Intracellular calcium stores are involved in growth hormone-releasing hormone signal transduction in rat somatotrophs.

In rat pituitary somatotrophs, the stimulation of growth hormone secretion by growth hormone-releasing hormone (GHRH) is a Ca(2+)-dependent event involving Ca2+ influx. The presence of calcium-induced calcium release (CICR) Ca2+ stores has been suggested in these cells. The aim of our study was to demonstrate the presence of CICR stores in rat somatotrophs and to determine their function in GHRH Ca2+ signalling. To this end we measured cytosolic free Ca2+ concentration ([Ca2+]i), using indo-1 in purified rat somatotrophs in primary culture, while altering intracellular Ca2+ stores. Ionomycin (10 ttM) or 4-bromo-A23187 (10 ItM), used to mobilise organelle-bound Ca2+, raised [Ca2+]i in the absence of extracellular Ca2+. Caffeine (5 to 50 mM), used to mobilise Ca2+ from CICR stores, transiently raised [Ca2+]i in 65% of cells tested. The response to 40 mM caffeine was abolished when Ca2+ stores were depleted, with 1 microM thapsigargin or with 10 microM ryanodine. All cells that responded to 40 mM caffeine responded to 10 nM GHRH. The [Ca2+]i response to 10 nM GHRH was reversible and repeatable. However, the second response was 38% smaller than the first. Ryanodine treatment abolished the reduction in the second [Ca2+]i response, while thapsigargin increased the reduction by 67%. We conclude that rat somatotrophs possess CICR Ca2+ stores and that they account for 30-35% of the GHRH-induced increase in [Ca2+]i, and that their partial depletion is involved in somatotroph desensitization.

Multiple cytosolic calcium signals and membrane electrical events evoked in single arginine vasopressin-stimulated corticotrophs.

The action of arginine vasopressin (AVP) on cytosolic free Ca2+ concentration ([Ca2+]i) was investigated in single rat pituitary corticotrophs using indo-1 microfluorimetry, in part in combination with the monitoring of membrane electrical events with the perforated patch-clamp technique. In corticotrophs showing the series of short-lived [Ca2+]i rises (transient pattern) in response to corticotropin-releasing factor, 100 nM AVP evoked either the transient pattern or a [Ca2+]i spike followed by a sustained plateau (spike/plateau pattern). Not all corticotrophs responded to changes in AVP concentration in the same manner. Some cells exhibited a concentration-dependent increase in [Ca2+]i transient activity, whereas others showing the spike/plateau at high AVP concentrations responded to low agonist concentrations by two [Ca2+]i responses: a slow rising step or two to three sinusoidal-like oscillations. Combined [Ca2+]i and patch-clamp recordings as well as manipulation of extracellular Ca2+ showed that both transient pattern and the plateau of spike/plateau response depended on Ca2+ entry mainly through voltage-gated, dihydropyridine-sensitive Ca2+ channels. By contrast, step, oscillations, and spike were due to Ca2+ release from internal stores. These Ca(2+)-mobilizing responses caused the activation of Ca(2+)-activated, apamin-sensitive K+ channels, which led to a membrane hyperpolarization. These results reveal cell-specific [Ca2+]i signals and associated electrical events in individual AVP-stimulated corticotrophs.

Somatostatin activates an inwardly rectifying K+ conductance in freshly dispersed rat somatotrophs.

1. Somatotrophs from enzymatically dispersed anterior pituitary glands of rats, enriched to greater than 94% purity by density gradient centrifugation, were studied within 16 h of isolation using patch clamp recording methods in the conventional whole-cell and the perforated-patch configurations. 2. Rhythmic oscillations of membrane potential gave rise to action potentials in thirty-six of fifty-two cells studied with the perforated-patch technique. Membrane potential oscillated between approximately -70 mV and approximately -25 mV with an average frequency (mean +/- S.D.) of 0.9 +/- 0.9 s-1. 3. The current-voltage (I-V) relationship of cells was linear at negative potentials with outward rectification at potentials positive to -40 mV. Evidence that the outward current was due to K+ channels came from the deactivation tail currents, which reversed direction close to the K+ equilibrium potential (EK). The reversal potential shifted 60 mV per tenfold change of external K+ concentration ([K+]o), as expected for K+ current. 4. Suppression of outward current by tetraethylammonium (TEA) provided additional evidence for K+ current. Cd2+ reduced outward current, suggesting the presence of Ca(2+)-activated K+ conductance. 5. Depolarizing commands elicited transient inward Na+ current and a sustained Ca2+ current (ICa). ICa was recorded in isolation with Cs+ and TEA in the recording pipette and 10 mM-Ba2+ as the charge carrier. Activation of ICa began at approximately -40 mV, with peak inward current at 0 to +10 mV. The half-inactivation potential was approximately -35 mV. In addition, ICa was blocked by nifedipine. These characteristics indicate the presence of L-type Ca2+ channels in somatotrophs. 6. Somatostatin caused hyperpolarization and suppressed the spontaneous bursts of action potentials. Under voltage clamp, somatostatin activated an inwardly rectifying current that reversed direction near EK. When EK was altered by elevation of [K+]o, the reversal potential of the somatostatin-induced current shifted 55 mV per tenfold change of [K+]o, as predicted for a K+ current by the Nernst relation. The somatostatin-induced conductance (gK) was greater at more negative potentials, and the activation range shifted positive with elevation of [K+]o. 7. We conclude that freshly isolated rat somatotrophs possess Na+, Ca2+ and K+ currents. A large proportion of the cells exhibit spontaneous bursts of action potentials. Somatostatin activates an inwardly rectifying K+ conductance, causing hyperpolarization and cessation of spontaneous action potential activity, actions that would contribute to suppression of growth hormone release.

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.

Free intracellular Ca2+ concentration ([Ca2+]i) and growth hormone release from purified rat somatotrophs. I. GH-releasing factor-induced Ca2+ influx raises [Ca2+]i.

This study was carried out to investigate the role of free intracellular Ca2+ ([Ca2+]i) in the action of GH-releasing factor (GRF) by determining whether GRF causes and increase in [Ca2+]i and whether this increase results from changes in Ca2+ influx/efflux and/or mobilization of intracellular Ca2+ stores. We used a purified preparation of normal rat somatotrophs and examined the changes in 45Ca uptake, [Ca2+]i measured with indo-1, intracellular cAMP, and GH release induced by GRF. GRF stimulated a concentration-related biphasic increase in [Ca2+]i. Both the GRF-dependent increase in [Ca2+]i and GH release were blocked by incubation in low Ca2+ medium and by the organic Ca2+ antagonists nifedipine and diltiazem. The measurement of 45Ca uptake, in both steady state and nonsteady state conditions, demonstrated directly that GRF stimulates Ca2+ influx into somatotrophs. These data demonstrate that the GRF-stimulated increase in [Ca2+]i is dependent on Ca2+ influx. Redistribution of intracellularly stored Ca2+ could not be detected, even though intracellular Ca2+ stores were present. Therefore, the increase is due to Ca2+ influx, and the biphasic nature of the increase in [Ca2+]i induced by GRF is due to a difference in the rate of activation of Ca2+ influx and Ca2+ removal from the cytosol.

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.

The effect of hepatic insulin influx on insulin retention: comparison of net flux and per cent extraction as indices of hepatic insulin retention.

We studied effects of changes in hepatic insulin influx on hepatic insulin retention. In order to compare insulin net flux (influx - efflux) with percentage (%) hepatic insulin extraction (net flux/influx X 100), as indices of hepatic insulin retention, we examined which index is better predicted by insulin influx to the liver. Electromagnetic hepatic blood flow measurements were made and blood was sampled from portal, hepatic and peripheral veins and femoral artery in anesthetized dogs. Insulin influx was perturbed by sequentially superimposed peripheral venous infusions of somatostatin, glucagon and insulin (which were subsequently discontinued one at a time). Although the somatostatin was biologically effective in inhibiting insulin and glucagon release we found no significant plasma flow changes on starting or stopping the infusion of somatostatin (800 ng X kg-1 X min-1). Net insulin flux was linearly related to influx (n = 56, r = 0.99, p less than 0.001). Hepatic glucose production was better correlated with net insulin flux (r = -0.66; p less than 0.01) than with % extraction (r = 0.08; N.S.), when mean glucagon influx was held constant (partial correlation). Although net insulin flux and % extraction are complementary indices, the former reflects influx changes more faithfully than the latter.

Hepatic insulin retention depends on influx in both diabetic and normal dogs.

Hepatic retention of endogenous and exogenous insulin was investigated in normal and alloxanstreptozotocin diabetic dogs. An hour's peripheral venous infusion of insulin was preceded and followed by saline infusion. Blood samples were drawn simultaneously from the femoral artery and portal, hepatic and peripheral veins. Blood flow was measured electromagnetically in the portal vein and hepatic artery. Hepatic plasma flow was lower in diabetic dogs (p less than 0.001) so that prior to insulin infusion (basal hour) basal mean hepatic venous flux was diminished in them. Hepatic glucose production was higher (p less than 0.05) and arterio-venous glucose difference lower (p less than 0.01) in diabetics basally, but post-insulin infusion, these became similar in diabetics and normals. Although basal hour arterial and peripheral venous insulin concentrations were similar, portal venous insulin was 3.5-fold lower in diabetics. Mean insulin influx (hepatic arterial + portal venous fluxes) was four-fold lower in diabetics. The insulin net flux (influx - hepatic venous flux) was lower in diabetics (p less than 0.05) as was % extraction (p less than 0.001). During the insulin infusion hour the values remained lower for diabetics (p less than 0.05) for influx, net flux and % extraction. During the post-insulin infusion hour the influx, net flux and % extraction became similar. When the basal hour data in normals was restricted to the range of diabetic insulin influx values less than or equal to 10 mU X min-1, the differences in net flux and % extraction between normals and diabetics were no longer present.(ABSTRACT TRUNCATED AT 250 WORDS)