Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells.

A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2- to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells.     

Covalent cross-linking of growth hormone-releasing factor to pituitary receptors

We have used a bifunctional cross-linker, disuccinimidyl suberate, to covalently attach [125I]human pancreatic GH-releasing factor (GHRF) (-1-40)OH to bovine pituitary membranes and rat anterior pituitary cells. Covalently radiolabeled membrane and cell preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. In the former case, we observed the specific labeling of a polypeptide with an apparent mol wt of 75,000 +/- 3,000. The labeling of this species was specific for GHRF, as evidenced by the fact that it was inhibited in a dose-dependent fashion with increasing concentration of unlabeled GHRF. Furthermore, the radiolabeling was inhibited in the presence of excess unlabeled GHRF analogs but not unrelated peptides such as insulin and rat GH. The size of the radiolabeled band was the same in both bovine pituitary membranes and rat anterior pituitary cells. The extent of radiolabeling was dependent on the amount of membrane or the number of cells present during the binding reaction. These observations indicate that the mol wt 75,000 species is a ligand-binding subunit of the GHRF receptor in the pituitary. Under nonreducing conditions, a species much larger than mol wt 200,000 was specifically radiolabeled, again in both bovine pituitary membranes and rat cells. This result suggests the possibility that the ligand-binding subunit might be disulfide-linked to other subunit(s) forming homo- and heterooligomers.     

Diminished pituitary responsiveness to growth hormone-releasing factor in aging male rats

The pattern of GH secretion undergoes substantial changes in the aging rat, resulting in decreased daily secretion of GH. In this study, the pituitary responsiveness to GH-releasing factor (GRF) was examined in young (2- to 5-month old) and aging (14- to 18-month old) male rats. In vivo studies were performed under sodium pentobarbital anesthesia. After injection of 250 ng GRF/100 g BW, young rats experienced more GH secretion [peak level, 544.5 +/- 209.5 (+/- SEM) ng/ml] than did 18-month-old rats (89.3 +/- 13.7 ng/ml). To investigate the locus of this insensitivity to GRF, anterior pituitary cells from young and aging rats were dispersed and placed in primary culture. While basal GH secretion from the cultured pituitary cells was similar in the two groups (49.7 +/- 3.5 vs. 47.8 +/- 2.7 ng/ml X 4 h for the 2- and 18-month old rats, respectively), the GH-releasing ability of GRF was partially but significantly impaired in cells derived from both 14- and 18-month old rats; 100 nM GRF stimulated the release of 96.7 +/- 1.8 ng/ml X 4 h in the 18-month old rats as opposed to 115.0 +/- 6.0 (P less than 0.05) ng/ml X 4 h in the 2-month-old rats. Since GRF stimulates GH release through the activation of adenylate cyclase, intracellular cAMP levels were measured in the cultured pituitary cells. GRF stimulated 65% less intracellular cAMP accumulation in the 18-month-old rats. In 14-month-old rats, the ability of forskolin and (Bu)2 cAMP to release GH was impaired, while phorbol ester-elicited GH secretion was unchanged. In conclusion, the GH response to GRF is blunted in aging rats. While much of the insensitivity to GRF may be mediated by the increased somatostatin tone reported in aging rats, a diminished pituitary cAMP response to GRF may also be an important etiological factor in the hyposomatotropinemia of the aging male rat.     

Alterations of pituitary growth hormone-releasing factor binding sites in aging rats.

This study was designed to determine whether growth hormone (GH)-releasing factor (GRF) binding sites are altered in parallel to the diminution of GH secretion that occurs in aging. Using [125I-Tyr10] hGRF (1-44)NH2 as radioligand, we performed cold saturation studies in 2, 8, 14 and 18-month-old Sprague-Dawley male rat pituitary homogenates. In young rats (2 months), analysis by Ligand revealed the presence of two distinct classes of binding sites (KDs = 0.86 +/- 0.15 and 400 +/- 210 nM; BMAXS = 269 +/- 47 fmol and 42 +/- 19 pmol/mg protein, respectively). In 8 and 14-month old rats, there was a concomitant decrease in capacity of the high affinity class of sites (P less than 0.01) and increase in capacity of the low affinity class of sites (P less than 0.05). In parallel, a transient increase in affinity of the high affinity class of sites was observed in 14-month-old rats (P less than 0.05). In old rats (18 months), the data were no longer statistically analyzed with a two site-model, indicating a severe blunting of the high affinity sites. As the GRF-induced GH secretion is still not diminished at 8 month of age in these animals, our results indicate: 1) that alterations of GRF binding sites precede the GH impairment, thus probably participate in the initiating of this phenomenon and 2) that the biological actions of GRF on GH secretion are likely mediated by the high affinity class of sites.     

Nucleotide regulation of growth hormone-releasing factor binding to rat pituitary receptors

The specific binding of a GRF radioligand, [His1,125I-Tyr10,Nle27]hGRF-1-32NH2, to rat pituitary homogenates is reduced by the addition of GTP and its nonhydrolyzable analogs 5'-guanylylimidodiphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). GDP and cAMP had no effect while the nonhydrolyzable ATP analogs 5'-adenylylimidodiphosphate and adenosine 5'-O-(3-thiotriphosphate) did elicit a significant reduction in GRF binding. The effect of GppNHp was half-maximal at 0.2 microM, and the maximum inhibition achieved was 85%. The effect of 0.1 microM GppNHp on GRF competitive displacement experiments indicated a significant reduction in affinity for the ligand (Kd = 0.51 +/- 0.11 nM in the absence of GppNHp and 2.1 +/- 1.1 nM in its presence) without an effect on receptor number. The GRF radioligand dissociates slowly from its receptor (t1/2 = 250 +/- 50 min), but the addition of 0.1 microM GppNHp converts approximately half of the receptors present to a more rapidly dissociating form (t1/2 = 9 +/- 10 min). These results are consistent with existing models for receptor-G-protein interactions, and thus, we conclude that transduction of the GRF response across the cell membrane involves a guanine nucleotide-binding protein, presumably Gs.     

Circulating growth hormone forms after stimulation of pituitary secretion with growth hormone-releasing factor in man

Human GH (hGH) in the circulation of acromegalic patients and pharmacologically stimulated normal subjects consists of several monomeric and oligomeric molecular forms. However, little is known about the nature of plasma hGH under physiological conditions. We examined the molecular composition of plasma hGH secreted in response to synthetic human pancreatic tumor GRF-(1-40) (hpGRF-40), a peptide closely resembling or identical to hypothalamic GRF. The peptide (10 micrograms/kg) was injected iv into six normal men, and blood was obtained 30 min later. Plasma hGH was characterized by gel filtration and by polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing after extraction from plasma by immunoadsorbent chromatography. At least 53% of hGH eluted as little (monomeric) hGH, 27% as big (dimeric) hGH, and 20% or less as big-big (oligomeric and spurious) hGH during gel filtration. Among the monomeric forms, the 22,000-dalton form was predominant (83%), with smaller quantities of the 20,000-dalton variant (11%), and one or more unidentified acidic forms (N alpha-acetylated, deamidated, or cleaved hGH) (6%) also present. The molecular composition of plasma hGH secreted in response to hpGRF-40 is similar to that released after pharmacological stimuli or that circulating in acromegaly.     

Insulin-like growth factor binding proteins in the bovine anterior pituitary

Insulin-like growth factor binding proteins (IGFBPs) were characterized in bovine anterior pituitary tissue, pituitary conditioned media, and serum collected during the preovulatory and early luteal phases of the estrous cycle. Effects of in vitro treatments of pituitaries with luteinizing hormone-releasing hormone (LHRH), estradiol, and progesterone on IGFBP secretion were also evaluated. Predominant IGFBPs detected in anterior pituitary tissue by immunoprecipitation, ligand blotting, and Northern blotting were IGFBP-5 (29 kDa), IGFBP-2 (32 kDa), and IGFBP-3 (36 and 39 kDa doublet). Conditioned culture media contained IGFBP-5, a slightly larger form of IGFBP-3 (33 kDa), the 36- and 39-kDa forms of IGFBP-3, and a more extensively glycosylated form of IGFBP-3 (44 kDa). In serum, IGFBP-5 was not readily detected, and IGFBP-3 (40- and 44-kDa doublet) and IGFBP-2 (34 kDa) were larger than in pituitary tissue. Levels of IGFBP-2,-3, and-5 in pituitary tissue decreased during the preovulatory period and were lowest in the early luteal phase. Treatment with LHRH increased IGFBP-2 levels in media twofold. Estradiol or progesterone did not alter IGFBP secretion in vitro. Predominant IGFBPs produced and released by anterior pituitary tissue were IGFBP-2,-3 and-5. The activity of IGFBPs fluctuates in the pituitary in association with changes in stage of estrous cycle, implicating IGFBPs as potential regulators of gonadotrope function. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Growth hormone-releasing factor does not stimulate phosphoinositides breakdown in primary cultures of rat and human pituitary cells

It has been reported that rat growth hormone releasing factor (rat GRF-43), similarly to the two human GRFs (GRF-40 and 44) stimulates adenylate cyclase activity in pituitary cells. Controversial findings have been presented by two different groups on the action of GRF on phosphoinositides (PI) metabolism, a phenomenon linked to Ca-- mediated intracellular mechanisms. In the work to be reported, we evaluated the accumulation of inositol phosphates induced by GRF exposure in primary cultures of rat and human pituitary cells. Addition of rat GRF-43 to rat pituitary cells at doses up to 1 microM had no effect on inositol phosphates accumulation, while already at a dose as low as 0.05 nM it increased growth hormone secretion in the incubation medium significantly. In the same cell system, TRH, a known activator of PI breakdown, significantly increased [3H]inositol phosphates. In primary cultures of human somatotrophs from acromegalic subjects as in rats, addition of hpGRF-40 and also of TRH did not elicit any modification in the accumulation of [3H]inositol phosphates. Consistent with in vivo findings, both peptides induced a significant release of GH in the medium. Our results show that the GH releasing effect of GRF does not involve the hydrolysis of phosphatidylinositol in normal rat as well as in tumoral human somatotrophs. In addition it appears that the anomalous response of TRH on adenomatous cells from acromegalic patients is differently mediated in respect to the action of the tripeptide on normal lactotrophs and thyrotrophs.     

Decreased pituitary growth hormone response to growth hormone-releasing factor in cafeteria-fed rats: dietary and obesity effects

The in vivo and in vitro growth hormone (GH) responsiveness to growth hormone-releasing factor [rGRF(1-29)NH2] was evaluated in a dietary obese rat model. Sprague-Dawley rats were divided into two groups after weaning. The control group received a semisynthetic defined diet, and the cafeteria-fed group was maintained on a mixed energy-rich palatable diet. After 2 months of diet, the cafeteria-fed rats were divided into two groups, according to their degree of weight gain compared to controls: group I: 0%; group II: 24%. After 5 months of diet, the weight increase was, respectively, in groups I and II, 12 and 41%, as compared to controls. Under pentobarbital anesthesia, rGRF(1-29)NH2 was injected intravenously in two consecutive doses of 0.8 and 4.0 micrograms/kg body weight into the control and cafeteria-fed rats. After 2 months of diet, a significant decrease of basal GH levels and GH peak response to the 4.0 micrograms/kg rGRF dose was observed in both cafeteria-fed rat groups as compared to the control group. After 5 months of diet, basal GH levels decreased in both cafeteria-fed groups. However, a significantly blunted GH response to both doses of rGRF occurred only in group II. After 5 months of diet, perifused anterior pituitary cells of control and cafeteria-fed rats were challenged with increasing concentrations of rGRF (6.25, 25 and 100 pM). The basal GH secretion was similar in all groups but the stimulated GH release in response to 25 and 100 pM GRF, expressed as peak value, was depressed in group II, compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of growth hormone-releasing factor and somatostatin on adenylate cyclase and growth hormone release in rat anterior pituitary

The interaction of growth hormone-releasing factor (GRF) and somatostatin (SRIF) on adenylate cyclase activity and growth hormone release was investigated in pituitary homogenates and 2-day cultured rat anterior pituitary cells. GRF stimulated growth hormone release by about 3-fold (ED50 1.6 X 10(-12) M) and caused a rapid 15-fold increase in cyclic AMP production (ED50 6.0 X 10(-12) M). The increase in cyclic AMP was due to direct stimulation of adenylate cyclase by GRF, which caused a 4-fold increase in the activity of the enzyme measured in anterior pituitary homogenates. GRF-induced cyclic AMP formation and GRF-stimulated adenylate cyclase activity were maximally inhibited to the extent of about 50% by 10(-8) M somatostatin. In contrast, GRF-stimulated growth hormone release was completely inhibited by somatostatin (ID50 3.2 X 10(-11) M), suggesting a second site of action of somatostatin. These studies demonstrate that GRF stimulates growth hormone release via activation of adenylate cyclase and a rise in intracellular cyclic AMP. In addition, these findings indicate that the inhibitory action of somatostatin on growth hormone release is exerted at two levels, one at the level of adenylate cyclase affecting the production of cyclic AMP, and the other beyond the formation of the nucleotide, at a site which modulates the release of growth hormone from the cell.     

Influence of sex steroid hormones on rat growth hormone-releasing factor and somatostatin in dispersed pituitary cells

The modulatory effects of glucocorticoid and sex steroid hormones on the effects of rat GH-releasing factor (GRF) and somatostatin (SRIF) on GH release and biosynthesis were studied in monolayer cultures of rat anterior pituitary cells with RIA and quantitative immunoprecipitation methods. Dexamethasone (10(-7) M), a potent synthetic glucocorticoid, increased both the sensitivity and maximum response of GH release stimulated by GRF. Progesterone (10(-7) M) also enhanced GH release stimulated by GRF. The stimulatory effects of dexamethasone and progesterone were dose dependent and required a latent period of at least 24 h to be evident. Testosterone, dihydrotestosterone, and 17 beta-estradiol showed no apparent influence on GRF-induced GH release under the same conditions. None of the hormones studied showed significant influences on basal or SRIF-suppressed GH release. Progesterone added to the maximally effective concentrations of dexamethasone had no additional effects on GRF-induced GH release. The effect of progesterone was attenuated by both 5 alpha-dihydronorethindrone, a progesterone antagonist and 17 alpha-methyltestosterone, a glucocorticoid antagonist. In terms of GH synthesis, stimulatory effects of GRF on GH synthesis were apparent only when pituitary cells were pretreated with dexamethasone. These results indicate that: pretreatment with glucocorticoid or progesterone enhances the effects of GRF on GH release and/or synthesis; these two steroids share at least one common step to enhance GRF effects; and steroid hormones have little influence on basal or SRIF-suppressed GH release.     

Steroids can modulate transdifferentiation of prolactin and growth hormone cells in bovine pituitary cultures.

Fluctuations in the proportions of pituitary acidophils (cells that release GH and PRL, either separately or concurrently) have been correlated with dynamic changes in the steroid hormone milieu. Since modulation of these acidophilic subtypes can occur without appreciable alterations in the total number of acidophils, it has been proposed that GH- and PRL-secreting cells can actually transdifferentiate (gain or lose the ability to release GH or PRL). To test this hypothesis, we examined the effects of steroids on the proportions of cells that released GH, PRL, or both hormones (i.e. mammosomatotropes) in bovine pituitary cell cultures. Specifically, anterior pituitary cells from castrated males were cultured for 6 days in the absence (controls) or presence of 17 beta-estradiol, cortisol, or progesterone. Reverse hemolytic plaque assays revealed that 6.64 +/- 1.2% of all pituitary cells released GH, while 68.8 +/- 5.3% were PRL secretors in control cultures. Cortisol and progesterone induced an increment in the proportions of GH-secreting cells (10 nM cortisol, +11.7 +/- 2.4%; 1000 nM cortisol, +10.5 +/- 4.7%; 1000 nM progesterone, +2.87 +/- 1.5%) above control values while decreasing the relative abundance of cells that released PRL (10 nM cortisol, -7.6 +/- 1.7%; 1000 nM cortisol, -6.6 +/- 1.2%; 1000 nM progesterone, -5.5 +/- 1.3%) below control values. However, 17 beta-estradiol was ineffective in this regard at doses of 0.1-1000 nM. A more critical examination of the steroid-induced changes revealed that they were attributable to increases in the proportions of cells that released GH alone and both hormones simultaneously along with a concomitant decrease in the fraction that secreted only PRL. Two lines of evidence discount the possibility that these effects were due to selective cell proliferation. First, the mitotic rate of cultures (assessed by immunofluorescent detection of bromodeoxyuridine incorporation) was only about 1% of all cells in both control and steroid-treated cultures. Second, blocking cell proliferation by the addition of cytosine arabinoside (100 microM) did not inhibit the cortisol-induced augmentation of GH-releasing cells. Taken together, these results substantiate the hypothesis that acidophilic subpopulations are capable of transdifferentiation given an appropriate hormonal signal.     

Food intake alters the serum growth hormone response to bovine growth hormone-releasing factor in meal-fed Holstein steers

The hypothesis that endocrine or nutritional factors related to feeding may affect pituitary responsiveness to an acute challenge with bovine GH-releasing factor (1-44)-NH2 (GRF) was examined in steers. In these experiments, either steers were trained to consume their total daily food allotment in a 2-h period (meal-fed) or food was withheld at the normal time of feeding (sham-fed). In the first of three experiments, the serum GH pattern was determined around the time of feeding in meal-fed and sham-fed steers. The temporal GH rhythm in both groups appeared to be synchronized to the time of feeding, with limited pulsatile GH activity occurring 2-3 h after feeding. Baseline secretion of GH and total area under the GH response curve were lower (P less than 0.01) in meal-fed compared with sham-fed steers. In the second experiment, 50 micrograms GRF was injected i.v. in meal-fed steers at -4, -2, 0, +2, +4, +6 and +8 h relative to the time of feeding. The number of steers responding to GRF (53%), the amplitude of the GH peak (15.8 micrograms/l) and the area under the GH response curve (0.6 arbitrary units) were lower (P less than 0.001) after than before feeding (90 +/- 6 (S.E.M.)%, 61.3 +/- 3.2 micrograms/l and 2.0 +/- 0.3 units respectively). Of those steers responding to GRF, the GH response was significantly reduced following feeding compared with before feeding. In the third experiment, 50 micrograms GRF was injected i.v. in sham-fed steers at -4, -2, 0, +4 and +6 h relative to the time of sham-feeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The absence of an age-related change in the pituitary response to growth hormone-releasing factor in rats

The capability of the anterior pituitary gland to secrete growth hormone (GH) in response to an intravenous injection of growth hormone-releasing factor, hpGRF-44, was evaluated in male rats ranging from 22 days to 24 months of age. The increase in GH concentrations following a submaximal dose of hpGRF-44 (0.5 microgram/kg) was similar among the different aged rats suggesting there is no age-related change in pituitary sensitivity to hpGRF-44. Likewise, the response to a maximal dose of this peptide (25 micrograms/kg) was not different in young and old animals indicating there is no decrease in the readily releasable pool of GH. Thus, the age-related changes in GH secretion observed in rats do not appear to be due to a change in the pituitary response to the hypothalamic growth hormone-releasing factor.     

Platelet activating factor induces inositol phosphate accumulation in cultures of rat and bovine anterior pituitary cells

The phospholipid platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) stimulated the accumulation of inositol phosphates in cultures of rat and bovine anterior pituitary cells. In response to PAF, inositol 1,4-bisphosphate showed the largest percent increase of the inositol phosphates in the presence of lithium chloride. PAF induced an increase of inositol 1,4,5-trisphosphate, the biologically active isomer responsible for mobilization of intracellular calcium. A characterization of the PAF response indicated that PAF, but not its biologically inactive enantiomer, induced the accumulation of inositol phosphates in the rat anterior pituitary. Further, the PAF receptor antagonist L652731 reduced PAF stimulation. The ED50 for PAF-induced inositol 1,4-bisphosphate accumulation was 0.4 nM. PAF induced a rapid response that did not persist beyond 20 min. While PAF treatment of anterior pituitary cells did not alter TRH-induced inositol phosphate accumulation, it did prevent a second exposure of PAF from inducing inositol phosphate accumulation. These data suggest that PAF induces a rapid stimulation of phospholipase C causing the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the generation of the second messengers, inositol 1,4,5-trisphosphate and diglyceride, in anterior pituitary tissue. This action is transient probably due to PAF receptor desensitization. The action of PAF on generation of inositol phosphates may account, in part, for PAF-induced secretion of PRL and GH.     

Effect of dietary restriction and repeated growth hormone-releasing factor injections on growth hormone response to growth hormone-releasing factor in obese subjects

The response of serum growth hormone (GH) to a test bolus of growth hormone-releasing factor (GRF) (1 micrograms/kg, IV) was examined in eight obese subjects (166% +/- 8% ideal body weight [IBW] during a weight-maintaining control period and after 2 weeks of hypocaloric feeding (750 kcal). During the period of hypocaloric feeding subjects received either repetitive GRF 1 micrograms/kg, IV (GRF group, n = 6) at 9 AM, 1 PM, and 5 PM three times a week, or saline injections (placebo group, n = 6) at the same intervals. Four subjects were studied twice, several months apart, each receiving GRF and placebo treatments. Nitrogen balance was determined daily throughout the study. We also examined GH response to the GRF test in seven normal-weight subjects (106% +/- 6% IBW]. During the control period, obese subjects demonstrated a blunted GH response to GRF expressed as peak height or GH area under the curve (0 to 120 minutes) compared with normal subjects (P less than .005). At the end of the hypocaloric feeding, mean GH peak height after the GRF test was unchanged in the placebo group compared with the control period. GH release was significantly increased only during the second hour of testing (P less than .025). However, the GRF group demonstrated an increase in both mean GH peak height (P less than .025) and GH release during the first and second hours of testing (P less than .025) compared with the control period. Weight loss and blood glucose, triglycerides, FFA, amino acids, insulin, and T3 changes were similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased stimulation of adenylate cyclase by growth hormone-releasing factor in the anterior pituitary of old rats

Adenylate cyclase activity was studied in anterior pituitary homogenates from young adult (6 months) and old (20 and 24 months) male rats. Basal, NaF-, GTP-, 5'-guanylimidodiphosphate (Gpp(NH)p)- and forskolin-stimulated activities were similar in the three groups, implying that the stimulatory guanyl nucleotide regulatory binding site (NS) and the catalytical unit were unaffected by aging. Vasoactive intestinal peptide (VIP)-stimulated adenylate cyclase activity was also identical in the three groups. The efficacy (i.e., the maximum effect) of growth hormone-releasing factor [GRF(1-29)-NH2] on adenylate activity was reduced by 45-49% in old and senescent rats with no change in peptide potency (i.e., the concentration required for half-maximal enzyme stimulation). These results suggest that aging induced a selective loss of functional GRF receptors but influenced neither the coupling between receptors, NS and the catalytic unit nor the efficacy of the catalytic unit per se.     

Calcium signalling in single growth hormone-releasing factor-responsive pituitary cells.

The release of pituitary GH appears to be critically dependent on alterations in the free intracellular Ca2+ concentration ([Ca2+]i). However, little is known about the nature of Ca2+ signalling within normal pituitary cells. We, therefore, examined [Ca2+]i patterns in individual cultured pituicytes of adult male rats under basal conditions and in response to GH regulatory agents, using the calcium-sensitive dye fura-2 together with digital imaging microscopy. Perfusion of cultured anterior pituitary cells with GH-releasing factor (GHRF) resulted in a marked increase in [Ca2+]i in specific pituitary cells. These cells did not respond to other hypothalamic secretagogues (GnRH, TRH, or CRF), and there was no evidence of desensitization on repetitive administration of GHRF. Somatotrophs (n = 134) exhibited spontaneous oscillations of [Ca2+]i in the basal state, with considerable heterogeneity of oscillatory patterns among cells. After application of a near-maximal stimulatory dose of GHRF (1 nM), there was a striking 2.2-fold increase in the amplitude of [Ca2+]i oscillations and only a modest increase in their frequency. Forskolin (1 microM) augmented somatotroph [Ca2+]i in patterns similar to those of GHRF. Somatostatin (10 nM) abolished the [Ca2+]i response to GHRF (n = 26); this reflected a marked reduction in the amplitude of [Ca2+]i oscillations and a slight reduction in their frequency. Ca(2+)-free medium or the Ca2+ channel antagonist nimodipine (0.1-1 microM) suppressed the Ca2+ stimulatory effect of GHRF. Conversely, the Ca2+ channel agonist BAY K8644 (1 microM) strikingly augmented the GHRF-induced rise in [Ca2+]i, with a major stimulatory effect on the amplitude of [Ca2+]i oscillations and no observed effect on their frequency. In summary, GHRF and other hypothalamic secretagogues increase [Ca2+]i in pituitary cells in a highly specific manner, consistent with the known specificity of their effects on hormone release. Somatotrophs exhibit spontaneous rhythmic oscillation of [Ca2+]i in the basal state. Known regulators of GH release markedly alter the [Ca2+]i oscillatory pattern in characteristic manners, exerting predominant effects on the amplitude of [Ca2+]i pulses and lesser effects on their frequency. These striking effects of GH regulatory agents on pituitary Ca2+ signalling are consistent with the concept that modulation of [Ca2+]i is a critical mediator of somatotroph function.