Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland

The possibility that prostaglandin E₂ (PGE₂) may play a role in luteinizing hormone (LH) release was examined using an model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE₂ or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE₂ content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood.It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Studies on growth hormone secretion IX. Prostaglandins do not act like ionophores

To gain further insight on the mechanism of GH secretion in general and on the stimulation of this process by prostaglandins in particular, we compared the effects of PGE₁ and PGE₂ on hormone release and cyclic nucleotide levels with those of the ionophores A23187 and X537A under a variety of experimental conditions. All these substances (in the presence but not in the absence of calcium) enhanced GH release in incubated rat anterior pituitaries , prostaglandins being considerably more potent than ionophores. However, while PGE₂ caused a dose-dependent rise in pituitary cyclic AMP levels (from doubling at 10⁻⁷ M to a two-hundred fold increase at 10⁻⁵ M), the ionophores had no effect on the concentrations of this nucleotide. Neither PGE₂ nor the ionophores had any measurable effect on cyclic GMP levels. Exposure of tissues to ionophores for 60 minutes rendered them refractory to subsequent stimulation by PGE₁ or to ionophores themselves, whereas preincubation with PGE₁ did not diminish GH responses during a second incubation period. On the other hand, 60-minute preincubation of hemipituitaries in the presence of ionophores (10⁻⁵ M) did not suppress subsequent PGE₁-promoted cyclic AMP accumulation. Metabolic blockers inhibited PGE₂ and A23187-promoted GH-release but failed to suppress GH-response to X537A. Verapamil partially inhibited PGE₂ but not ionophore induced GH secretion. Ionophores particularly X537A, accelerated 45Ca efflux while PGE₁ did not influence this. Electronmicroscopy revealed extensive vacuolization localized chiefly at the Golgi apparatus when tissues were incubated with X537A. PGE₁ and A23187 had no such morphological effect. It is concluded that prostaglandins E and ionophores promote GH secretion by different mechanisms.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

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.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis

The cyclic AMP response to prostaglandin E₁ (PGE₁) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE₁ induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10⁻⁶M) of PGE₁ tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE₁. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

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.     

Stimulation of guanosine 3',5'-cyclic monophosphate accumulation in rat anterior pituitary gland in vitro by synthetic somatostatin

Synthetic somatostatin stimulated cyclic GMP accumulation with dose dependency (10 ng/ml – 10 μg/ml in a dose examined) in rat anterior pituitary gland in vitro. The stimulation of cyclic GMP levels in the gland was observed after 2 min incubation with somatostatin. Cyclic AMP production induced by TRH or PGE₁ was supressed by this GH release inhibiting factor, while cyclic GMP concentration in the gland was elevated. The present results seem to suggest that inhibitory effect on GH release by somatostatin in anterior pituitary gland is mediated through change in concentration of cyclic AMP and cyclic GMP in the target cells.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration

Five min following a single iv injection of PGE₂ into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE₂ injection. Plasma LH level increased at 2 min after PGE₂ and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Somatocrinin (growth hormone releasing factor) in vitro bioactivity; Ca++ involvement, cAMP mediated action and additivity of effect with PGE2

The release of GH induced by purified hypothalamic GRF or native or synthetic tumor-derived GRF is antagonized by the presence of CoCl²; it is simulated by 8Br .cAMP, IBMX, cholera toxin, forskolin, with identical maximal effects (E_max). Somatocrinin (GRF) stimulates the efflux of cAMP by the pituitary cells in parallel to the release of GH. Addition of either 8Br .cAMP, IBMX, cholera toxin or forskolin to a maximally stimulating dose of GRF does not increase the response which remains GRF-E_max. In contradistinction with these results PGE₂ releases GH with a dose-response curve different from that of GRF, and the combination of PGE₂ + GRF produces an E_max far greater than that due to either agonist alone; showing a true additivity. The name $\text{somatocrinin}$ is proposed to replace the acronym GRF.     

Cholecystokinin octapeptide releases growth hormone from the pituitary in vitro

Cholecystokinin-octapeptide (CCK-8)(10^?6 to 10^?8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH₃ pituitary tumor cells. Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion $\text{in vitro}$. In the GH₃ cell line, CCK-8 (10^?7M) reversed the inhibitory effect of somatostatin (10^?5M) on GH release. As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor.     

Studies on growth hormone secretion VIII effects of α,β-methylene-ATP on prostaglandin induced GH release and cyclic AMP accumulation in rat anterior pituitary glands

α, β-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.     

Prostaglandin stimulation of adenosine 3′,5′-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cycle AMP level in cultured chondrocytes were examined. Prostaglandin E₁ at 0.4 to 30 μM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D₂ and E₂ also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E₁. Prostaglandin A₁ had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E₁, D₂ or E₂ was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E₁, E₂, or D₂ on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E₁, E₂, and D₂ increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists; cyclic AMP and corticosterone production

Isolated adrenal cortex cells respond to the addition of ACTH_1–39 or analogs with increased production of cyclic AMP and corticosterone. It is estimated that cyclic AMP production need proceed at less than 20% of maximum to induce maximum corticosterone production. ACTH_1–24, [Lys¹⁷, Lys¹⁸]ACTH_1–8 amide, and ACTH_1–16 amide induce a maximum rate of cyclic AMP and of corticosterone production equal to those of ACTH_1–39. The relative potencies as determined by cyclic AMP and by corticosterone production are in excellent agreement. The analog, ACTH_5–24, induces maximum cyclic AMP production equal to 45% of that of the natural hormone, but as predicted, induces maximum corticosterone production equal to that of ACTH_1–39. The derivative, [Trp(Nps)⁹]ACTH_1–39 induces 77% of maximum corticosterone production and less than 1% of maximum cyclic AMP production. The fragment ACTH_11–24 is a competitive antagonist of ACTH_1–39 for both cyclic AMP and corticosterone production. The observations on agonists, a partial agonist and a competitive antagonist are in harmony with the “second messenger” role assigned to cyclic AMP. A provisional model, based on the fit of the experimental observations to a set of equations, provides expressions of “intrinsic activity,” “receptor reserve”, “sensitivity”, and “amplification” in terms of maximum cyclic AMP production, concentration of ACTH which induces $\text{1}\text{2}$ maximum cyclic AMP production and concentration of cyclic AMP which induces $\text{1}\text{2}$ maximum corticosterone production.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the $\text{in vitro}$ release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all $\text{in vitro}$ experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

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.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

The effect of various secretagogues on accumulation of cyclic AMP and secretion of α-amylase from rat exocrine pancreas

The relationship between accumulation of cyclic AMP and the secretion of α-amylase was investigated in the rat pancreas $\text{in vitro}$. Theophylline and secretin induced an increase in tissue cyclic AMP levels, however, only secretin stimulated secretion of α-amylase. Pancreozymin caused a release of α-amylase and had a biphasic effect on nucleotide levels — stimulation followed by inhibition. Carbachol, which induced a secretory response in the rat pancreas, reduced tissue levels of the cyclic nucleotide.