Calcium homeostasis in bovine somatotrophs: calcium oscillations and calcium regulation by growth hormone-releasing hormone and somatostatin.

The free intracellular calcium ion concentration ([Ca2+]i) was measured in single cells of a population containing 65-80% somatotrophs, using the fluorescent Ca(2+)-indicator Fura-2 and digital imaging microscopy. Spontaneous oscillations in [Ca2+]i ranging in frequency up to 1.5 oscillations per minute were observed in 30% of somatotrophs. These Ca2+ oscillations were blocked by the Ca2+ channel blocker CoCl2 and were thus proposed to be the result of influx of Ca2+ into the cell, possibly as the result of spontaneous electrical activity. GHRH (10-100 nM) increased [Ca2+]i in 61% of the cells studied, although the amplitude and dynamics of the response varied from cell to cell. Typically [Ca2+]i rose from 170 +/- 26 nM to 321 +/- 44 nM (n = 13) in response to a challenge with 66 nM GHRH. GHRH also increased the frequency of Ca2+ oscillations in a number of cells, and some previously quiescent cells showed Ca2+ oscillations following addition of GHRH. Forskolin, which raises cAMP levels in bovine anterior pituitary cells, also stimulated a sustained rise in [Ca2+]i in 10 out of 14 cells tested. Somatostatin (SS) (10-80 nM) rapidly reduced basal [Ca2+]i, blocked Ca2+ oscillations, and blocked the [Ca2+]i response to GHRH. The Ca2+ channel blocker CoCl2 (4 mM) had similar actions on [Ca2+]i to those of SS. These results suggest that GHRH and SS may regulate GH release by modulating Ca2+ entry into the cell through the cell membrane. The [Ca2+]i oscillations seen in a proportion of the somatotrophs were modulated in frequency by GHRH and SS, and are probably generated by influx of Ca2+ through channels in the cell membrane. Thus GH secretion may be regulated by changes in the mean level of [Ca2+]i, which in turn, may be influenced by the frequency of [Ca2+]i oscillations in bovine somatotrophs.     

Morphological effects of somatostatin on rat somatotrophs previously activated by growth hormone-releasing factor

Summary Correlative morphological and physiological analysis was carried out in order to clarify the role of somatostatin in the inhibition of the secretion of growth hormone (GH) from somatotrophs of the rat anterior pituitary gland in vivo. Transmission electron microscopy combined with immunogold labelling showed an increased number of exocytotic GH-containing secretory granules in somatotrophs fixed between 2 and 10 min after injection of GH-releasing factor (GRF). Injection of GRF also induced the appearance of immunopositive material in cisternae of the Golgi apparatus, many coated vesicles and multivesicular bodies. Microtubules were observed more frequently throughout the cytoplasm, particularly in and near the Golgi region. At 2 and 10 min after injection of somatostatin (SRIF), both the number of exocytotic figures in the somatotrophs previously stimulated by GRF and the amount of radioimmunoassayable GH in the plasma were clearly decreased. Undulation of the plasma membrane (PM) induced by GRF rapidly disappeared, and the number of granules just beneath the plasma membrane was significantly reduced. After injection of SRIF, parallel bundles of microfilaments were often observed in the space between the granules and the plasma membrane. SRIF did not cause a noticeable decrease in the amount of immunopositive material, coated vesicles and multivesicular bodies in the Golgi areas or any significant changes in the distribution of microtubules. SRIF therefore appears to inhibit hormone release mainly at the level of the plasma membrane, probably through changes in the distribution of microfilaments.     

Somatostatin prevents the desensitizing action of growth hormone-releasing factor on growth hormone release

We have investigated the effect of prior exposure to somatostatin (SRIF) alone or in combination with growth hormone-releasing factor (GRF) on the subsequent cyclic AMP and GH responses to GRF in rat anterior pituitary cells in primary culture. The maximal 4.5-fold stimulation of GH release induced by a 3-hr incubation with GRF is reduced by 60% following a prior 3-hr exposure to 30 nM GRF. A 3-hr preincubation with GRF in the presence of 30 nM SRIF doubles spontaneous GH release while the maximal amount of GH released during a subsequent 3-hr exposure to GRF is similar to that measured in cells pretreated with control medium, thus completely preventing the loss of GH responsiveness induced by prior exposure to GRF. The prevention by SRIF of the desensitizing action of GRF on GH release is not observed on the cyclic AMP response which remains almost completely inhibited in GRF-pretreated cells. Similar protective effects are obtained when SRIF is incubated with prostaglandin E2 (PGE2), thus completely preventing the desensitizing action of PGE2 on GH release. Prior treatment with pertussis toxin completely prevents the protective action of SRIF on GH responsiveness. Pretreatment with GRF + SRIF increases by 85 and 60% the maximal amount of GH release induced by cholera toxin and 8-bromoadenosine 3',5'-monophosphate, respectively. The post-SRIF rebound effect on GH release occurs mainly during the first 30 min following withdrawal of the tetradecapeptide. The present data demonstrate that simultaneous preincubation with SRIF and GRF prevents the marked inhibition of GH release during subsequent exposure to GRF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release in chickens

The effects of synthetic somatostatin (SRIF) on serum growth hormone (GH) concentrations stimulated by exogenous administration of synthetic thyrotropin-releasing hormone (TRH) and/or human pancreatic GH-releasing factor (hpGRF) were investigated in 4-week-old cockerels. In addition, the additive effects of TRH and hpGRF on serum GH were examined. TRH and hpGRF, when given in combination intravenously, produced an additive effect on serum GH concentration that peaked 10 min after the injection. The somatostatin did not significantly affect basal GH concentrations when given alone, but did significantly decrease the magnitude of the GH response to hpGRF. In contrast, SRIF did not significantly decrease the stimulatory effects of TRH on GH release. These results suggest that TRH and hpGRF are potent GH releasers in vivo and that their stimulating effects on GH release are additive, suggesting different mechanisms for their stimulation. The results obtained from the combination studies suggest that the main site of the stimulatory action of hpGRF is at the pituitary, and that SRIF significantly inhibited the rise in serum GH induced by a synthetic hpGRF, but not that induced by TRH.     

The effects of testosterone and estrogen on the pituitary growth hormone response to growth hormone-releasing factor

The effects of testosterone and estrogen on the pituitary growth hormone response to hypothalamic growth hormone-releasing factor (GRF) were evaluated in vivo using male and female rats and in vitro using a pituitary cell monolayer culture system. In vivo the increase in plasma growth hormone (GH) concentration in response to a 500 ng/kg dose of GRF was similar in gonadectomized male and female rats. Pretreatment of intact and gonadectomized male rats with testosterone caused significant enhancement of the pituitary GH response to GRF, whereas pretreatment of gonadectomized female rats with 17 beta-estradiol did not alter the response. The GH response to GRF was not different between prepubertal (i.e., 30-day-old) male and female rats. However, following puberty (i.e., by 60 days of age), the response in male rats was significantly greater than that observed in female rats. The in vitro preincubation of anterior pituitary cells with either testosterone or 17 beta-estradiol did not cause any shift in the dose-response curve between GRF and GH. These results demonstrated that androgens play an active role in modulating the pituitary response to GRF in vivo.     

Response of growth hormone release to human growth hormone-releasing factor and its analogs in the bovine

Responses of growth hormone (GH) release to synthetic human growth hormone-releasing factor (hGRF)-44-NH2 analogs were determined, and the GH-releasing potency based on dose per kg of body weight (bw) was compared with that of hGRF-44-NH2 in female dairy calves. Four- and 12-month-old calves were injected intravenously with 0.25 microgram of hGRF-44-NH2 or its analogs per kg of bw. Blood samples were collected before, and during 180 min after each injection, and plasma GH concentrations were measured by radioimmunoassay. Areas under the GH response curves for 180 min after injection of hGRF-44-NH2 and its analogs were used as an index of the GH-releasing potency of each peptide. The GH-releasing potency of hGRF(1-26)-NH2 was significantly lower than that of hGRF-44-NH2 (P less than 0.05). On the other hand, hGRF(1-29)-NH2 possessed similar potency to hGRF-44-NH2. [D-Tyr1]-hGRF-44-NH2 showed prolonged GH-releasing activity, though its potency was similar to that of hGRF-44-NH2. Also, [D-Ala2]-hGRF(1-29)-NH2 exhibited prolonged GH-releasing activity, and its potency was 2.5 (P less than 0.05) and twice (P less than 0.05) as great as that of hGRF-44-NH2 and hGRF(1-29)-NH2, respectively. These results demonstrate that the N-terminal 29 amino acid residues of hGRF possess the activity site required for full GH release in vivo, and [D-Ala2]-hGRF(1-29)-NH2 has longer and greater activity, on a dose basis, than hGRF-44-NH2 in the calves.     

Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils.

We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function.     

Effect of growth hormone-releasing hormone and clonidine on growth hormone release in type 1 diabetic patients

We administered growth-hormone releasing hormone (GHRH), clonidine or thyrotropin-releasing hormone (TRH) as intravenous boli each in three different randomized mornings to nine well-controlled Type 1 diabetic men and to six age-matched healthy men who served as controls. GHRH and clonidine evoked a prompt and brisk GH release both in diabetic and in control subjects with no significant difference being evident between the two groups. Only one diabetic subject showed a paradoxical GH release after TRH when he was under long-term poor metabolic control. These results indicate that in insulin-dependent patients with good control of the metabolic disease the response of somatotropes to pituitary- or central nervous system-directed stimuli is normal. These data are supportive of the idea that altered GH secretion in Type 1 diabetes rather than reflecting a primary hypothalamic and/or pituitary alteration may be a state-dependent phenomenon related to the metabolic state of the disease.     

Role of aging on growth hormone and prolactin release after growth hormone-releasing hormone and domperidone in man

Growth hormone (GH) and prolactin (PRL) secretion after GH-releasing hormone (GHRH) and domperidone (DOM), an antidopaminergic drug which does not cross the blood-brain barrier (BBB), was evaluated in 8 healthy elderly men (65-91 years) and in 7 young adults (23-40 years). All received in random order at 2-day intervals: GHRH(1-40) (50 micrograms i.v.) bolus, DOM (5 mg/h) infusion, GHRH(1-40) (50 micrograms i.v.) plus DOM (5 mg/h i.v.), saline solution. In elderly men GH increase after GHRH was significantly lower than in young men. DOM alone did not change GH secretion in either of these groups, whereas it increased the GH response to GHRH only in young adults. PRL levels increased in both young and elderly men during both DOM and GHRH plus DOM, but the PRL release was more marked in young than in elderly men. Both integrated secretion of GH after GHRH and of PRL after DOM were inversely correlated to chronological age. Our data show an impairment of GH rise after GHRH and of PRL after DOM in elderly adults. It is also stressed that peripheral blockade of dopamine receptors by DOM is unable to amplify the GH response to GHRH only in elderly men. A reduction in GH release after GHRH might be related to aging, perhaps through a reduction of dopaminergic tonus.     

Effects of growth hormone-releasing hormone on somatotrophs in anterior pituitary gland allografts in hypophysectomized,orchidectomized hamsters

Summary We investigated the influences of growth hormone-releasing hormone (GHRH) on the percentage, size, and shape of somatotrophs in ectopic anterior pituitary tissue. Entire pituitary glands removed from 7-week-old male hamsters were placed beneath the renal capsules of 12-week-old hamsters that had been hypophysectomized and castrated 3 weeks previously. Beginning 6 days after each host had received a single allograft, each was injected subcutaneously twice daily with 4 g GHRH in 100 l of vehicle or 100 l of vehicle for 16 days. Six hosts in each group were killed by decapitation on day 17, 16 h after the last injection. Nine normal male hamsters were also decapitated and their pituitary glands were removed. Sections of anterior pituitary tissue were stained for GH and with hematoxylin. The percentage of anterior pituitary cells that stained for growth hormone was similar in the 3 groups. In contrast, somatotrophs in grafts had a smaller mean cross-sectional area than those observed in glands in situ. This effect was reversed by GHRH. Analysis of the shape of somatotrophs in both groups of grafts disclosed that they were less circular in cross-section than those in glands in situ. The results suggest that GHRH may not play a role in maintaining the percentage of somatotrophs among anterior pituitary cells, but that it does play a role in maintaining their size.     

Effects of calcium channel antagonists on the corpora allata of adult male loreyi leafworm Mythimna loreyi: juvenile hormone acids release and intracellular calcium level

The effects of voltage-dependent calcium channel (VDCC) antagonists and the non-specific calcium channel antagonists on both juvenile hormone acids (JHA) release and cytosolic free calcium concentration ([Ca2+]i) are investigated in the corpora allata (CA) of the adult males loreyi leafworm Mythimna loreyi. The VDCC antagonists used in this study are: the L-type antagonists diltiazem, nifedipine, and verapamil, the N-type antagonist omega-Conotoxin (CgTx) GVIA, the N- and P/Q-type antagonist omega-CgTx MVIIC, and the T-type antagonist amiloride. The non-specific calcium channel antagonists used in this study were cadmium (Cd2+), cobalt (Co2+), nickle (Ni2+), and lanthanum (La3+). The results show that both the DHPs-sensitive L-type antagonist nifedipine and the N-type antagonist omega-CgTx GVIA were able to inhibit JHA release, but only omega-CgTx GVIA was able to reduce the [Ca2+]i. Among the non-specific calcium channel antagonists, Cd2+ is the most potent in reducing JHA release but without obvious effect on the [Ca2+]i, La3+ significantly increases the [Ca2+]i but without effect on JHA release.     

Effect of solubilization on adenosine 5'-triphosphate induced calcium release from purified sarcoplasmic reticulum calcium adenosinetriphosphatase

ATP-induced Ca2+ release from the purified sarcoplasmic reticulum Ca2+-ATPase has been monitored in several different ATPase environments. Arsenazo III was used as a Ca2+ indicator in stopped-flow experiments and was shown to detect the early burst in Ca2+ transport, slower steady-state transport, and release of Ca2+ from fragmented sarcoplasmic reticulum. ATP-induced rapid release of Ca2+ followed by a slower rebinding step could be demonstrated for purified Ca2+-ATPase in leaky vesicles if the reaction was slowed by lowering the pH to 6.1 and by including dimethyl sulfoxide in the reaction medium. At a dodecyl octaoxyethylene glycol monoether (C12E8) to protein weight ratio of 0.2, a detergent concentration too low for solubilization to occur, ATP-induced Ca2+ release occurred more rapidly than for native leaky membranes, whereas the rebinding step was slower. In contrast, no Ca2+ release was observed for any soluble preparation. The kinetics of Ca2+ release was studied under conditions where the ATPase was monomeric or aggregated, and also in the presence of added phospholipid. The ATPase was shown to be monomeric by sedimentation equilibrium measurements in the presence of Ca2+, ADP, and beta, gamma-methylene-ATP at a C12E8 to protein weight ratio of 2.0. It is concluded that solubilization of the Ca2+-ATPase may result in uncoupling of ATP hydrolysis from ATP-induced Ca2+ release.     

Characteristics of phorbol ester stimulated growth hormone release: inhibition by insulin-like growth factor I, somatostatin, and low calcium medium and comparison with growth hormone releasing factor

TPA (12-O-tetradecanoylphorbol 13-acetate) is one of a class of compounds known as tumor promoters which perturb the inositol phosphate pathway in a number of cells. We have used TPA in a dispersed rat adenohypophysial cell system to probe the characteristics of growth hormone (GH) release. In this system we have found that the cells release GH in response to low concentrations of TPA: the EC50 was 0.23 +/- 0.05 nM (n = 6) and the maximal concentration was 5 nM. However, the maximal TPA-induced GH release was only 34 +/- 5% (n = 7) of the GH released by maximal growth hormone releasing factor (GRF) suggesting TPA releases a subpool of stored GH. Both somatostatin and insulin-like growth factor I inhibit GH release stimulated by TPA to the same extent as that stimulated by GRF, showing that the normal inhibitory control mechanism of release is not altered. Incubation in a low calcium medium that totally blocks GRF-stimulated GH release also inhibits TPA-stimulated GH release. The calcium channel blockers nifedipine and diltiazem both partly inhibit GRF- and TPA-stimulated GH release, showing some component of the calcium necessary for GH release arises from influx across the cell membrane.     

Multiple conductance states of the purified calcium release channel complex from skeletal sarcoplasmic reticulum.

The CHAPS-solubilized and purified 30S ryanodine receptor protein complex from skeletal sarcoplasmic reticulum (SR) was incorporated into planar lipid bilayers. The resulting electrical activity displayed similar responses to agents such as Ca2+, ATP, ryanodine, or caffeine as the native Ca2+ release channel, confirming the identification of the 30S complex as the Ca2+ release channel. The purified channel was permeable to monovalent ions such as Na+, with the permeability ratio PCa/PNa approximately 5, and was highly selective for cations over anions. The purified channel also showed at least four distinct conductance levels for both Na+ and Ca2+ conducting ions, with the major subconducting level in NaCl buffers possessing half the conductance value of the main conductance state. These levels may be produced by intrinsic subconductances present within the channel oligomer. Several of these conductances may be cooperatively coupled to produce the characteristic 100 +/- 10 pS unitary Ca2+ conductance of the native channel.     

Release of insulin-like growth factor carrier proteins by osteoblasts: stimulation by estradiol and growth hormone

Osteoblast-like rat calvaria cells release specific insulin-like growth factor (IGF) carrier proteins (CPs). As analyzed by SDS-PAGE under nonreducing conditions, Western blotting and detection by 125I-IGFs, CPs migrating with the IGF-binding subunits of the major CP species of rat serum (42/45/49 kDa) accumulate in cell culture medium. Treatment of the cells with growth hormone and estradiol increases the abundance of this glycosylated CP species. Since the two hormones were previously found to stimulate osteoblast replication via an IGF I-dependent mechanism, the data indicate that hormones may control local IGF action not only by regulating synthesis of IGFs and their receptors but also their presentation by CPs.     

Effects of platelet-activating factor on the release of arachidonic acid and prostaglandins by rabbit iris smooth muscle. Inhibition by calcium channel antagonists.

Addition of physiological concentrations (10(-12)-10(-8)M) of platelet-activating factor (PAF) to rabbit iris muscle induced a rapid release (in 15s) of prostaglandin (PG)E2 and 6-oxo-PGF1 alpha, measured by radioimmunoassay and rapid release of 14C-labelled arachidonate and PGE2 in muscle prelabelled with [14C]arachidonic acid, measured by radiochromatography. These PAF actions are concentration- and time-dependent. The effect of PAF on PG release is not mediated through the cyclo-oxygenase pathway. The studies on the properties and mechanism of arachidonate release from phosphatidylinositol and other phospholipids in prelabelled irides by PAF suggest the involvement of a phospholipase A2. This conclusion is supported by the findings: (a) that both the removal of arachidonate and formation of lysophosphatidylinositol, from phosphatidylinositol, by PAF occur concomitantly in a time-dependent manner, (b) that Ca2+ is required for the agonist-induced release of arachidonate and PGE2, and (c) that in contrast to the rapid release of [3H]myo-inositol phosphates by carbachol and other Ca2+-mobilizing agonists previously reported in the iris muscle [Akhtar & Abdel-Latif (1984) Biochem. J. 224, 291-300], PAF (10(-12)-10(-8)M) did not appreciably enhance the release of [14C]myo-inositol phosphates and 32P labelling of phosphatidate and phosphatidylinositol in this tissue. Ca2+-channel antagonists, such as nifedipine, verapamil, diltiazem and manganese inhibited PAF-induced arachidonate and PGE2 release in a dose-dependent manner. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not increase the release of arachidonate and PGE2. The ability of Ca2+ antagonists to inhibit arachidonate release by PAF in this tissue probably reflects interference with PAF binding to its receptor. The PAF-induced release of arachidonate and PGE2 occur independently of the cyclo-oxygenase and lipoxygenase pathways. Whether the PAF-induced release of arachidonate and PG in the iris muscle is involved in the pathogenesis of inflammatory and/or physiological reactions in the eye, and how much the inhibitory effects of Ca2+-entry blockers on the PAF actions contribute to the therapeutic use of these drugs, remain to be established.     

In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release.

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50-60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20-240 min after the start of the perifusion and was stimulated by depolarization induced by high K+ concentration. The release of GRF was inhibited by somatostatin at concentrations of 10(-11) to 10(-8) M with maximum inhibition to 52.5% of the basal release at a concentration of 10(-9) M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.     

In vitro release of growth hormone-releasing factor from rat hypothalamus: effect of insulin-like growth factor-1

The release of growth hormone-releasing factor (GHRF) from rat hypothalamus was investigated in vitro. After 60 min preincubation the released GHRF from sliced rat hypothalamic fragments during 60 min incubation was detected by a highly specific and sensitive radioimmunoassay for rat GHRF. The release of GHRF was Ca2+-dependent and enhanced by high concentration of K+. Insulin-like growth factor-1 (IGF-1) significantly decreased GHRF release to 65% and 84% of the control at concentrations of 10(-8) M and 10(-7) M, respectively. These results suggest that this in vitro system is useful for the investigation of the mechanism of GHRF release from the hypothalamus and that IGF-1 is probably involved in the feedback inhibition of growth hormone secretion by attenuating GHRF release from the hypothalamus besides countering the effect of GHRF on the pituitary.     

Dynamics of secretory granules in somatotrophs of rats after stimulation with growth hormone-releasing factor: a stereological analysis

The anterior pituitary tissue of male rats injected with growth hormone-releasing factor (GRF) was either processed for stereology at the light-and electron-microscopic levels, or homogenized for growth hormone (GH) assay 2–60 min after GRF injection. Secretory granules of somatotrophs became smaller but increased in numerical density 2 min after GRF injection. Their volume density began to increase at 5 min. The frequency of exocytosis of the granules was most prominent as early as 2 min after GRF injection and reduced thereafter. GH levels in the tissue were lowest at 2–5 min, and returned to the control value by 60 min. Serum GH levels were highest at 15 min; even at 60 min, this value was higher than in the controls. These findings suggest that secretory granules in somatotrophs are stimulated to divide by GRF, resulting in a decrease in size and an increase in number. The discrepancy between the earlier formation of new secretory granules and the later restoration of intracellular GH levels implies that GRF first stimulates the synthesis of constituents of granules other than GH, and only later the synthesis of GH, and that newly formed small secretory granules contain less GH. From the clearance rate of serum GH and the frequency of granule exocytosis, it can be estimated that about a half million granules are released to maintain 1 ng/ml of serum GH in rats.