Pituitary immediate release pools of growth hormone and prolactin are preferentially refilled by new rather than stored hormone

Pituitary stores of rat GH (rGH) and PRL (rPRL) are divisible into immediately releasable and more stable compartments representing either compartmentalized hormone within individual cells of a homogeneous population or responses of specialized cell subsets in a functionally heterogeneous population. In addition, newly synthesized rGH and rPRL can be processed either into intracellular storage or toward direct release. Fractional assignment of new hormone to these two paths can be influenced in the somatotroph by GHRH and may also represent either intracellular processes or functional heterogeneity of cells. We investigated the source, newly synthesized or stored, of hormone refilling the somatotroph and lactotroph immediately releasable pools (IRP) after their discharge by 21 mM potassium ion, 1 mM (Bu)2cAMP, 3 nM human GHRH-44, or 3 microM prostaglandin E1. Experiments were performed using perifused pituitary fragments exposed sequentially to [14C]- and [3H]leucine in association with stimulation by two 30-min pulses of the same secretagogue. Therefore, only [14C]hormone was available for release by the first stimulus, whereas both [14C]- and [3H]hormone were available for release by the second stimulus. Analysis was by specific immunoprecipitation. The first episode of stored [14C]rGH release exceeded the second episode of stored [14C]rGH release in response to each secretagogue. However, release of newly synthesized [3H]rGH in response to the second episode of stimulation exceeded the simultaneous release of stored [14C]rGH while matching or exceeding the [14C]rGH release that had occurred in the same experiment in response to the first episode of stimulation. Refilling both GH and PRL IRP stores drew primarily upon newly synthesized hormone, but with different secretagogue-specific patterns. These data confirm differential handling of new and stored rGH and rPRL within the pituitary. They are consistent with either (1) the enhanced shunting of newly synthesized hormone to IRPs within cells that are capable of compartmentalized intracellular hormone storage, or (2) the relatively complete discharge of a subset of somatotrophs and lactotrophs that are specialized to deliver pulsed hormone release, after which they are refilled by newly synthesized hormone.     

Growth hormone-releasing factor-44 specificity for components of somatotroph and lactotroph immediate release pool substructures

Rat somatotroph and lactotroph hormone storage is divisible into at least two functional compartments: an immediate release pool (IRP) and a pool that responds to prolonged stimulation. An IRP substructure has been defined by release in response to potassium ion (K+), prostaglandin E1 (PGE1), and Bu2cAMP. The somatotroph IRP is expandable; the lactotroph IRP is fixed in size. The present experiments examined which IRP components contribute to the rapid release of stored GH in response to GH-releasing factor-44 (GRF). Release of stored PRL was monitored for comparison. In vitro prelabeling defined stored rat (r) GH and rPRL. Release in response to 21 mM K+, 3 microM PGE1, 1 mM Bu2cAMP, and/or 3 nM GRF was monitored with a perifusion-immunoprecipitation system. After 120 min of basal perifusion, tissue was exposed to one of the four secretagogues for 90 min. During a second 90-min period a second secretagogue was added while exposure to the first secretagogue continued. We demonstrated that 21 mM K+ reduces peak rGH release in response to 3 nM GRF by 52%, whereas GRF does not reduce rGH release in response to K+; 3 microM PGE1 reduces rGH release in response to GRF by only 19% although GRF reduces rGH release in response to PGE1 by 88%; 1 mM Bu2cAMP reduces rGH release in response to GRF by 87%, and GRF eliminates rGH release in response to Bu2cAMP (1.2% of control value); combined K+ plus Bu2cAMP reduce rGH release in response to GRF to 2.5% of the control value, whereas after GRF pretreatment rGH release in response to combined K+ plus Bu2cAMP is 93% of the control value; and combined PGE1 and Bu2cAMP reduce the response to GRF to 17% of the control value. Effects on rPRL release are qualitatively similar. We conclude that immediate GRF-stimulated release of stored rGH originates in the somatotroph IRP components defined by responses to PGE1 and Bu2cAMP; it derives only slightly, if at all, from the IRP component defined by the response to K+. The smaller GRF-stimulated release of IRP rPRL is similarly derived.     

Growth hormone release: interaction of increased extracellular calcium and somatostatin

These studies were designed to examine the effects of extracellular calcium ion (Ca++) concentration upon basal and dibutyryl (db) cAMP or potassium ion (K+)-stimulated release of growth hormone (GH) and to determine whether increased extracellular Ca++ can overcome somatostatin (SRIF)-inhibited release of stored rGH in parallel with its reported effect upon SRIF inhibition of stimulated insulin and glucagon release. Experiments were performed in vitro using prelabeled rat pituitary fragments in a perifusion, specific immunoprecipitation system designed to limit observations to release of stored hormone from viable cells. Increased (up to 5.4 mM) extracellular Ca++ inhibits basal and dbcAMP-stimulated release of stored, prelabeled [3H]rGH in parallel with the effects of SRIF: post-inhibition rebound, dose responsivity, and differential effect upon early and late dbcAMP-stimulated release of stored [3H]rGH. Increased (21 mM) extracellular K+ interferes with both Ca++- and SRIF-inhibited early dbcAMP-stimulated release of stored [3H]rGH. The combination of increased extracellular Ca++ and SRIF inhibits basal release of stored [3H]rGH more than either agent alone and during dbcAMP stimulation, rebound release of stored [3H]rGH follows withdrawal of either inhibitor despite continuation of the other. This rebound release is enhanced when both inhibitors are withdrawn simultaneously. Conclusions: (a) the inhibition of stored rGH release induced by increased extracellular Ca++ and SRIF occurs through at least partially independent mechanisms, and (b) increased extracellular Ca++ does not reverse SRIF inhibition of stimulated rGH release from prelabeled intracellular storage, in contrast with observations in the pancreatic islet.     

Effect of growth hormone-releasing factor-44 upon release of concurrently synthesized hormone by perifused rat pituitary tissue

We previously reported the differential stimulation of stored and newly synthesized rat (r)GH release by human GH-releasing factor-44 (hGRF-44). Those studies were performed over a 3-h period in a static in vitro incubation system. The present experiments focus on hGRF-44 effects upon release of new hormone (synthesized during tissue stimulation by the secretagogue) and were performed in in vitro perifusion to study the time course of the response. A double label ([14C], [3H]), immunoprecipitation protocol defined hormone release in relation to time of synthesis: intracellular stores of hormone were prelabeled with [14C]; subsequent exposure of prelabeled tissue to continuous concurrent [3H]leucine and 3 nM hGRF-44 defined newly synthesized hormone and associated it with the secretagogue. In a parallel set of experiments, 1 mM (Bu)2cAMP was substituted for hGRF-44. Prolonged exposure to hGRF-44 in perifusion stimulated an initial surge of stored [14C]rGH release which was followed by a rate of [14C]rGH release which declined rapidly but remained suprabasal. [14C]rGH release in response to (Bu)2cAMP was also biphasic, but the initial surge was delayed and the later stimulatory period was better sustained in comparison to responses to hGRF-44. Stimulation of stored [14C] rPRL release by hGRF-44 was observed in perifusion, confirming our observation in the static system. Release of newly synthesized, 3H-labeled rGH was immediately stimulated by either hGRF-44 or (Bu)2cAMP, and that stimulation was maintained throughout exposure to either secretagogue. In contrast, whereas newly synthesized [3H]rPRL release was stimulated by (Bu)2cAMP, its release in response to hGRF-44 resembled that in control experiments. No effect upon hormone synthesis was observed during the 3h of exposure to hGRF-44. In conclusion, these experiments confirm that hGRF-44 differentially stimulates release of both newly synthesized and stored rGH, and demonstrate differential dynamics in the response as well. Specifically, hGRF-44 stimulation of new rGH release is sustained while its effect on stored hormone simultaneously wanes. Further, when expressed as a percent of intracellular hormone available for release, new hormone is released more than 10 times faster than stored hormone. These observations argue for the existence of separate intracellular paths along which newly synthesized and stored hormone are released by the somatotroph. Finally, these data confirm that hGRF-44 stimulates release of stored rPRL without altering release of newly synthesized rPRL.     

Release of stored, pre-labeled growth hormone and prolactin from perifused rat pituitary: effect of human pancreatic growth hormone-releasing factor-44

Human pancreatic growth hormone-releasing factor-44 (hpGRF-44) differentially stimulates release of stored and newly synthesized rGH without altering rGH synthesis over 3 h in static in vitro incubation; hpGRF-44 also stimulates release of stored, but not newly synthesized, rPRL. To study the time course of pre-labeled, stored hormone release without pharmacologically interrupting synthesis, the current experiments were performed in perifusion. Fifteen minute pulses of 0.1 to 10 nM hpGRF-44 stimulated stored [3H]rGH release (to 890% of base); 1.0 to 10 nM hpGRF-44 stimulated stored [3H]rPRL release (to 440% of base). Pulses of 0.1 to 1.0 mM (Bu) 2cAMP also stimulated release of [3H]rGH (to 570% of base) and [3H]rPRL (to 410% of base). However, peak [3H]rGH and [3H]rPRL responses to hpGRF-44 required 10 min, while peak responses to (Bu) 2cAMP required 25 min. Continuous hpGRF-44 stimulated an initial surge of stored [3H]rGH release which was not sustained; the diminishing release was not explained by hpGRF-44 degradation. Total radioimmunoassayable (RIA) hormone release roughly paralleled release of stored immunoprecipitable (IPn) hormone. Conclusions: in pituitary perifusion, hpGRF-44 stimulates release of both stored rGH and rPRL as shown in static incubation, but the response is biphasic: initial rapid release is followed by a progressively lesser response; and the response is both more acute and less well sustained than that resulting from exposure to (Bu) 2cAMP.     

Basal and dibutyryl cyclic AMP-stimulated release of newly synthesized and stored growth hormone from perifused rat pituitaries.

The basal rate of pre-labeled stored 14C-labeled rat growth hormone ([14C]rGH) release from perifused rat pituitary explants is a constant fraction of pituitary GH content, suggesting random release from the storage pool. Basal release of newly synthesized [3H]rGH occurs in two phases: 1) immediate and associated with [3H]rGH synthesis, and 2) late (delayed by 60 min) and independent of concurrent [3H]rGH synthesis. Dibutyryl cyclic AMP (10(-2)M)-stimulated release of stored [14C]rGH is characterized by an initial acute rise followed by a second phase of continuous rapid release. Immediate and late release of new [3H]rGH is increased by dibutyryl cyclic AMP, and the late phase of [3H]rGH is less delayed. Simultaneous exposure of pituitary explants to [3H]alanine and [14C]leucine resulted in the release of immunoprecipitable rGH whose ratio of incorporated 3H and 14C varied with time. The observed changes suggest that after it is synthesized, a GH molecule may either be released directly or be processed into the somatotroph's storage compartment. In addition, stored GH is composed of two pools, one of which is immediately releasable. The differential incorporation of [3H]alanine and [14C]leucine into "big" and "small" rGH, together with the ability to differentially displace 3H-labeled "big" and 14C-labeled "small" rGH from the GH antibody suggest that "big" rGH is a heterogenous molecule including "small" rGH and another peptide rather than simply a dimer of "small" rGH.     

Functional substructure of the rat somatotroph immediate release pool: definition by responses to N6,2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate, potassium ion, and/or prostaglandin E1

Previous results from our laboratory suggest that stored rat GH (rGH) in the pituitary is divisible into at least two functional compartments. An immediate release pool (IRP) responds quickly and can be exhausted. A larger and less labile pool responds continuously to long term stimulation. We previously demonstrated that the sum of IRP rGH discharged by (Bu)2cAMP and potassium ion (K+) in separate experiments exceeds by one third the amount released by the two agents administered simultaneously. This overlap suggested an IRP substructure. We used prelabeled rat pituitary fragments in an in vitro perifusion-immunoprecipitation system to define intracellular hormone storage and to track release of stored rGH and rat PRL (rPRL). We tested three secretagogues: K+ to induce release without altering pituitary cAMP levels, (Bu)2cAMP to introduce cAMP into cells without activating adenylate cyclase, and prostaglandin E1 (PGE1) to produce a temporary, localized cAMP increase through adenylate cyclase activation. Prelabeled tissue in basal perifusion was first exposed to one secretagogue for 90 min. Then, while the first secretagogue was continued, a second secretagogue was added for a second 90-min period. Demonstrable alterations in tissue responses to secretagogues included: K+ diminished PGE1-induced rGH release from the IRP by 69% but had a mixed effect on the response to (Bu)2cAMP; (Bu)2cAMP enhanced K+-induced rGH release from the IRP by 71% but reduced PGE1-induced rGH release by 72%; PGE1 diminished K+-induced rGH release by 13% and (Bu)2cAMP-induced rGH release by 23%; combined K+ and (Bu)2cAMP reduced the rGH response to PGE1 stimulation by 81% whereas prior PGE1 enhanced the response to subsequent combined K+ and (Bu)2cAMP by 16%. We conclude that the somatotroph IRP consists of a K+-sensitive portion which overlaps with, but is not identical to, a (Bu)2cAMP-sensitive portion. The PGE1-sensitive portion of the IRP appears to be roughly equivalent to the shared fraction of the K+- and (Bu)2cAMP-sensitive portions of the IRP. These agents define a similar rPRL compartmentalization. However, the K+-sensitive portions of the somatotroph and lactotroph IRP differ in that the former is larger and expandable, whereas the latter is smaller and appears to be of limited capacity.     

Combined effects of human growth hormone (GH)-releasing factor-44 (GRF) and somatostatin (SRIF) on post-SRIF rebound release of GH and prolactin: a model for GRF-SRIF modulation of secretion

Somatostatin (SRIF) and GRFs play key roles in regulating GH secretion. We previously presented a model of SRIF-cAMP interaction; SRIF blocks rat (r) GH release without preventing its accumulation in a potentially releasable pool. This phenomenon may represent a mechanism whereby tonic SRIF inhibition and its subsequent reduction or withdrawal can modulate the magnitude if not the initiation of rGH pulses. Herein we test that model using human GRF-44 (hGRF-44). Tritium-prelabeled rat anterior pituitary fragments were perifused until stored [3H]rGH and [3H]rPRL release rates were stable. SRIF (10 or 25 nM), with and without hGRF-44 (3 or 10 nM), was added in short (1-h hGRF-44) and long (3-h hGRF-44) protocols; SRIF was then withdrawn while hGRF-44 was continued. Release of stored prelabeled [3H]rGH and [3H]rPRL was assessed by immunoprecipitation. Effects on PRL release were followed for comparison. SRIF-induced inhibition of release was only partially reversed by hGRF-44. At these concentrations and so long as SRIF was present, hGRF-44 could not stimulate the rate of hormone release to values above pre-SRIF basal rates. On the other hand, the amplitude of post-SRIF rebound release was increased by prolonging exposure to SRIF alone, by including hGRF-44 with SRIF, by increasing the amount of hGRF-44 included with SRIF, by prolonging exposure to hGRF-44 plus SRIF, and by using a smaller concentration of SRIF during exposure to hGRF-44. Interaction of hGRF-44-SRIF effects generated peak rates of hormone release after SRIF withdrawal which exceeded the maximum rates achieved using hGRF-44 alone in this system. Lactotroph responses were much smaller, but qualitatively resembled somatotroph responses. We conclude that the interplay of simultaneous hGRF-44 and SRIF effects can regulate the amplitude of rGH pulses. Although GRF can initiate physiological GH release, and GRF antisera can block GH pulses, we suggest that the surge of release that follows reduction of SRIF-induced inhibitory tone in vitro represents a potential mechanism that could contribute to the initiation of some pulses of release. Finally, we also present a theoretical model of secretagogue interactions at the cellular level to explain our results. The model is compatible with either a homogeneous cell population in which each secretory cell has multiple capabilities or a heterogeneous cell population composed of cell subgroups with complementary secretory abilities.     

Monensin influences basal and human growth hormone-releasing hormone 44-induced release of stored and new rat growth hormone and prolactin

When previous data suggested a growth hormone-releasing factor (GRF)-sensitive branch in intracellular hormone processing, the monensin-sensitive Golgi apparatus seemed a likely candidate. We examined monensin's effect on basal and GRF-stimulated release of newly synthesized and stored rat growth hormone (rGH) and rat prolactin (rPRL). 14C-Pre-labeled, perifused rat pituitary fragments were exposed to [3H]leucine in 0-10 microM monensin; a pulse of 3 nM GRF assessed subsequent secretory responsivity. Monensin dose-dependently reduced basal release of stored [14C]rGH and [14C]rPRL. GRF-stimulated release of stored [14C]hormone was doubled after 0.03 microM and 0.1 microM monensin; higher concentrations diminished stored hormone release. Low concentrations of monensin accelerated basal (0.03 microM and 0.1 microM) and GRF-stimulated (0.03 microM) [3H]rGH and [3H]rPRL release without altering recovery; higher monensin concentrations (greater than or equal to 1 microM) reduced basal, and abolished GRF-stimulated, new hormone release and reduced total [3H]rGH and [3H]rPRL recovery. These data are consistent with a GRF-sensitive and monensin-influenced branch in intracellular hormone processing that regulates the fraction of new hormone exiting the cell without prior immersion in storage compartments.     

Differential spontaneous and stimulated in vitro release of newly synthesized or stored rGH and rPRL by pituitaries from rats with hypothalamic lesions

These experiments were designed to examine which aspects of basal and responsive somatotroph and lactotroph synthesis and release behavior in vitro are functions of in vivo tonic hypothalamic environment. Although we cannot specifically define in vivo hypothalamic tone, we show that spontaneous rates of hormone synthesis and release in vitro, as well as release in response to a secretagogue, are influenced by altered in vivo hypothalamic tone. This work combines in vivo destruction of hypothalamic (ventromedial [VMN] or dorsomedial [DMN]) nuclei with in vitro double-label perifusion to track hormone synthesis and release of newly synthesized and stored hormone. We demonstrate that hormone synthesis rates are greater in DMN-lesioned (DMNL) or sham-operated (SHAM) animals than in VMN-lesioned (VMNL) animals and that DMNL and SHAM synthesis rates fall with time outside the hypothalamic environment. We show that basal release of newly synthesized rGH by DMNL tissue exceeds that of SHAM, while release from VMNL tissue is less than that of SHAM. Accidental placement of small bilateral lesions between and not impinging on either the DMN or VMN nuclei did not alter newly synthesized rGH release but accelerated newly synthesized rPRL release. Although basal fractional release of stored growth hormone and prolactin was the same for the three groups, potassium ion-induced release of stored hormone was similar in DMNL or SHAM tissue, but depressed in VMNL tissue. Thus, the creation of definable hypothalamic damage in a living animal produced specific modifications in in vitro pituitary synthetic/secretory behavior, presumably by reproducibly altering hypothalamic secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive intestinal polypeptide and thyrotropin-releasing hormone stimulate newly synthesized, not stored, prolactin

Experiments were designed to determine whether vasoactive intestinal polypeptide (VIP), reported to stimulate basal PRL secretion, affects PRL processing by lactotrophs. Initially, rat anterior pituitary quarters were incubated for 2 h with [3H]leucine, with and without 10(-5) M VIP, and immunoreactive and immunoprecipitable rPRL were measured during 56 mM KCl perifusion to determine total and 3H-labeled PRL, respectively. Inclusion of VIP increased immunoreactive PRL (P less than 0.05), decreased immunoprecipitable PRL (P less than 0.01), and, therefore, decreased the specific activity of labeled PRL (P less than 0.001). These results suggested an enhanced release of newly synthesized PRL before KCl depolarization, thus decreasing the release of labeled PRL. To discriminate between the two PRL pools, newly synthesized and storage, pituitary quarters were incubated with and without 10(-5) M VIP for 4 h with [14C]leucine, 2 h in cold medium and 2 h with [3H]leucine. Immunoprecipitable PRL was measured during perifusion with 56 mM KCl. Data were depicted as the 3H/14C disintegrations per min ratio of PRL released/3H/14C disintegrations per min of total tissue to account for any differences in tissue labeling. This ratio was greater for tissue labeled in the presence of VIP (P less than 0.002). To determine whether VIP, as a secretagogue, differentiates between the newly synthesized and storage pools, VIP was added after pulse chase, as previously described. No preferential release was observed between the two groups. Finally, using the same [3H]- and [14C]leucine-labeling protocol with and without 10(-5) M VIP, tissue was perifused with medium 199 for 1 h, with 10(-5) M TRH for 30 min, with medium 199 for 30 min, and with 56 mM KCl for 1 h. Inclusion of VIP increased the 3H/14C released/3H/14C total tissue ratio during basal perifusion (P less than 0.04) and TRH exposure (P less than 0.05). Within the control group, the TRH ratio was greater than basal (P less than 0.003). These experiments suggest that newly synthesized PRL is preferentially secreted over stored PRL from tissue incubated with VIP during pulse-chase labeling; however, addition of VIP as a secretagogue did not affect either PRL pool preferentially.     

Fractional reduction of somatostatin concentration interacted with rat growth hormone releasing hormone to titrate the magnitude of pulsatile growth hormone and prolactin release in perifusion

Growth hormone (GH) pulses in vivo are associated with increased hypothalamic portal growth hormone releasing hormone (GH-RH) concentration and can be prevented by GH-RH antisera. GH pulses are also associated with prior reduction of portal somatostatin (SRIF) concentrations, although SRIF antisera do not abolish GH pulses. In vitro, pulses of GH-RH as well as SRIF withdrawal are followed by pulses of GH release; the presence of GH-RH enhances post-SRIF GH release. We asked four questions: (1) During combined GHRH-SRIF exposure in vitro, must SRIF withdrawal be complete to produce a pulse of GH release, or is there a threshold diminution of SRIF which permits it? (2) When pulsatile GH release does occur, is it an all-or-none phenomenon, or is it titratable by fractional reduction of SRIF? (3) Does varying the GH-RH concentration while administering SRIF systematically alter GH release in response to fractional SRIF reduction? (4) Given a small but distinct effect of GH-RH on release of stored prolactin (PRL) in this system, does fractional SRIF reduction alter PRL release in parallel? Rat pituitary tissue whose hormone stores had been prelabeled with tritium was perifused for 120 min in combined 25 nM SRIF and 3 or 10 nM rat GH-RH (rGH-RH). Then, while maintaining rGH-RH concentrations, the SRIF concentration was left unchanged (control) or was reduced to 20, 15, 10, 5, or 0 nM for 60 min. Release of stored rGH and rPRL was assessed by immunoprecipitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of ethanol with signal transduction mechanisms mediating growth hormone release by rat pituitary cells in vitro.

The effects of ethanol on signal transduction mechanisms of rat GH (rGH) release were investigated in primary culture of rat anterior pituitary cells. Ethanol (30, 100, and 300 mM) had no significant effect on basal rGH release or cell content after a 4-h incubation or on intracellular cAMP levels at 30 min. Ethanol did not alter rGRH (10(-11) M)-stimulated rGH release, but at concentrations of 100 and 300 mM it inhibited rGRH (10(-9) M)-stimulated rGH release by 12% (P less than 0.05) and 54% (P less than 0.01). In contrast, a dose-dependent stimulatory effect was observed on rGRH-induced cAMP accumulation. Ethanol enhanced the inhibitory effect of SRIH (10(-11) and 10(-9) M) on rGH release by up to 24% (P less than 0.01). Stimulation of rGH release by cAMP derivatives and forskolin was partially inhibited by ethanol, as was cAMP accumulation after forskolin treatment. Cholera toxin-stimulated rGH release was also inhibited by ethanol, whereas cAMP accumulation was increased. At the higher concentrations, ethanol enhanced rGH release after protein kinase-C activation by phorbol ester and after stimulation of calcium influx with Ca ionophore. No significant ethanol effect was noted on prostaglandin E2-stimulated rGH release, and ethanol did not alter rGH mRNA levels or proliferation of a pituitary somatomammotroph cell line. The results indicate that ethanol exerts multiple effects on systems mediating GH release from the pituitary in vitro. However, the inhibitory influence of ethanol on GH secretion is related primarily to the adenylate cyclase-cAMP pathway, which represents the major signal transducing system in the somatotroph.     

Structural correlates of stimulated and inhibited secretion: electron microscopic observations of somatotrophs in perifused rat pituitary

The in vitro release of stored intracellular rat growth hormone (rGH) in response to several secretagogues suggests the functional division of rGH storage into at least two 'compartments' or 'pools'. The first is an immediately releasable compartment whose response is brief. The second is a compartment which responds to more prolonged secretory demands. These observations are consistent with either a single, homogeneous population of somatotrophs, each of which exhibits functional compartmentalization of storage, or with heterogeneous populations of somatotrophs, each family of which provides one of the observed responses. We sought anatomical correlates of this functional compartmentalization using a perifusion/morphometric system which permitted examination of the first model while not excluding the second model. We selected for study an established perifusion protocol whose behavior was consistent and whose previous results suggested phases of both filling and emptying of the immediate release pool. Five parallel perifusions of pituitary fragments were run. The fifth perifusion was used to monitor rGH release and to confirm that the experiment had behaved in standard fashion. The first pituitary chamber was dismantled during basal perifusion to obtain tissue for microscopy, the next during exposure to 25 nM SRIF, the third during exposure to both 25 nM SRIF and 1 mM (Bu)2cAMP, and the fourth shortly after the rapid release which followed SRIF withdrawal. Somatotroph granulation was decreased by 54% in the presence of SRIF, and then increased by 45% with the addition of (Bu)2cAMP. The intracellular distribution of granules also fluctuated in relation to the stimulatory and inhibitory secretagogues. In addition, secondary lysosomes increased by 549% during SRIF-induced inhibition of rGH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of rat growth hormone binding by membrane disulfide reduction

The effect of disulfide reduction on the binding of [125I]rat GH (rGH) to rat liver plasma membranes and hepatocytes was studied to determine the role of disulfide bonds in the binding of GH to its receptor. The total amount of [125I] rGH bound to the liver receptors increased severalfold in the presence of dithiothreitol and mercaptoethanol. The nonspecific binding also increased at higher concentrations of the reductant, but the amount specifically bound was still greater in the presence of disulfide reductant. In contrast, the disulfide reductant inhibited [125I] human GH (hGH) binding and enhanced its displacement from hypophysectomized female rat hepatocytes. This was similar to the effect of reductants on [125I]hGH binding to normal female rat hepatocytes. The effect of the disulfide reductants on [125I]rGH binding could be prevented or reversed by the simultaneous or subsequent addition of an oxidizing agent such as NAD or oxidized glutathione. Sulfhydryl-reactive agents such as iodoacetamide prevented additional binding of [125I]rGH when added at 30 min of the incubation. The additional [125I] rGH bound in the presence of disulfide reductant was displaceable by excess unlabeled rGH. Both rGH and hGH exhibited similar degrees of disulfide reduction in the presence of mercaptoethanol. The disulfide reductant produced effects on binding at concentrations that resulted in less than 10% reduction of the GH disulfides. We conclude that: 1) the disulfides and sulfhydryls of the hepatocyte membrane are intimately involved in the binding of GH to hepatic receptors; 2) the locus of the disulfides and sulfhydryls may be in the subunit structure of the membrane receptor, but this will require verification using soluble receptors; and 3) the effect of disulfide reducing agents reveals basic differences in the mechanism of binding of rGH and hGH to somatotropic hormone receptors on the hepatocytes.     

Sex difference in the induction of lactogenic receptors in rat livers.

The relationship between serum levels of growth hormone (rGH), prolactin (rPRL), luteinizing hormone (rLH), follicle-stimulating hormone (rFSH), corticosterone, oestrogen, (oestradiol-17 beta) and testosterone and the hepatic binding sites specific to [125I]human-prolactin (h-PRL) were investigated in normal rats, in rats bearing the GH- and PRL-secreting tumour (GH3), and in rats 14 days after tumour removal. The presence of GH3 tumour elevated serum levels of rGH and rPRL and concomitantly increased the hepatic binding of [125I]h-PRL; the male rats had a greater increase than the female rats. The increased binding was due to an increase in the specific membrane binding sites, whereas the affinity constant (Ka) was not changed. In both male and female rats, there was a significant positive correlation between ser-m rGH levels (P less than 0.001) or serum rPRL (P less than 0.02) and specific binding of [125I]h-PRL in female rats only (P less than 0.02). In male rats, there was a significant negative correlation between serum testosterone levels and specific bindings of [125I]h-PRL (P less than 0.05). These results suggest that rGH and rPRL regulates the hepatic h-PRL receptors in female rats, and testosterone predominantly inhibits the induction of the hepatic lactogenic receptors in male rats.     

The loss of 45Ca2+ associated with prolactin release from the tilapia (Oreochromis mossambicus) rostral pars distalis

The relationship between tritium 3H-labeled prolactin (PRL) release and the loss of tissue-associated 45Ca2+ was examined in the tilapia rostral pars distalis (RPD) using perifusion incubation under conditions which inhibit or stimulate PRL release. Depolarizing [K+] (56 mM) and hyposmotic medium (280 mOsmolal) increased both the release of [3H]PRL and the loss of 45Ca2+. The responses to high [K+] were faster and shorter in duration than those produced by reduced osmotic pressure. The depletion of Ca2+ from the incubation medium with 2 mM EGTA suppressed the [3H]PRL response evoked by high [K+] or reduced osmotic pressure. Exposing the tissues to Ca(2+)-depleted medium in the absence of high [K+] or reduced osmotic pressure produced a sharp, but brief, increase in 45Ca2+ loss. Cobalt (10(-3) M), a competitive inhibitor of calcium-mediated processes, inhibited the [3H]PRL response to hyposmotic medium and to high [K+]. Cobalt also diminished the increased loss of 45Ca2+ evoked by exposure to reduced osmotic pressure, but was ineffective in altering responses to high [K+]. Methoxyverapamil (D600; 10(-5) M), a blocker of certain voltage-sensitive Ca2+ channels, did not alter either the [3H]PRL or the 45Ca2+ responses to high [K+] and reduced osmotic pressure. Taken together with our earlier studies, the present findings suggest that exposure to high [K+] or hyposmotic medium produces rapid changes in the Ca2+ metabolism of the tilapia RPD that are linked to the stimulation of PRL secretion. Nevertheless, the increased 45Ca2+ loss, but not [3H]PRL release, upon exposure to Ca(2+)-depleted media suggests that Ca2+ loss may not always reflect intracellular events that lead to PRL release.     

SRIF-sensitive compartmentalization of stored rGH is abolished by hypothyroidism

Rat adenohypophyses lose immuno- and bioassayable growth hormone in hypothyroidism. We examined whether the somatotroph also loses mechanisms for intracellular hormone compartmentalization during hypothyroidism. A series of identical perifusions was performed using pituitary tissue from thyroidectomized rats before and after thyroxine replacement. Somatostatin (SRIF), (Bu)2cAMP and potassium ion were employed to produce a wide range of hormone release responses. Growth hormone synthesis diminished with hypothyroidism and increased with thyroid hormone replacement. Growth hormone release was therefore expressed as a percent of pituitary content to circumvent effects of variable content. Post-somatostatin rebound release was lost in hypothyroidism: it fell progressively after thyroidectomy (day 7 = 45% of control; day 14 = 11%; day 71 = 3%) and was restored by thyroxine replacement (day 2 = 24%; day 5 = 50%; day 9 = 102%). In conclusion, hypothyroid somatotrophs lose the ability to sequester stored hormone in a SRIF-sensitive compartment. Thyroxine replacement restores that capability. Thus, SRIF-sensitive rGH compartmentalization is thyroid hormone dependent.     

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia.

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03-3 mM) or forskolin (0.1-10 microM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [D-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01-1.0 microM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01-1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release.(ABSTRACT TRUNCATED AT 250 WORDS)