GRF-induced Growth Hormone Release Research Articles

Short Communication: Growth Hormone Response to Somatostatin-28 and Growth Hormone-Releasing Factor in Dairy Heifers

In an attempt to investigate the role of somatostatin-28 (SRIF-28) in modulating growth hormone (GH)-releasing factor (GRF)-induced GH release in dairy cattle, we assigned Holstein heifer calves (n=12) to receive 0, 10, and 20μg of SRIF-28/100kg of BW (0, 10, 20) in conjunction with administration of 3μg of GRF/100kg of BW. Administration of 10 or 20μg of SRIF-28 reduced GRF-induced GH release, because areas under the response curves during 30min after GRF administration were less when cows received those dosages compared with receiving 0. Some evidence of a dose response was observed because serum GH concentrations increased from 15 to 20min after GRF administration when animals received 10μg, but continued to decrease when animals received 20μg of SRIF-28. We conclude that, in contrast to studies with SRIF-14, pituitary GH release is very sensitive to inhibitory effects of SRIF-28 when used in as little as a fivefold molar excess. Additional experimentation is necessary to determine whether equimolar concentrations of SRIF-28 are effective in altering GRF-induced GH release. Further, these results give rise to speculation that SRIF may be involved in physiological mechanisms mediating well-documented genetic influences on GH concentrations in dairy cattle.

Growth hormone (GH)-releasing factor differentially activates cyclic adenosine 3',5'-monophosphate- and inositol phosphate-dependent pathways to stimulate GH release in two porcine somatotrope subpopulations.

Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.

Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells.

Previous results demonstrate that porcine somatotropes can be separated by density gradient centrifugation into low density (LD) and high density (HD) subpopulations. In rat, two analog somatotrope subpopulations differ morphologically and functionally. In an attempt to determine whether morphological differences were also present within LD and HD porcine somatotropes, we undertook a quantitative electron microscope study of the subcellular organelles of immunoidentified LD and HD somatotropes. In addition, to test for the existence of functional differences, cultures of separated HD and LD subpopulations were treated for 4 h with or without 10 microM GRF-(1-29) and/or 100 microM somatostatin (SRIF), and porcine GH release and intracellular content were evaluated using a homologous enzyme immunoassay. Morphometric results demonstrate that LD somatotropes are smaller in size (P < 0.05) and contain fewer secretory granules (P < 0.05) and more rough endoplasmic reticulum (P < 0.05) than HD somatotropes. In terms of secretion, LD somatotropes showed a classical response; GRF increased GH release 1.7-fold (n = 6; P < 0.05) over the control value, whereas treatment with SRIF alone did not affect basal GH release in this subpopulation, but partially blocked GRF-induced GH release. HD somatotropes responded to GRF with a similar 1.7-fold increase in GH release. However, SRIF administered alone or in combination with GRF exerted a paradoxical stimulatory effect on HD somatotropes (2.15- and 2.12-fold over control value, respectively; n = 6; P < 0.05). These results demonstrate that the porcine somatotrope population is composed of two major subpopulations that display a distinctive pattern of ultrastructural organization and a markedly divergent secretory response to in vitro SRIF treatment.

Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells

Previous results demonstrate that porcine somatotropes can be separated by density gradient centrifugation into low density (LD) and high density (HD) subpopulations. In rat, two analog somatotrope subpopulations differ morphologically and functionally. In an attempt to determine whether morphological differences were also present within LD and HD porcine somatotropes, we undertook a quantitative electron microscope study of the subcellular organelles of immunoidentified LD and HD somatotropes. In addition, to test for the existence of functional differences, cultures of separated HD and LD subpopulations were treated for 4 h with or without 10 microM GRF-(1-29) and/or 100 microM somatostatin (SRIF), and porcine GH release and intracellular content were evaluated using a homologous enzyme immunoassay. Morphometric results demonstrate that LD somatotropes are smaller in size (P < 0.05) and contain fewer secretory granules (P < 0.05) and more rough endoplasmic reticulum (P < 0.05) than HD somatotropes. In terms of secretion, LD somatotropes showed a classical response; GRF increased GH release 1.7-fold (n = 6; P < 0.05) over the control value, whereas treatment with SRIF alone did not affect basal GH release in this subpopulation, but partially blocked GRF-induced GH release. HD somatotropes responded to GRF with a similar 1.7-fold increase in GH release. However, SRIF administered alone or in combination with GRF exerted a paradoxical stimulatory effect on HD somatotropes (2.15- and 2.12-fold over control value, respectively; n = 6; P < 0.05). These results demonstrate that the porcine somatotrope population is composed of two major subpopulations that display a distinctive pattern of ultrastructural organization and a markedly divergent secretory response to in vitro SRIF treatment.

In vivo evidence of a positive role for somatostatin to optimize pulsatile growth hormone secretion.

Despite convincing evidence that somatostatin (SRIF) and growth hormone (GH)-releasing factor (GRF) individually play crucial roles in GH regulation, the nature of the interplay between these two hypothalamic hormones is far from clear. In the present study, we used the long-acting SRIF analogue, octreotide, as a probe in both the normal and mutant dwarf (dw) rat 1) to further elucidate the temporal nature of the SRIF-GRF interaction in vivo and 2) to define possible mechanisms of action of SRIF in generating pulsatile GH secretion. Normal free-moving adult male rats pretreated with octreotide (25 and 50 micrograms iv) and subsequently challenged with GRF (1 micrograms iv) exhibited a markedly blunted GH response to exogenous GRF 1 h after treatment. In contrast, preexposure to octreotide for 3 h produced a two- to threefold augmentation in GH responsiveness to GRF. Compared with normal saline-pretreated controls, 3-h pretreatment with octreotide produced a 14- to 16-fold augmentation in the postinhibitory rebound release of GH after the coadministration of native SRIF-14 and GRF (P < 0.001). In dw rats, which show a selective reduction in GH synthesis and storage, 3-h preexposure to octreotide failed to significantly alter GRF-induced GH release. These results demonstrate that, in the normal male rat, a 3-h period of exposure to the SRIF analogue octreotide is sufficient to enhance GH responsiveness to GRF. Our findings suggest that this effect is due to a SRIF-mediated buildup of pituitary GH stores in a readily releasable poo.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

Clonidine potentiates the growth hormone response to a growth hormone releasing hormone challenge in hypothalamic growth hormone releasing hormone deficient rats.

This study was designed to further investigate our postulate regarding the inhibitory role played by central alpha 2-adrenergic pathways on hypothalamic somatostatin (SS) release in rats. The growth hormone (GH) responses to exogenous GH-releasing factor (GRF; 3 micrograms/kg i.v.) or clonidine (CLO; 100 micrograms/kg i.v.), either given alone or in combination, were tested in 3-month-old male rats made GH-releasing hormone (GH-RH) deficient neonatally by administration of monosodium glutamate (MSG; 4 mg/g body weight s.c.). To prevent the presumable decrease in the pituitary GH content in these animals from leading to an erroneous interpretation of the results obtained, half of these rats were given GRF (MSG-GRF rats; 30 micrograms/kg s.c.) for 3 days immediately prior to GH testing. The other half of MSG-treated and non MSG-treated rats received saline during these days (MSG-S and controls, respectively). To establish the efficiency of GRF priming, the pituitary GH content was measured in other MSG-GRF, MSG-S, and control animals. The mean (+/- SEM) GH peaks in response to GRF challenge were significantly higher in controls than in MSG-GRF rats (125.2 +/- 28.5 vs. 67.5 +/- 19.4 micrograms/l; p < 0.05), while no significant GRF-induced GH release was observed in the MSG-S group. Most likely these results are related to the different pituitary GH content, significantly (p < 0.01) higher in controls than in MSG-GRF rats, and in the latter higher than in MSG-S animals (p < 0.05). CLO administration did not evoke a significant GH release in MSG rats, whether primed with GRF or not.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens modulate growth hormone-releasing factor-induced GH release from bovine anterior pituitary cells in static culture

Static primary cultures of bovine anterior pituitary (AP) cells were utilized to study the effect of sex steroids on basal growth hormone (GH) and GH-releasing hormone (GRF)-stimulated release of GH. The AP cells (5 × 10 5 cells/well) were allowed to attach for 72 hr and become confluent before treatments were imposed. Cells were incubated for an additional 24, 48 or 72 hr with either estradiol-17β (E 2, 10 −11 to 10 −8 M), testosterone (T, 10 −8 to 10 −5 M), dihydrotestosterone (DHT, 10 −9 to 10 −6 M) or 5α-androstane-3α, 17β-diol (3α-diol, 10 −11 to 10 −8 M). Media were collected every 24 hr and GH concentrations determined by RIA. Incubation of calf AP cells with gonadal steroids did not affect (P>0.05) basal GH released at 24, 48, or 72 hr. In another experiment, calf AP cells were incubated with the same concentrations of the steroids for 24 hr, media harvested, cells washed and challenged in serum-free media for 1 hr with bovine GRF 1–44-NH 2 (10 −8 M). In non-steroid treated wells, GRF increased (P<0.05) GH from 58 to 134 ng/ml. Incubation with E 2 or 3α-diol did not affect (P>0.05) GRF-induced GH release; however, preincubation with T (10 −5 M) and DHT (10 −9, 10 −8 and 10 −7 M) increased (P<0.05) GRF-induced GH release above control concentrations (195, 235, 190 and 185 ng/ml, respectively). At the doses tested, sex steroids did not affect basal release of GH, but androgens increased responsiveness of somatotropes to GRF.

Time-dependent reduction and potentiation of growth hormone (GH) responsiveness to GH-releasing factor induced by exogenous GH: role for somatostatin.

Exogenous GH is known to exert a negative feedback effect on its own responsiveness to GH-releasing factor (GRF); however, the mechanism is not known. In the present study we examined the time course of effects of a single sc administration of recombinant human (rh) GH on GH responsiveness to GRF and investigated the possible involvement of somatostatin (SRIF) in this response. Free-moving adult male rats were administered 200 micrograms rhGH, sc, at 0800 h and subsequently challenged with 1 microgram GRF-(1-29)NH2, iv, at times of spontaneous peaks (1100 and 1500 h) and troughs (1300 h) in GH secretion during a 6-h (1000-1600 h) sampling period. H2O-injected control rats exhibited the typical cyclic responsiveness to GRF stimulation, with GRF-induced GH release significantly greater during peak compared to trough periods of the GH rhythm. Pretreatment with rhGH 3 h before GRF injection markedly inhibited the GH response to GRF at a peak time [integrated GH release over 30 min, 1135 +/- 271 vs. 6372 +/- 1185 ng/ml.30 min in H2O-injected controls (mean +/- SE); P less than 0.01]. In striking contrast, 5 h after rhGH administration, there was a 6-fold augmentation of GH responsiveness to GRF compared to that in H2O-injected controls at a trough time (7032 +/- 1622 vs. 1128 +/- 216 ng/ml.30 min; P less than 0.01). High GH responsiveness to GRF was preserved 7 h after rhGH injection. Passive immunization of rhGH-treated rats with SRIF antiserum reversed the rhGH-induced blunted GH response at 3 h (7985 +/- 366 vs. 1705 +/- 431 ng/ml.30 min in rhGH-treated control rats given normal sheep serum; P less than 0.01) and completely restored GH responsiveness to levels as high as those in H2O-injected controls. These results demonstrate that 1) a single sc injection of rhGH markedly attenuates GH responsiveness to GRF acutely for about 3 h, but subsequently enhances somatotroph sensitivity to the stimulatory actions of GRF; and 2) the short term blunting of GRF-induced GH release by rhGH is due at least in part to increased release of endogenous SRIF. The subsequent potentiation of GH responsiveness to GRF is probably due to a SRIF-mediated build-up of pituitary GH stores in a readily releasable pool. Such a mechanism of GH autofeedback may play a physiological role in the genesis of pulsatile GH secretion.

Pituitary Response to Growth Hormone–Releasing Hormone in IDDM: Abnormal Responses to Insulin and Hyperglycemia

In poorly controlled insulin-dependent diabetes mellitus (IDDM), hyperglycemia fails to inhibit the pituitary response to growth hormone-releasing factor (GRF). To evaluate whether this derangement is reversed by a simultaneous elevation of circulating insulin, 0.3 micrograms/kg i.v. GRF 1-40 was administered to nine poorly controlled IDDM subjects (HbA1 greater than 11.1%) with and without concomitant infusion of insulin. In the absence of insulin, the poorly controlled IDDM subjects demonstrated a growth hormone response to GRF similar to that of nondiabetic subjects, despite marked hyperglycemia (approximately 16.8 mM). When insulin was infused into these same patients (insulin clamp) to produce combined hyperinsulinemia (528 +/- 90 pM) and hyperglycemia (16.5 +/- 1.98 mM), the GRF-induced growth hormone rise was markedly exaggerated (65 +/- 11 vs. 20 +/- 4 micrograms/L without insulin infusion, P less than 0.001). This enhancement of GRF-stimulated growth hormone release by insulin was strikingly attenuated (22 +/- 7 micrograms/L) in five well-controlled diabetic subjects studied under conditions of similar hyperinsulinemia (486 +/- 84 pM) and hyperglycemia (16.41 +/- 0.95 mM). In contrast, in nondiabetic subjects, acute hyperinsulinemia reduced the growth hormone response to GRF. We conclude that the failure of hyperglycemia to block the pituitary response to GRF in poorly controlled diabetes is not attributable to the lack of a coincident increase in circulating insulin. The paradoxical stimulatory effect of insulin on GRF-induced growth hormone release may contribute to the high spontaneous growth hormone levels characteristically seen in poorly controlled insulin-treated patients, and its attenuation after intensive insulin therapy may contribute to the reversal of growth hormone hypersecretion in well-controlled diabetic patients.

Effect of Antiserum to Rat Growth Hormone (GH)- Releasing Factor on Physiological GH Secretion in the Female Rat

To further study the physiological role of GH-releasing factor (GRF), we examined the effect of antiserum to rat hypothalamic GRF on spontaneous GH secretion in the normal female rat. Two groups of six conscious female rats were passively immunized with either nonimmune rabbit serum (NRS) or antirat GRF serum via a chronic indwelling atrial catheter. The secretory profiles of GH were observed by collecting blood samples at 15-min intervals for 1 h before and 4 h after administration. The NRS-treated rats showed a characteristic female pattern of spontaneous GH secretion. GH pulses were of low amplitude (mean +/- SEM, 26.8 +/- 2.4 ng/ml) and occurred irregularly at a frequency of 4.2 +/- 0.2/5 h, while interpeak through levels of GH were relatively high, with nadir values of 8.6 +/- 0.7 ng/ml. Synthetic rat GRF, given iv at a dose of 1 microgram/kg BW after the last blood sampling, stimulated GH release to a peak level of 153 +/- 37 ng/ml in the control rats. Administration of GRF antiserum caused a profound suppression of both pulse and trough components of GH secretion. This effect occurred rapidly, within 15 min after injection of antiserum, and GH secretion decreased uniformly to very low levels (3.4 +/- 0.1 ng/ml), with little or no fluctuation throughout the observation period. GRF antiserum also abolished the synthetic rat GRF-induced GH release, indicating sufficient potency of immunoneutralization. These results demonstrate that both GH pulses and troughs are dependent upon hypothalamic GRF in normal female rats, thereby substantiating earlier observations in male rats which demonstrated the physiological role of GRF in GH secretion.

Differential effects of T4 and T3 on TRHand GRF-induced GH secretion in the domestic fowl

The in vivo growth hormone (GH) response of immature domestic fowl to thyrotrophin-releasing hormone (TRH) and GH-releasing factor (GRF) was suppressed in birds fed diets supplemented (1 ppm) with triiodothyronine (T3) or given bolus intraperitoneal (ip) injections (100 micrograms/kg for 10 d) of T3. Supplementation (ppm) of the diet with T4 had no effect on secretagogue- induced GH release. Exogenous T3 or T4 suppressed basal, TRH- and GRF-induced GH release 2 h after daily ip administration (100 micrograms/kg for 10 d). 24 h after the last injection, only T3 was effective in inhibiting basal and stimulated GH secretion in vivo. The systemic administration of T3 was followed 2 h and 24 h later by a downregulation of pituitary TRH binding sites. T4 administration had no effect on pituitary TRH binding. When chicken pituitary glands were incubated in vitro, basal GH release was unaffected by the addition of 10(-9)-10(-5) M T3 or T4 to the incubation media. The in vitro GH response to TRH (10(-6) M) or GRF (10(-6) M) challenge was, however, suppressed in a dose-related manner by T3 but was unaffected by the coincubation of T4. These results demonstrate inhibitory effects of T3 and T4 on basal and secretagogue-induced GH secretion in fowl. T4 is less active than T3 and probably exerts some of its effects via T3-independent mechanisms.

Effect of long-term administration of human growth hormone-releasing factor and (or) thyrotropin-releasing factor on hormone concentrations in lactating dairy cows

Fifteen cows (87 ± 8 d in lactation; 641 ± 33 kg BW) were randomly assigned to treatment and then subjected for 182 d to daily sc injection (1000 hr), in the cervical area, of saline (control), thyrotropin-releasing factor (TRF; 1 μg/kg BW), growth hormone-releasing factor (1–29)NH 2 (GRF; 10 μg/kg BW) or GRF plus TRF (10 and 1 μg/kg BW, respectively) according to a 2 × 2 factorial design. On days 1, 31, 88 and 179, jugular blood samples were collected from 2 hr before to 6 hr after injection. Samples were also collected for 5 consecutive days after cessation of treatment. GRF always induced growth hormone (GH) release (600 vs 7925 ng·min/ml) with augmentation of response with time (interaction GRF ∗ day; P<.001). TRF did not affect (P>.25) GH release; there was no interaction (P>.25) with time. There was no significant interaction (P>.25) between GRF and TRF on GH release. However, the amount of GH release with GRF plus TRF was always greater than with GRF alone (9419 vs 6431 ng·min/ml). TRF induced a significant release of prolactin (23769 vs 42175 ng·min/ml) but GRF reduced the amount of prolactin release on the last day of sampling. TRF induced thyroid stimulating hormone (TSH) release only on the first day of injection while triidothyronine (T 3) and thyroxine (T 4) continued to respond to TRF throughout the treatment period. Concentrations of T 3 and T 4 fell below control levels after cessation of TRF injection. In conclusion, GRF-induced GH release and TRF-induced Prl and thyroid hormone release were maintained over a 6-mo treatment period. TRF induced TSH release only on the first day of injection. Overall, these results raised the possibility of a direct effect of TRF on the thyroid gland.

The effect of thyroid hormone and glucocorticoids on carp growth hormone-releasing factor (GRF)-induced growth hormone (GH) release in rainbow trout ( Oncorhynchus mykiss)

1. 1. The effect of thyroid hormone and glucocortieoids on carp growth hormone-releasing factor (GRF)-induced growth hormone (GH) secretion was studied on rainbow trout using a dispersed pituitary cell culture system. 2. 2. A combined administration of lower doses (0.01 μM) of 3,5,3'-trhodo- l-thyronine (T 3) and dexamethasone (Dex) significantly increased spontaneous as well as carp GRF-induced GH release. 3. 3. Lower doses of Dex alone had no effect, and T 3 had a marginal effect on GH release. Higher doses of either Dex or T 3 potentially reduced GH release. 4. 4. This study indicates an important role of thyroid hormone and/or glucocortieoids in the hypothala-mic regulation of GH secretion in fish.

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

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

Influence of catecholamines, prostaglandins and thyroid hormones on growth hormone secretion by chicken pituitary cells in vitro

In young chickens plasma concentrations of growth hormone (GH) are depressed by prostaglandins (PG) E 1 and E 2, epinephrine, norepinephrine, α 2 and β agonists or thyroid hormones. A primary culture of chicken adenohypopyseal cells was used to examine the direct effects of these agents at the level of the pituitary as evaluated by GH release in the presence and absence of growth hormone releasing factor (GRF). Following collagenase dispersion and culture (preincubation, 48 hr) cells were exposed (incubation, 2 hr) to test agents, except for thyroid hormones which were added during the preincubation, and incubation period. Growth hormone release was increased (P<.05) in the presence of PGE 1 (10 −8M by 34%; 10 −7M by 54%), PGE 2 (10 −8M by 29%; 10 −7M by 29%), PGF 2α (10 −8M by 28%), and the β agonist isoproterenol (10 −7M by 46%). Basal GH release from chicken pituitary cells was not affected by dopamine, norepinephrine, epinephrine, thyroxine (T 4), triiodothyronine (T 3), or α adrenergic agonists. Growth hormone releasing factor stimulated GH release was not affected by the presence of prostaglandins E 1, E 2 or F 2α in the incubation media. However, GRF stimulated GH release was reduced by high doses of catecholamines: dopamine (10 −6M by 34%), norepinephrine (10 −6M by 74%), epinephrine (10 −8M by 47%; 10 −7M by 41%; 10 −6M by 89%), and by the α 1 adrenergic agonist, phenylephrine (10 −7M by 52%), the α 2 agonist, clonidine (10 −8M by 34%; 10 −7M by 83%) and the β agonist, isoproterenol (10 −7M by 64%). Similarly, GRF-induced GH release was decreased by coincubation with 10 or 100 ng/ml of T 3 by 75% and 89%, respectively. In contrast, T 4 did not affect GH release evoked by GRF although T 3 depressed GH release in the presence of both thyrotropin releasing hormone (TRH) and GRF. To determine the temporal relationship for T 3 inhibition of GRF stimulated GH release, T 3 (1.3 × 10 −8M) was added during either the preincubation, incubation period or both. T 3 did not affect basal GH release during any time period, however, T 3 supplementation during the preincubation (48 hr) or both the preincubation (48 hr) and incubation (2 hr) period did suppress GRF stimulated GH release. These data suggest that the decrease in plasma concentration of GH observed in young chickens injected with catecholamines or T 3 may involve direct effects on the pituitary gland. However, the inhibitory effects of PGE 1, PGE 2 and PGF 2α on in vivo GH secretion appears to be mediated at another site, presumably the hypothalamus.

Effects of chronic GRF treatment on lambs having low or normal birth weight

The effects of a long term treatment with human GRF(1–29)NH2 on plasma growth hormone (GH), somatomedin C (Sm-C), histomorphometric parameters of bone growth and body composition were investigated in normal and low birthweight male lambs. The animals were divided into two groups according to their birthweight: 24 normal birthweight (NBW) lambs weighing more than 4 kg and 22 low birthweight (LBW) lambs weighing less than 2.5 kg at birth. Half of the animals in each group received two daily subcutaneous injections (8 μg/kg body weight) of hGRF(1–29)NH2 (GRF) from birth to slaughter at 45 or 90 days of age. The other animals received the solvant only. At the beginning and at the end of the treatment, plasma GH and serum Sm-C concentrations were measured in all groups. After slaughter, a histomorphometric study was performed on undecalcified sections of metacarpal growth plates, and the remaining of the carcass was pulverized to study the chemical body composition. GRF induced GH release in both GRF-treated groups. However, plasma GH reached higher (P<.001) concentrations and the GRF-induced GH peak lasted longer in LBW than in NBW lambs. At day 45, the GRF treatment increased (P<.05) serum Sm-C concentrations in LBW. Most of histomorphometric parameters reflecting the metacarpal growth in length, were not statistically modified under GRF treatment. However, the size of degenerative cells was smaller (P<.05) in LBW treated lambs as compared to controls. Consequently, the cell production in the growth plate was increased (P<.05) under GRF treatment. In both NBW and LBW groups at 45 days of treatment, GRF treatment reduced the amounts of lipids (P<.025) and energy (P<.05), while increased (P<.01) phosphorus deposition in the body. In contrast, there was no effect of GRF treatment on protein content. We conclude from this experiment that the induction of GH secretion by a chronic treatment with GRF is able to modify some patterns of growth. However, most of the effects of GRF were observed in LBW lambs and after 45 days of treatment only. This suggests that treatment with GRF may serve to compensate for growth in growth-retarded animals. Further studies with different mode of GRF administration should indicate whether it is as much effective in normal animals.

Protein Kinase C Is not Essential for Growth Hormone (GH)-Releasing Factor-Induced GH Release from Rat Somatotrophs*

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

Paradoxical Enhancement of Pituitary Growth Hormone (GH) Responsiveness to GH-Releasing Factor in the Face of High Somatostatin Tone*

Pituitary GH secretion is regulated by a delicate interplay between stimulatory (GRF) and inhibitory [somatostatin (SRIF)] hypothalamic hormones, although the nature of the GRF/SRIF interaction remains to be elucidated. In the present study, we documented a significant elevation of plasma SRIF-like immunoreactivity in 72-h fasted rats compared to that in fed controls (129.0 +/- 17.9 vs. 38.2 +/- 5.8 pg/ml; P less than 0.01) and used this model of high SRIF tone to further delineate the interrelation between GRF and SRIF in physiological regulation of pulsatile GH secretion. We examined pituitary GH responsiveness to GRF, both in vivo and in vitro, after 72-h exposure to nutritional deprivation and high SRIF secretion. In vivo, GRF-induced GH release was markedly enhanced in the face of high circulating SRIF; freely moving, starved rats released 4- to 8-fold more GH than fed controls in response to rat GRF iv. In vitro, both basal and human GRF-induced GH release were augmented 2- to 4-fold in perifused dispersed anterior pituitary cells of starved rats compared to those in fed controls, and this enhanced responsiveness persisted in the presence of 10(-9) M SRIF. These results demonstrate that SRIF not only inhibits GH secretion stimulated by GRF, but that under different temporal conditions SRIF may act in a paradoxically positive manner to sensitize pituitary GH responsiveness to GRF. Such a cooperative interaction of the two peptides may be necessary to optimize pulsatile GH release. Our findings provide support for the hypothesis that the temporal patterning of hypothalamic GRF/SRIF signals to pituitary somatotrophs may be the major determinant for pulsatile GH secretion and, ultimately, body growth.

Triiodothyronine inhibition of thyrotropin-releasing hormone- and growth hormone-releasing factor-induced growth hormone secretion in anesthetized chickens

The ability of triiodothyronine (T 3) to reduce basal and secretagogue-induced growth hormone (GH) release was examined in anesthetized young and adult male chickens. Infusion of T 3 had no effect on basal plasma concentrations of GH in either young or adult chickens. However, GH secretion following challenge with either thyrotropin-releasing hormone (TRH) or growth hormone-releasing hormone (GRF) was reduced, in a dose-dependent manner, by the infusion of T 3. In vivo sensitivity to T 3 inhibition was greater with TRH-than GRF-stimulated GH release in either young (ED 50 for TRH-induced GH release, 0.34 μg T 3/kg/min; ED 50 for GRF-induced GH release, 0.49 μg T 3/kg/min) or adult chickens (Ed 50 for TRH-induced GH release, 0.11 μg T 3/kg/min; ED 50 for GRF-induced GH release 1.89, μg T 3/kg/min). Moreover, there was an increase in sensitivity of TRH-induced GH release to T 3 with age.