Growth hormone-releasing peptide-2 (GHRP-2) does not act via the human growth hormone-releasing factor receptor in GC cells

Effect of growth hormone-releasing peptide-2 (GHRP-2) on ovine somatotrophs is abolished by a growth hormone-releasing factor (GRF) receptor antagonist, which raises the possibility that GHRP-2 may act on GRF receptors. In the present study, we used rat pituitary GC cells with or without stable transfection of cDNA coding for the human GRF receptor (GC/R⁺ or GC/R⁻) to determine whether or not GHRP-2 acts via the GRF receptor. Northern blot analysis indicated that GRF receptor mRNA was undetectable in GC/R⁻ cells, whereas a high level of expression occurred in GC/R⁺ cells that were transfected by GRF receptor cDNA. In GC/R⁻ cells, incubation with up to 10⁻⁷ M of either hGRF or GHRP-2 did not alter the intracellular cAMP, [Ca²⁺]i, or GH secretion. In GC/R⁺ cells, hGRF (10⁻¹¹–10⁻⁷ M) increased cAMP levels in a concentration-dependent manner up to 20-fold. This increase in cAMP levels was blocked by a GRF receptor antagonist, [Ac-Tyr¹, d-Arg²]-GRF 1–29, but not by a Ca²⁺ channel blocker, NiCl₂ (0.5 mM). GH secretion and [Ca²⁺]i were, however, not increased by hGRF. Incubation of the transfected cells with 10⁻¹¹–10⁻⁸ M GHRP-2 did not modify intracellular cAMP levels. This result suggests that GHRP-2 does not act through the GRF receptor.     

Interaction of glucose and pyridostigmine on the secretion of growth hormone (GH) induced by GH-releasing hormone (GHRH)

In order to investigate the mechanisms by which hyperglycaemia induces an inhibition of GHRH-induced GH release, we gave the following treatments to seven normal men: a) GHRH 100 micrograms iv; b) pyridostigmine (PD) 120 mg po 60 min before GHRH; c) glucose 250 mg/kg iv as a bolus (10 min before GHRH) plus 10 mg/kg/min until the end of the test; d) glucose pyridostigmine and GHRH as above. Glucose significantly reduced GHRH-stimulated GH levels, whereas PD significantly enhanced them. When PD and glucose were given together, the effect on GHRH-stimulated GH secretion was not different from the algebraic sum of the single effects of the two substances. Thus glucose seems to be able to exert its inhibition, at least partially, also when pyridostigmine is coadministered.     

Exogenous GH-releasing hormone increases GH and LH secretion in growing mithuns (Bos frontalis)

To investigate the effects of growth hormone-releasing hormone (GHRH) administration on the patterns of GH and LH secretion in growing female mithuns, 12 mithuns within the age group of 10-12 months of age were divided into two groups (treatment and control groups) of six each in such a way that average body weight between the groups did not differ significantly (P>0.05). Both the groups were administered i.v. either with synthetic bGHRH [bGHRH (1-44)-NH2] at 10 microg/100 kg body weight (treatment group) or equal volume of normal saline (control group). Blood samples collected prior to and after GHRH challenge at -60, -45, -30, -15, -10, -5 min and 5, 10, 15, 30 min, and thereafter, at an interval of 15 min up to 8 h post-injection were assayed for plasma GH and LH. Plasma progesterone was estimated in twice-a-week samples collected for six consecutive weeks preceding GHRH challenge to assess whether either group has begun ovarian cyclicity. Body weight of all animals was recorded once in a week during the period. A peak of GH was registered in all animals within 5-25 min post-GHRH administration with a mean peak of 443.5+/-25.32 ng/ml at 15 min post-administration, which was much higher than in any other bovines reported following GHRH challenge. The patterns of LH secretion were pulsatile in nature in both the groups. Interestingly, the hormone concentrations exhibited higher pulsatility with greater amplitude after GHRH challenge in GHRH-treated than in control mithuns. The GHRH-treated mithuns averaged 0.44 pulses/h (4 pulses/9 h) and the rate was 0.20/h (2 pulses/9 h) in controls. The rate of pulse frequency and amplitude differed significantly with time of sampling. The mean plasma LH levels after GHRH administration were significantly higher in treatment group than those recorded in control mithuns. The mean plasma progesterone was similar (P>0.05) in both the groups and no animal from either group had begun ovarian cycle. In conclusion, exogenous GHRH significantly increases plasma GH and also LH pulse frequency and amplitude with higher mean post-GHRH LH levels in growing mithuns suggesting thereby its possible use for enhancement of maturity process in this unique meat animal.     

Sex differences in growth hormone (GH) secretion by rats administered GH-releasing hexapeptide

The purpose of this study was to compare GH secretion after the administration of GH-releasing hexapeptide (GHRP-6) in conscious male and female rats. Plasma GH was significantly elevated in female rats (six of six) compared to male rats (three of six) 15 min after administration of a single sc injection of GHRP-6 (0.5 mg/kg). In male rats, GHRP-6 administration was associated with suppression of episodic GH secretion and desensitization to a second injection administered 6 h later, whereas in female rats, GH secretion occurred after both GHRP-6 injections. After 14 consecutive days of administering GHRP-6 twice per day, mean plasma GH concentrations in males decreased from 110 +/- 91 to 2.8 +/- 0.6 ng/ml (P less than 0.05) and in females increased from 170 +/- 53 ng/ml to 361 +/- 81 ng/ml (P less than 0.05). Desensitization to GHRP-6 in conscious male rats was not observed in pentobarbital-anesthetized male rats, suggesting that GHRP-6 administration enhanced somatostatin release in the conscious state. After 14 consecutive days of GHRP-6 administration, the mean pituitary GH concentration in female rats was significantly lower than that in male rats (5.1 +/- 0.2 vs. 12.9 +/- 1.2 micrograms/mg, respectively). Lower pituitary GH concentrations in females correlated with higher GH secretion after GHRP-6 administration. Desensitization to GHRP-6 in male rats is attributed to neurohumoral factors producing their unusual pattern of episodic GH secretion, and the response is probably not typical of other species.     

Inhibition by gastrin-releasing peptide of growth hormone (GH) secretion induced by human pancreatic GH-releasing factor in rats

Intravenous injection of synthetic human pancreatic GH-releasing factor (1-44) [hpGRF(1-44)] (50 ng-1 micrograms/100 g BW) resulted in a dose-related increase in plasma GH levels in urethane-anesthetized male rats. Pretreatment with cysteamine (30 mg/100 g BW, sc, 4 h previously) or anti-SRIF rabbit serum (0.5 ml/rat, iv, 1 h previously) raised basal plasma GH levels and markedly exaggerated the plasma GH response to hpGRF (1-44) (80 ng/100 g BW, iv). The intraventricular injection of gastrin-releasing peptide (GRP) (1 micrograms/rat) completely inhibited the increase of plasma GH induced by hpGRF (80 ng/100 g BW, iv) in control rats. However, in the rats treated with cysteamine or anti-SRIF rabbit serum, the inhibitory effect of GRP on hpGRF-induced GH release was significantly attenuated. hpGRF (10(-11)-10(-8) M) stimulated in a dose-related manner GH release from rat anterior pituitary cells superfused in vitro. GRP (10(-5) M) did not affect pituitary GH release induced by hpGRF (10(-9) M) in vitro. These results indicate that hpGRF stimulates rat GH secretion by acting at the pituitary level and the hypothalamic SRIF interacts with the action of GRF, and that GRP inhibits GH secretion, at least in part, by stimulating SRIF release from the hypothalamus.     

Phorbol ester pretreatment attenuates the growth hormone (GH) response to GH-releasing factor in cultured rat pituitary cells

The effect of phorbol ester pretreatment on rat (r) GH release induced by GH-releasing factor (GRF) or 8-bromo-cyclic (c)AMP was investigated using rat pituitary cells cultured in monolayers. Pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 h significantly suppressed the rGH release induced by GRF, but not that by 8-bromo-cAMP 20 h later; this suppressive effect of TPA was concentration-dependent from 8 to 160 nmol/l, and complete suppression was observed after pretreatment with 80-160 nmol TPA/l. Production of cAMP by pituitary cells stimulated with GRF was similarly attenuated in TPA-pretreated cells. The rGH responsiveness to GRF of these cells was fully recovered on prolonged culture (40 h), suggesting that the inhibitory effect of TPA is reversible. In contrast, pretreatment with GRF (5 nmol/l) resulted in suppression of the rGH response to subsequent exposure to GRF (5 nmol/l) or 8-bromo-cAMP (10 mmol/l), but not to TPA. These observations suggest that pretreatment with TPA modifies the rGH response to GRF at steps before the formation of cAMP.     

Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells.

The involvement of nitric oxide (NO) in human GH-releasing hormone (hGHRH)-induced GH secretion was studied with freshly dissociated male rat pituitary cells. The cells were packed in a column of Bio-Gel-P2 and continuously perifused at 37 C. Hemoglobin (Hb; 10 microM), which is known to strongly bind NO, potentiated 0.01, 0.1, and 1 nM hGHRH-induced GH secretion by 73%, 52%, and 39%, respectively, without affecting the basal secretion of GH. As reported previously, 1-nM or higher concentrations of hGHRH elicit an increase in GH secretion during the application of hGHRH (on-response) and also a transient increase after the cessation of hGHRH application (off-response). It was found that Hb potentiated only the off-response in 1 nM hGHRH-induced GH secretion, and the same concentration of Hb had no effect on 10 nM hGHRH-induced GH secretion. N-Methyl-L-arginine (MeArg; 500 microM), a competitive inhibitor of NO synthase, also potentiated both the on- and off-responses of 1 nM hGHRH-induced GH secretion by 39% without affecting basal GH secretion. Since cAMP is thought to be an intracellular messenger of hGHRH action, the effects of Hb and MeArg on 1 mM (Bu)2AMP-induced GH secretion were examined. Their actions were found to be greater than those in hGHRH-induced GH secretion. Excess K+ (15 and 50 mM)-induced GH secretion, which does not involve cAMP, however, was not affected by either Hb or MeArg. In contrast, 3 mM sodium nitroprusside, which releases NO, suppressed the 1 nM hGHRH-induced off-response by 18%. The same concentration of sodium nitroprusside had no effect on excess K(+)-induced GH secretion. The effect of 8-bromo-cGMP on hGHRH-induced GH secretion was also examined, since NO is thought to exert its action through cGMP by activating guanylate cyclase in neural tissue. The application of 8-bromo-cGMP, however, did not affect 1 nM hGHRH-induced GH secretion. These observations suggest that hGHRH stimulates the synthesis of NO at least partly through cAMP, thereby partially inhibiting hGHRH-induced GH secretion.     

Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor

BACKGROUND AND AIMS: Polyamines are essential for the normal postnatal development, maintenance, and function of gastrointestinal epithelia. The extracellular Ca(2+) (Ca(2+)(o)/nutrient)-sensing receptor is expressed on both luminal and basolateral membranes of colonocytes, and, in other cell systems, this receptor has been shown to respond to polyamines. Thus, the Ca(2+)-sensing receptor could provide a mechanism for modulation of colonocyte function by dietary and systemic extracellular polyamines. In the present study, we investigated the interaction of polyamines, particularly spermine, and extracellular Ca(2+) on second messenger generation by, and on function of, rat distal colonic crypts. METHODS: Calcium-sensing receptor activation was assessed in colonic epithelial cells and intact crypts freshly isolated from distal colon by monitoring intracellular IP(3) and Ca(2+) accumulation using radioimmunoassay and Fluo-3 fluorometry, respectively. Interactions of extracellular Ca(2+) and spermine on regulation of both basal and forskolin-stimulated fluid transport were measured in crypts microperfused in vitro. RESULTS: Polyamine (spermine > spermidine > putrescine)-mediated enhancement of intracellular D-myo-inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) accumulation required extracellular Ca(2+), and the EC(50) for extracellular Ca(2+)-mediated activation of the calcium-sensing receptor was reduced by polyamines. Extracellular spermine modulated both basal and forskolin-stimulated fluid secretion in perfused colonic crypts, and the EC(50) for spermine-induced reduction in forskolin-stimulated fluid secretion was inversely dependent on extracellular Ca(2+) (Ca(2+)(o)). CONCLUSIONS: The interactions of extracellular Ca(2+) and polyamines on second messenger accumulation and fluid secretion support a role for the luminal and basolateral calcium-sensing receptors in mediating some of the effects of polyamines on distal colonic epithelial cells.     

Hypoxia exacerbates Ca-handling disturbances induced by very low density lipoproteins (VLDL) in neonatal rat cardiomyocytes

It is known that myocardium suffers serious alterations under ischemic conditions such as lipid overloading and electrophysiological alterations. However, it is unknown whether intracellular lipid accumulation and calcium dysfunction share common pathophysiological mechanisms under ischemia. The aims of this study were 1) to analyze the effect of normal and high doses of very low density lipoproteins (VLDL) on lipid content and calcium handling; 2) to investigate whether hypoxia modulates the effect of high VLDL doses; and 3) to identify potentially underlying mechanisms in cardiomyocytes. For this purpose, neonatal rat ventricular myocytes cultures were prepared from hearts of 3-4-day-old rats. High doses of VLDL that induced cholesteryl ester (CE) and triglyceride (TG) accumulation strongly reduced sarco(endo)plasmic reticulum Ca ATPase-2 (SERCA-2) expression, calcium transient amplitude and sarcoplasmic reticulum (SR) calcium loading. Interestingly, hypoxia, by upregulating VLDL-receptor expression (4.5-fold at 16 h) increased CE (1.5-fold) and TG (3-fold) cardiomyocyte content and exacerbated the negative effect of VLDL on SERCA-2 expression. Functionally, the hypoxic exacerbation of VLDL-mediated SERCA-2 downregulation was translated into a stronger decrease in calcium transient amplitude and SR calcium loading in myocytes exposed simultaneously to hypoxia and high VLDL. In conclusion, high VLDL doses alter calcium handling in cardiomyocytes and SERCA-2 play a pivotal role in the hypoxic exacerbation of VLDL-mediated effects on cardiac calcium handling. Potentiation of VLDL's effects under hypoxia is explained, at least in part, by hypoxic upregulation of the expression of VLDL-receptor.     

Perinatal growth hormone (GH) physiology: effect of GH-releasing factor on maternal and fetal secretion of pituitary and placental GH

To study regulation of the secretion of human pituitary GH (hGH) and placental GH (hPGH) in the pregnant woman and human fetus, the GH-releasing factor Sermorelin [GRF-(1-29)-NH2] was administered to pregnant women at term (n = 5), just before elective cesarean section; saline was administered in control studies (n = 5). The effects of GRF-(1-29)-NH2 administration on maternal and fetal serum concentrations of hGH and GRF-(1-29)-NH2 and maternal serum levels of hPGH were evaluated at birth. The mean time span between injection and birth was 20 min (range, 15-25 min). Cord serum hGH concentrations were similar in infants of GRF-(1-29)-NH2-injected mothers and control infants. GRF-(1-29)-NH2 elicited a consistent but small rise in maternal hGH serum concentrations (P = 0.08), whereas hPGH concentrations remained unaltered. Finally, GRF-(1-29)-NH2 concentrations were undetectable in cord serum, but readily detectable in concomitantly obtained maternal serum. In conclusion, these data suggest that hGH secretion in the pregnant woman at term is suppressed at the pituitary level, that GRF does not affect hPGH secretion, and that fetal hGH secretion is independent of circulating maternal GRF, probably because of lack of transplacental GRF passage.     

Inhibitory effect of hypothalamic stimulation on growth hormone (GH) release induced by GH-releasing factor in the rat

The object of the present experiments was to clarify the topography of hypothalamic areas related to the suppression of GH release induced by human pancreatic GH-releasing factor (hpGRF). One week before the experiments, bipolar concentric stimulating electrodes were implanted in the organum vasculosum of the lamina terminalis (OVLT), the periventricular nucleus of the hypothalamus (Pe), the ventromedial nucleus of the hypothalamus (VMH), the lateral hypothalamic area (LHA), or the ventral premammillary nucleus (PMV). At the same time, the jugular vein was cannulated for blood sampling, and lesion of the anterior Pe was performed with a cathodal current. One and one half hours before the first blood sampling the rats were anesthetized with pentobarbital to prevent spontaneous GH bursts. One minute after the first blood sampling, 10 micrograms hpGRF dissolved in 0.3 ml saline was injected iv through the cannula. The blood samples were collected at 10, 20, 30, and 60 min after the zero-time sample. The above mentioned hypothalamic nuclei were electrically stimulated for the first 10 min. Injection of hpGRF increased the plasma GH level from 44.2 +/- 7.3 ng/ml (mean +/- SE) to 981.4 +/- 59.8 ng/ml in 10 min, and the plasma GH level gradually decreased to 50.8 +/- 8.8 ng/ml by 60 min. The hpGRF-induced GH release was most effectively suppressed by the stimulation of the Pe. After the stimulation of the OVLT, the VMH, the LHA, or the PMV, the suppression of hpGRF-induced GH release was weaker than that of the Pe. This stimulation-induced suppression was fully or partly prevented in Pe-lesioned animals. These results indicate that the stimulation of the Pe, the OVLT, the VMH, the LHA, or the PMV exerts an inhibitory effect on GH release. For this inhibitory effect, the Pe was essential. The results are discussed with regard to the topography of immunoreactive SRIF neuronal cell bodies and processes in the hypothalamus.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Calcium tolerance of isolated rat heart cells

Freshly isolated adult rat heart cells, which initially show the elongated, rod-shaped morphology typical of heart cells in situ, are almost quantitatively converted to rounded contracture forms by exposure to 1 m Ca²⁺. These Ca²⁺-sensitive cells became Ca²⁺-tolerant following a short period of metabolic activity in a low-Ca²⁺ medium, in that they retain their rod-shaped configuration when challenged with Ca²⁺ after this preincubation step. Tolerance to Ca²⁺ develops in parallel with the establishment of low Na⁺/K⁺ ratios in these cells and both processes are sensitive to ouabain. The initial net uptake of Ca²⁺ is greater in Ca²⁺-sensitive than in Ca²⁺-tolerant cells. These results suggest that contracture in the Ca²⁺-sensitive cells is a consequence of the rapid entry of excessive amounts of Ca²⁺ in exchange for internal Na⁺.     

Nitric oxide and hydroperoxide affect islet hormone release and Ca(2+) efflux

We have investigated the influence of the intracellular free radical donors hydroxylamine (giving nitric oxide [NO]) and tert-butylhydroperoxide (giving hydroperoxide [“H₂O₂”]) on glucose- and cyclic adenosine monophosphate (cAMP)-induced transduction signaling in islet hormone release. Both donors dose dependently inhibited glucose-stimulated insulin release and induced modest (hydroxylamine) or profound (tert-butylhydroperoxide) suppression of ⁴⁵Ca²⁺-efflux from perifused islets. By contrast, both donors stimulated glucagon release. Similar effects on hormone release were displayed after K⁺-depolarization. Insulin and glucagon release stimulated by activation of the cAMP system through isobutylmethylxanthine (IBMX) at basal glucose was modestly potentiated by low concentrations of both donors. These effects were still observed, although less pronounced, in K⁺-depolarized islets. In vitro as well as in vivo, the NO-synthase inhibitor N^G-nitro-L-arginine methyl ester inhibited IBMX-induced glucagon release, but did not affect insulin release. The results suggest that NO and hydroperoxide inhibit glucose-stimulated insulin release by perturbing Ca²⁺ fluxes and probably acting through S-nitrosylation (NO) or oxidation (hydroperoxide) of thiol groups critical to the secretory process. These effects are largely independent of depolarization events. By contrast, both NO and hydroperoxide can potentiate cAMP-stimulated hormone release presumably at a distal site in the stimulus-secretion coupling.     

The effects of thyroid hormone deprivation in vivo and in vitro on growth hormone (GH) responses to human pancreatic (tumor) GH-releasing factor (1-40) by dispersed rat anterior pituitary cells

Anterior pituitary cells from euthyroid and hypothyroid male rats have been cultured as monolayers for 3 days with or without 5 nM T3 and stimulated with either human pancreatic GH-releasing factor 1-40 (hpGRF), TRH, or the Ca2+ channel ionophore A23187. Basal GH secretion was reduced in the hypothyroid cultures (P less than 0.001) and basal TSH secretion increased (P less than 0.001). Culture with T3 increased GH secretion and intracellular GH content in euthyroid and hypothyroid cultures but suppressed TSH secretion with no effect on intracellular TSH content in either euthyroid or hypothyroid cultures. hpGRF released more GH from euthyroid [3.52 +/- 0.2 (SE) micrograms/6 h X 10(5) cells] than hypothyroid cultures of (0.17 +/- 0.01 micrograms/6 h X 10(5) cells, P less than 0.001) without a change in ED50 (approximately 0.02 nM). The reduction in hpGRF-induced GH release remained significant when corrected for the reduced intracellular GH content in the hypothyroid cultures. hpGRF-induced GH release also declined relative to A23187-induced GH release in hypothyroid cultures. Culture with 5 nM T3 doubled maximum hpGRF-induced GH release in euthyroid cultures and increased maximum release 10-fold in hypothyroid cultures without altering the ED50 of hpGRF action. In contrast, T3 suppressed TRH-induced TSH release in euthyroid cultures but was without effect on TRH-induced TSH release in the hypothyroid cultures. T3 did not effect the ED50 of TRH action (2-5 nM). In summary, hypothyroid rat anterior pituitary cells in culture have a reduced maximal GH response to hpGRF, but the same ED50. hpGRF activity can be partially restored by physiological concentrations of T3 in vitro.     

Influence of interleukin-2 on Ca2+ handling in rat ventricular myocytes

In the present study, we examined the effect of interleukin-2 (IL-2) on cardiomyocyte Ca(2+) handling. The effects of steady-state and transient changes in stimulation frequency on the intracellular Ca(2+) transient were investigated in isolated ventricular myocytes by spectrofluorometry. In the steady state (0.2 Hz) IL-2 (200 U/ml) decreased the amplitude of Ca(2+) transients induced by electrical stimulation and caffeine. At 1.25 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), when the stimulation frequency increased from 0.2 to 1.0 Hz, diastolic Ca(2+) level and peak intracellular Ca(2+) concentration ([Ca(2+)](i)), as well as the amplitude of the transient, increased. The positive frequency relationships of the peak and amplitude of [Ca(2+)](i) transients were blunted in the IL-2-treated myocytes. The effect of IL-2 on the electrically induced [Ca(2+)](i) transient was not normalized by increasing [Ca(2+)](o) to 2.5 mM. IL-2 inhibited the frequency relationship of caffeine-induced Ca(2+) release. Blockade of sarcoplasmic reticulum (SR) Ca(2+)-ATPase with thapsigargin resulted in a significant reduction of the amplitude-frequency relationship of the transient similar to that induced by IL-2. The restitutions were not different between control and IL-2 groups at 1.25 mM [Ca(2+)](o), which was slowed in IL-2-treated myocytes when [Ca(2+)](o) was increased to 2.5 mM. There was no difference in the recirculation fraction (RF) between control and IL-2-treated myocytes at both 1.25 and 2.5 mM [Ca(2+)](o). The effects of IL-2 on frequency relationship, restitution, and RF may be due to depressed SR functions and an increased Na(+)-Ca(2+) exchange activity, but not to any change in L-type Ca(2+) channels.     

Involvement of the cAMP messenger system and extracellular Ca(2+) during hyposmotically-induced prolactin release in the Mozambique tilapia

In accord with its role in freshwater osmoregulation, prolactin (PRL) release from the tilapia pituitary is stimulated by small, physiologically relevant reductions in plasma osmolality, a response that is mediated by an acute influx of intracellular Ca²⁺ through stretch-activated Ca²⁺channels. In the present study, the role of the calcium and cyclic AMP (cAMP) messenger system in the transduction of a response to a hyposmotic stimulus was examined using dispersed PRL cells and PRL cell membrane preparations from freshwater-acclimated tilapia. When PRL cells were treated with the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (100 μM), significant increases in cAMP levels and PRL release were observed at 1 h. Exposure to reduced medium osmolality (300 mOsmolal) in the presence of IBMX further augmented PRL release. Depletion of Ca²⁺ from the incubation medium blocked PRL release even in the presence of IBMX. By contrast, exposure of PRL cells to cholera toxin (CTX), an activator of adenylyl cyclase (AC), stimulated PRL release and cAMP accumulation in both the presence and absence of extracellular Ca²⁺. On the other hand, treatment with the Ca²⁺ ionophore A23187, which elicits a large rise in intracellular free Ca²⁺, reduced cAMP accumulation. Likewise, the AC activity of a PRL cell membrane preparation was reduced as extracellular Ca²⁺ concentration increased from 0.1 to 1 μM. These results indicate that: (1) the stimulation of PRL release and cAMP formation by a fall in extracellular osmolality are Ca²⁺-dependent; (2) large increases in intracellular Ca²⁺ attenuate cAMP formation; (3) direct agonists of cAMP messenger system, such as cholera toxin, however, stimulate PRL release independently of the extracellular Ca²⁺. These findings add to the evidence that the osmosensitive response of the tilapia PRL cell is mediated through a Ca²⁺-dependent mechanism. Nevertheless, the present findings also suggest that tilapia PRL cells have the ability to rapidly augment release PRL both via a Ca²⁺-dependent manner and via a cAMP-dependent pathway in the absence of extracellular Ca²⁺.     

Regulation of growth hormone (GH) secretion by GH-releasing factor, somatostatin, and insulin-like growth factor I in ovine fetal and neonatal pituitary cells in vitro

Serum GH concentrations in the ovine fetus are much higher than those in the neonate, and the maximal GH response induced by GRF is 5-fold greater in the fetus than in the neonate. To clarify these in vivo observations further, we studied the effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on primary cultures of fetal and neonatal ovine pituitary cells. GH secretion from fetal ovine pituitary cells increased from 148 +/- 34 to 950 +/- 130 ng/10(5) cells.3 h in response to 1 nM GRF, whereas GH secretion from neonatal pituitary cells rose from 113 +/- 26 to 1221 +/- 129 ng/10(5) cells.3 h, a significantly greater response (P less than 0.001). This greater GRF-induced GH response in neonatal than fetal cells differs from the response in vivo and suggests that the increased in vivo response in the fetus is not due to inherently increased sensitivity of pituitary cells to GRF. SRIF (10 nM) decreased maximal GRF-induced GH secretion by 37 +/- 3% in fetal cells compared with 59 +/- 8% in neonatal cells (P less than 0.01). This may explain in part the decreased in vivo sensitivity to SRIF in the ovine fetus compared to that in the neonatal lamb. In fetal pituitary cells, 10 nM GRF increased ovine (o) GH mRNA from 100 +/- 14% to 145 +/- 40%, SRIF decreased oGH mRNA to 84 +/- 3%, and GRF and SRIF in combination increased fetal oGH mRNA to 126 +/- 24%. Values in neonatal pituitary cell cultures were similar (control, 100 +/- 17%; GRF, 132 +/- 6%, SRIF, 85 +/- 15%; GRF plus SRIF, 105 +/- 26%). Pretreating fetal cells with 100 nM IGF-I for 3 days reduced GRF-stimulated GH secretion from 1049 +/- 38 to 232 +/- 8 ng/10(5) cells.3 h (P less than 0.001). Similarly, IGF-I pretreatment of neonatal cells reduced GRF-stimulated GH secretion from 810 +/- 18 to 419 +/- 16 ng/10(5) cells.3 h (P less than 0.001). The mean secreted IGF-I was 0.58 U/ml (36 nM) in culture medium from neonatal cells and was unchanged by incubation for 3 days with 5 micrograms/ml hGH. Secreted IGF-I in medium from fetal cells was 0.87 U/ml (54 nM) without GH and 0.81 U/ml (51 nM) after incubation with human GH. IGF-I mRNA was present in neonatal pituitary and brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Somatostatin-secreting islet cell tumor (somatostatinoma): suppression of growth hormone (GH) release induced by GH-releasing hormone

A patient with a somatostatin (SRIH)-secreting islet cell tumor, whose only symptoms were dyspepsia and anemia, is described. The diagnosis of somatostatinoma was based on high plasma SRIH concentrations and immunocytochemical findings. The pancreatic exocrine response to secretin was decreased, whereas the insulin and/or glucagon responses to glucose and arginine were normal. Although the basal plasma GH concentration was normal, the plasma GH response to GHRH was subnormal. Gel permeation chromatography studies indicated that SRIH-14 was the predominant form of SRIH in plasma as well as in tumor tissue.