Cytosolic protein kinase A mediates the growth hormone (GH)-releasing action of GH-releasing factor in purified rat somatotrophs.

The growth hormone (GH)-releasing action of GH-releasing factor (GRF) is known to be cAMP-dependent. However, definitive proof for the involvement of the cAMP-dependent enzyme protein kinase A (PKA) is still lacking. In this study, we characterized the PKA system in purified rat somatotrophs and examined its role in mediating GRF-stimulated GH release under static incubation conditions. PKA enzyme activity was detected only in the cytosolic, but not the particulate fraction of rat somatotrophs. This cytosolic PKA activity exhibited the characteristic cAMP dependence (with ED50 of 0.1 microM), ability to phosphorylate kemptide (a synthetic peptide with a PKA phosphorylation site), and susceptibility to inhibition by the bovine heat-stable PKA inhibitor. GRF treatment (1 pM-1 nM) stimulated the cytosolic PKA activity and GH release from rat somatotrophs in a dose-dependent manner. Time-course studies also demonstrated that activation of cAMP synthesis and PKA activity preceded the GH response to GRF. Stimulation of cytosolic PKA activity in rat somatotrophs by the adenylate cyclase activator forskolin (10 nM-1 microM) and membrane permeant cAMP analog db.cAMP (5 microM-0.5 mM) mimicked the GH-releasing effect of GRF. In contrast, Rp.cAMP, a cAMP antagonist for PKA regulatory subunits, blocked both the cytosolic PKA activity as well as GRF-induced GH release. Similar inhibitions were also observed when an inhibitor for PKA catalytic subunits, H89, was used. Somatostatin (SRIF) (1 nM), the physiological GH-release inhibitor, suppressed the GH response to GRF without affecting the basal or GRF-stimulated PKA activity. SRIF at a higher dose (10 nM) abolished the GH-releasing effect of GRF. In this case, SRIF also induced a small but significant inhibition of GRF-stimulated PKA activity. Taken together, the present study provides direct evidence that PKA enzyme activity is localized only in the cytosol of rat somatotrophs and constitutes an essential component of the signal transduction mechanism for GRF-stimulated GH release. This cytosolic PKA system, however, does not appear to be a major target for the GH-release inhibiting action of SRIF.

Growth hormone-releasing factor does not activate protein kinase C in somatotrophs.

The purpose of this study was to investigate the involvement of protein kinase C in growth hormone-releasing factor (GRF) action by directly measuring the effect of GRF on protein kinase C activity in purified male rat somatotrophs. Somatotrophs were incubated with GRF (10(-7) M) for 0.33, 1, 3, 10, 30 and 90 min. Protein kinase C present in soluble and particulate fractions was partially purified using DEAE-cellulose chromatography, and protein kinase C activity was assayed. In control experiments, to insure protein kinase C activity could be activated, two known protein kinase C activators, phorbol 12-myristate 13-acetate (PMA) and dioctanoyl-rac-glycerol (diC8) were added for 3 min. Protein kinase C activity is present in somatotrophs. Under basal conditions the majority of the enzyme activity is located in the cytosol (approximately 90%). The protein kinase C activators caused a significant translocation of protein kinase C activity from soluble to particulate fractions at 3 min. GRF did not cause a translocation of protein kinase C activity even though GH release was significantly increased by 3 min. GRF did not significantly alter the specific activity of protein kinase C in the soluble or particulate fractions, except for a small (approximately 10%) increase in soluble activity at 90 min. We conclude that protein kinase C is present in the somatotrophs of the anterior pituitary. Protein kinase C, however, does not mediate the action of GRF and its role in signal transduction in somatotrophs awaits elucidation.

A comparison of the biological activities of authentic rat GRF(1-43)OH with the analogue rat GRF(1-29)NH2.

The purpose of this study was to characterize the biological activity of the synthetic rat growth hormone releasing factor analogue rGRF(1-29)NH2 and to compare its action on growth hormone (GH) release to that of authentic rGRF(1-43)OH. We first compared the concentration-response characteristics of the two peptides in static incubation, and then examined the reversibility and repeatability of the GH response in a perifusion system. Authentic rGRF(1-43)OH was significantly more potent in static incubation (EC50 = 3 x 10(-11) M) than the analogue (5 x 10(-11) M), whereas the reverse held true in perifusion. The shapes of the GH responses were similar for both peptides in the perifusion system. However, while the GH response to authentic rGRF was repeatable, the prior administration of rGRF(1-29)NH2 significantly reduced (greater than 50%) the GH response to the subsequent administration of either rGRF(1-29)NH2 or rGRF(1-43)OH. Thus authentic rGRF and the synthetic fragment may have different actions at the level of the GRF receptor or at a postreceptor (second messenger) step.

Free intracellular Ca2+ concentration ([Ca2+]i) and growth hormone release from purified rat somatotrophs. II. Somatostatin lowers [Ca2+]i by inhibiting Ca2+ influx.

This study was carried out to investigate the role of Ca2+ in the somatostatin (SRIF)-induced inhibition of GH release. We examined the effect of SRIF on basal and GH-releasing factor (GRF)-induced increases in Ca2+ influx and free intracellular Ca2+ concentration ([Ca2+]i) in normal somatotrophs and examined the effect of SRIF on 45Ca uptake, [Ca2+]i measured with indo-1, and GH release. SRIF inhibited basal and GRF-induced GH release concurrently with a reduction in steady state 45Ca uptake. In nonsteady state experiments, SRIF also decreased basal 45Ca uptake. SRIF decreased baseline [Ca2+]i in a concentration-dependent manner and inhibited the GRF-induced biphasic increase in [Ca2+]i, but in a differential fashion. Low concentrations of SRIF abolished the peak (first phase) without affecting the plateau (second phase), while at high concentrations, both phases were inhibited. SRIF blocked the GRF-induced increase in [Ca2+]i regardless of whether it was applied before or during GRF stimulation. These data indicate that the SRIF-dependent decrease in 45Ca uptake is due to a decrease in Ca2+ influx. This is further supported by the fact that the GRF-dependent increase in [Ca2+]i, which is dependent on Ca2+ influx, is blocked by SRIF. The reported ability of SRIF to reduce the activation rate of Ca2+ currents, decrease Ca2+ conductance, and hyperpolarize the cell would explain the differential effect of SRIF on the GRF-induced [Ca2+]i increase. The inhibitory effect of SRIF on GH release would then be dependent on the ability of SRIF to decrease, or prevent, an increase in [Ca2+]i.

Free intracellular Ca2+ concentration and growth hormone (GH) release from purified rat somatotrophs. III. Mechanism of action of GH-releasing factor and somatostatin.

GH-releasing factor (GRF)-stimulated GH release is dependent on a biphasic increase in free intracellular Ca2+ concentration [( Ca2+]i), resulting from an influx of Ca2+ into somatotrophs, while the inhibitory action of somatostatin (SRIF) on basal and GRF-induced GH release results from its ability to lower [Ca2+]i by inhibiting Ca2+ influx. This study was carried out to investigate the mechanism by which GRF and SRIF regulate [Ca2+]i to control GH release. The roles of ion channels, cAMP-dependent processes, and protein kinase-C (PKC) were investigated by measuring changes in [Ca2+]i, 45Ca influx, and GH release when purified rat somatotrophs were exposed to high K+, cAMP analogs, prostaglandin E2, as well as the PKC activators 1,2-dioctanoyl-glycerol and phorbol 12-myristate 13-acetate. High K+ depolarization produced a rapid and transient increase in [Ca2+]i, while cAMP and prostaglandin E2 led to a sustained elevated [Ca2+]i. PKC activators produced a transient increase in [Ca2+]i, followed by a decrease to below baseline. All secretagogues tested raised [Ca2+]i by stimulating Ca2+ influx through L-type voltage-sensitive Ca2+ channels (VSCC), since the increases in [Ca2+]i were blocked by incubation in Ca2(+)-free medium and by the dihydropyridine Ca2+ antagonist nifedipine. SRIF lowered [Ca2+]i by blocking the Ca2+ influx stimulated by all of these GH secretagogues except high K+. These results are consistent with the model in which GRF initiates its action by increasing Na+ conductance to depolarize the somatotroph via cAMP. This depolarization would stimulate Ca2+ influx through VSCC, which would result in the first phase of the GRF-dependent increase in [Ca2+]i. This increase in [Ca2+]i would stimulate Ca2+ removal from the cytosol by activating Ca-ATPase via Ca-calmodulin and/or PKC. This would result in the lowering of [Ca2+]i to the plateau level of the second phase of the GRF response. SRIF prevents the GRF-induced increase in [Ca2+]i by increasing K+ conductance and, thus, hyperpolarizing the cell. Hyperpolarization would close VSCC, leading to a decrease in Ca2+ influx, with a subsequent drop in [Ca2+]i.

Effect of growth hormone-releasing factor on phosphoinositide hydrolysis in somatotrophs.

We studied the role of the phosphatidylinositol system in the action of growth hormone-releasing factor (GRF). We asked whether GRF stimulates the activity of phospholipase C by determining GRF-induced changes in 32P labeling of the individual phosphoinositides and inositol phosphates in purified rat somatotrophs. The somatotrophs were challenged with GRF (10(-7)M) for 0.33, 1, 3, 10, 30, and 90 min. GRF did not significantly or consistently alter 32P incorporation into phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol monophosphate (PIP), or phosphatidylinositol (PI), except for a small reduction in PIP labeling at 90 min. In general the level of 32P incorporation into the inositol phosphates did not increase but instead decreased with GRF. There was a small but significant reduction of labeling of inositol trisphosphate (IP3) at 90 min of GRF incubation. There were also small but significant decreases in 32P incorporation into inositol bisphosphate (IP2) at 0.33, 3, and 30 min. GRF did not significantly alter 32P labeling of inositol monophosphate (IP). These results indicate that GRF does not stimulate phospholipase C activity in somatotrophs. We conclude that the phosphatidylinositol second messenger system does not play an essential role in the action of GRF.

Protein kinase C is not essential for growth hormone (GH)-releasing factor-induced GH release from rat somatotrophs.

To examine the role of protein kinase-C in the mediation of GH release we used acutely dispersed purified somatotrophs in static incubation and acutely dispersed adenohypophyses in perifusion. In static incubation, activation of protein kinase-C by phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8) resulted in an increase in GH release and a concurrent concentration-dependent increase in cAMP accumulation. The GH response to diC8 in perifusion was reversible and repeatable. On the other hand, the GH response to PMA was not repeatable. The lack of repeatability is most likely due to the depletion of protein kinase-C by prolonged treatment with PMA. This assumption is strengthened by the observation that 1 h of perifusion with PMA left the somatotrophs refractory to a subsequent application of diC8. When graded pulses of GRF were applied during treatment with PMA, the GH response to GRF was not altered. Somatostatin reduced (in static incubation) or blocked (in perifusion) the release of GH induced by diC8 and PMA, but the accumulation of cAMP was not affected. We conclude that 1) activation of protein kinase-C in normal somatotrophs results in GH release which may not be completely independent of the cAMP pathway; 2) activation of protein kinase-C is not essential for GRF-induced GH release; and 3) SRIF acts at a site distal to or independent of cAMP to inhibit GH release induced by activators of protein kinase-C.

Release of growth hormone from purified somatotrophs: effects of the calcium channel antagonists diltiazem and nifedipine on release induced by growth hormone-releasing factor.

We examined the effect of the voltage-sensitive Ca2+ channel antagonists, diltiazem and nifedipine, on basal and stimulated growth hormone (GH) release from purified somatotrophs. Our aim was to ascertain whether an influx of Ca2+ from the extracellular to the intracellular compartment is essential for augmented release. Basal release was decreased in a concentration-dependent manner by both diltiazem and nifedipine, while cAMP accumulation was unaffected. The release of GH induced by 29 mM K+ was blocked by diltiazem and nifedipine, at 10(-7) and 10(-8) M, respectively. Again cAMP was unaffected. The release of GH induced by growth hormone-releasing factor was significantly reduced by 10(-4) M diltiazem and completely blocked by nifedipine at a concentration of 10(-6) M or greater. Where the antagonists were effective, the growth hormone-releasing factor induced increase in cAMP accumulation was augmented. We conclude that an influx of Ca2+ from the extracellular compartment is essential for stimulated GH release.

Effect of withdrawal of somatostatin and growth hormone (GH)-releasing factor on GH release in vitro.

The secretion of GH, in vivo, is pulsatile. We have proposed that the timing of the episodic bursts of GH secretion is set by somatostatin (SRIF) withdrawal, while the magnitude of the bursts is set by the amount of GH-releasing factor (GRF) impinging on the somatotrophs, before and during SRIF withdrawal. We have now used an in vitro model of perifused rat pars distalis cells to further examine the interaction between GRF and SRIF on the magnitude of the burst of GH release that follows SRIF withdrawal. We first characterized the GH response, with time, to constant perifusion with GRF. The initial burst, followed by a rapid decrease in GH release induced by constant perifusion is due to a loss of GRF bioactivity in the perifusion medium and not to a decreasing responsiveness of the somatotrophs. This was followed by studies on the interaction between GRF and SRIF. The burst of GH release after cessation of perifusion with SRIF (10(-9) M) plus GRF (10(-10) M) can be blocked by the administration of SRIF during the burst. Also, the magnitude of the burst is proportional to the concentration of GRF preceding the withdrawal of SRIF. It is likely that similar relations apply in vivo, where SRIF withdrawal sets the timing and duration of the episodic burst of GH release, while GRF sets the magnitude.

Neuropeptide Y does not inhibit the release of alpha-MSH from the pars intermedia of the rat adenohypophysis.

Neuropeptide Y in concentrations from 10(-8) to 10(-6) M inhibits the release of alpha-MSH from the frog (Rana pipiens) pituitary in a reversible, sustained, and concentration-related manner. However, it does not inhibit the release of alpha-MSH from the rat pars intermedia. Thus, while neuropeptide Y may play a role in the control of alpha-MSH release in amphibia, it appears not to be a regulatory peptide for the mammalian pars intermedia.

Failure of growth hormone (GH) to feed back at the level of the pituitary to alter the response of the somatotrophs to GH-releasing factor.

GH feeds back at the level of the central nervous system to alter the release of somatostatin and GRF, resulting in altered GH release. The purpose of this study was to see whether the concentration of GH, impinging directly on the somatotrophs of the adenohypophysis, would alter the responsiveness of the somatotrophs to GRF. Using a perifusion system and dispersed pituitary cells, we found that the GH response to a pulse of GRF is unaltered over a wide range of GH concentrations. We conclude that GH does not feed back at the level of the adenohypophysis to alter the responsiveness of the somatotrophs to GRF.

Effect of somatostatin withdrawal and growth hormone (GH)-releasing factor on GH release in vitro: amount available for release after disinhibition.

The secretion of GH is strikingly episodic. We have suggested that the timing of the episodic bursts of GH secretion is set by somatostatin (SRIF) withdrawal, whereas the magnitude of the bursts is determined by the amount of GH-releasing factor (GRF) impinging on the somatotrophs before and during SRIF withdrawal. We have now used an in vitro model of perifused rat pars distalis cells to examine the interaction of SRIF and GRF on GH release and, in particular, to examine the effect of GRF on the magnitude of the burst of GH release that follows SRIF withdrawal. After 30 min of perifusion with SRIF (10(-9) M), there follows an immediate but small burst of GH release. The burst of GH release following concurrent perifusion with SRIF plus GRF (10(-10) M) is increased, with a 7.5- to 9.5-fold increase in the peak secretion rate. When GRF is maintained after the withdrawal of SRIF, the peak secretion rate is not different from that seen after simple withdrawal of both SRIF and GRF, but the duration of the burst is increased. These data demonstrate that the presence of GRF during SRIF perifusion, while not altering basal release, does strikingly increase the post-SRIF release of GH. We propose that a similar relation applies in vivo, where SRIF withdrawal sets the timing of the episodic bursts of GH release, whereas GRF determines the magnitude.

Release of growth hormone (GH) from purified somatotrophs: interaction of GH-releasing factor and somatostatin and role of adenosine 3',5'-monophosphate.

An acutely dispersed and purified preparation of somatotrophs obtained from rat adenohypophyses was used to study the mechanism of action of GH-releasing factor (GRF). Synthetic GRF [human pancreatic, hpGRF-(1-40)-OH] stimulated the immediate (within 4 min) release of GH in a dose-related manner, with a preceding or concurrent increase in cAMP in the somatotrophs. Somatostatin, at concentrations as low as 1.0 ng/ml, completely blocked the GRF-induced increase in GH release, with only a partial reduction in the GRF-induced accumulation of cAMP in the somatotrophs. 3-Isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, potentiated the action of GRF in increasing cAMP in the somatotrophs, with subsequent GH release. These results along with those of previous studies suggest that cAMP is an intracellular mediator in the action of GRF and that somatostatin has a major effect on blocking GH release at a step subsequent to cAMP accumulation.

Release of pro-opiomelanocortin-derived peptides from the pars intermedia and pars distalis of the rat pituitary: effect of corticotrophin-releasing factor and somatostatin.

The parenchymal cells of the pars intermedia (PI) and corticotrophs of the pars distalis (PD) synthesize pro-opiomelanocortin (POMC), which, through posttranslational processing, gives rise to a group of structurally related peptides, including MSHs, ACTH, CLIP, LPHs and endorphins. We investigated the control of release of these peptides using an in vitro system. We perifused either intact neurointermediate lobes (NI) or PD halves obtained from rats. Perifusion medium and tissue extracts were subjected to a battery of bioassays (BA) and radioimmunoassays (RIA) (including MSH-BA, alpha-MSH-RIA, ACTH-BA, ACTH-RIA, LPH-RIA) and a receptor-binding assay for morphine-like activity (MLA). The relative amounts of released peptide activities were examined under basal conditions and after challenging with synthetic ovine corticotrophin-releasing factor (CRF) and somatostatin. CRF stimulated the release of all assayed peptides from both the PD and PI in a dose-related manner. Stimulated release was immediate (within 3 min), constant, reversible and repeatable. Somatostatin (up to 100 ng/ml) did not alter basal release from either PD or PI. Somatostatin did block CRF-induced release from the PI but not from the PD. These observations support an action of both CRF and somatostatin in the control of secretion of POMC-derived peptides from the PI.

Secretory bursts of growth hormone secretion in the dog may be initiated by somatostatin withdrawal.

In 28 6-h experiments on 10 conscious resting trained male dogs, plasma growth hormone (GH) was determined at 5-min intervals by radioimmunoassay. For all experiments, the basal GH concentration in plasma was 0.80 +/- 0.06 ng mL-1. In each experiment, 1-3 secretory bursts of GH occurred, raising plasma GH 2.4 to 15.3 times basal concentrations (for all 43 bursts, 6.6 +/- 0.4 times the basal value). Metabolic clearance rates (MCR) and apparent distribution volumes (V) were determined, using stepwise infusions of canine GH. The MCR (3.99 +/- 0.30 mL kg-1 min-1) and V (57.9 +/- 5.5 mL kg-1) were used to transform the GH concentration versus time data into GH secretion rates, using a single compartment approach. Basal GH secretion rates for all 28 experiments were 3.12 +/- 0.24 ng kg-1 min-1. The secretory bursts yield peak GH secretion rates of 9.4 +/- 0.8 times basal secretion and these steep-sloped bursts last 25.1 +/- 1.2 min. Six-hour infusions of 0.15 microgram kg-1 min-1 of somatostatin (SRIF) abolished all secretory bursts but did not lower basal secretion rates. In five of seven SRIF infusion experiments in which samples were taken after the infusion ceased a secretory burst was seen in the hour following cessation of infusion (in four cases within 10 min). These secretory bursts lasted 23.0 +/- 2.9 min and were similar to those seen in control experiments. Infusions of SRIF at 0.05 microgram kg-1 min-1 had no effect. These results imply that during basal GH secretion, a surfeit of SRIF impinges on the somatotrophs, as extra SRIF does not further lower basal secretion. However, during secretory bursts, very little SRIF must be present, as exogenous SRIF blocks these bursts. The bursts are similar in duration to overshoots provoked in perifused dispersed rat somatotrophs by removal of an SRIF signal. It seems likely that their cause in vivo is similar. (All values are means +/- SEM.)

Effects of sexual activity on luteinizing hormone and testosterone levels in the adult male rabbit.

Sexually mature New Zealand White male rabbits were subjected to various sexual stimulation procedures. Plasma samples were obtained through an indwelling catheter in the central ear artery and analysed for LH and testosterone using specific radioimmunoassays. Coitus and exposure to females occasionally led to increases in testosterone levels which were usually preceded by a rise in LH. Increases in hormone concentration were not significantly different from those of rabbits which did not mate or which were isolated from females. These results suggest that coitus in the male rabbit does not cause the massive increase in LH concentration which is known to occur in the female.

Variations in peripheral levels of LH and testosterone in adult male rabbits.

Seasonal, diurnal and episodic patterns of LH and tests-sterone secretion in sexually mature male New Zealand rabbits were studied. Blood samples were obtained from the central ear artery by puncture or through an indwelling catheter, and were assayed for hormones using radioimmunoassay. Testosterone values appeared to be lower in the summer months while LH showed no seasonal cyclicity. There were no significant fluctuations when samples were taken at 10-min intervals, but specimens taken every hour for 24 or 36 hr revealed an episodic pattern of release. Peaks of both hormones occurred every 4 to 5 hr in most animals. Testosterone levels ranged from 0.5 to 10 ng/ml and LH from 15 to 200 ng/ml of WP360A standard. In general, a rise in LH preceded or coincided with an increase in testosterone. No specific diurnal rhythm could be demonstrated and the patterns appeared to be unrelated to external stimuli.