Prostaglandin E₂ (3·10^?7 M) stimulated cellular cyclic AMP concentrations, but did not affect cyclic GMP levels. We conclude that thyroliberin in the GH₄C₁ ccell strain stimulates cyclic GMP formation, in addition to elevate cyclic AMP concentrations. The stimulatory effect on cyclic GMP is probably not secondary to the rise in cyclic AMP concentration, since prostaglandin E₂ elevates only cyclic GMP is involved in the action of thyroliberin on prolactin, the present results suggest a role on hormone synthesis.     

Effects of intraventricular injection of gastrin on release of LH,prolactin, TSH and GH in conscious ovariectomized rats

Conscious ovariectomized (OVX) rats bearing a cannula implanted in the 3rd ventricle were injected with 2 μl of 0.9% NaCl containing varying doses of synthetic gastrin and plasma gonadotropin, GH and TSH levels were measured by RIA in jugular blood samples drawn through an indwelling silastic catheter. Control injections of saline iv or into the 3rd ventricle did not modify plasma hormone levels. Intraventricular injection of 1 or 5 μg gastrin produced significant suppression of plasma LH and prolactin (Prl) levels within 5 min of injection. Injection of 1 μg gastrin had no effect on plasma GH, but increasing the dose to 5 μg induced a progressive elevation, which reached peak levels at 60 min. By contrast, TSH levels were lowered by both doses of gastrin within 5 min of injection and the lowering persisted for 60 min. Intravenous injection of gastrin had no effect on plasma gonadotropin, GH and TSH, but induced an elevation in Prl levels. $\text{In}$$\text{vitro}$ incubation of hemipituitaries with gastrin failed to modify gonadotropin, GH or Prl but slightly inhibited TSH release at the highest dose of 5 μg gastrin. The results indicate that synthetic gastrin can alter pituitary hormone release in unrestrained OVX rats and implicate a hypothalamic site of action for the peptide to alter release of a gonadotropin, Prl and GH. Its effect on TSH release may be mediated both via hypothalamic neurons and by a direct action on pituitary thyrotrophs.     

Identification of two forms of glutamine synthetase in barley (Hordeum vulgare).

Hepatocytes of 14-day-old rats have no detectable glucokinase activity $\text{in}$$\text{vivo}$, but it was induced by insulin (10^?8M) in primary cultures of these hepatocytes. The glucokinase induced by insulin was separated by electrophoresis on a cellulose acetate membrane and identified by its low affinity for glucose. This precocious induction of glucokinase was completely prevented by the presence of either actinomycin D or cycloheximide. Glucagon also inhibited its induction by insulin. Dexamethasone and testosterone, which alone had no inductive effect, strongly enhanced the induction by insulin. When hepatocytes of 14-day-old rats were cultured with 10^?7M insulin, 10^?6M dexamethasone and 10^?7M testosterone for 48 hr, their glucokinase activity increased to the non-induced level in hepatocytes of adult rats. Estrogen, thyroxine or growth hormone did not induce glucokinase precociously. Testosterone did not enhance induction of glucokinase by insulin in cultured hepatocytes of adult rats.