The inhibition of growth hormone release by gastrin-releasing peptide involves somatostatin release

Injection of gastrin-releasing peptide-27 (GRP) into the third ventricle (IVT) has been shown previously to lower plasma GH levels and block the GH release induced by GRF, suggesting that GRP might act via stimulation of the release of somatostatin (SRIF) into hypophysial portal vessels. Several experiments were performed to test this hypothesis. In the first experiment rat median eminence (ME) fragments were incubated in medium containing concentrations of GRP ranging from 1 pM to 1 microM, and SRIF levels were measured after the 30-min incubation period. GRP significantly stimulated SRIF release at doses of 0.1 nM to 1 microM. Microinjection of SRIF antiserum (3 microliters) IVT prevented GRP (2 micrograms, IVT) from inhibiting the GH surge induced by GRF (1 microgram/kg, iv). A slight but significant decrease in basal plasma GH levels was observed after GRP administration even in the presence of SRIF antiserum. Finally, to rule out a GRP-GRF interaction at the pituitary level, tubes containing dispersed rat pituitary cells (2.5 x 10(5) cells/tube) were incubated for 1.5 h in medium containing various concentrations of GRF (0.4-40 nM) alone or with 0.1 microM GRP. The addition of GRP to the medium had no significant effect on the dose-dependent stimulation of GH release by GRF. The results of these studies demonstrate that GRP can directly stimulate SRIF release in vitro. They further suggest that SRIF is a component of the mechanism whereby GRP inhibits GH release in vivo. Finally, the possibility that GRP acts at the pituitary level to inhibit GH release by blocking GRF receptors on somatotrophs has been ruled out.     

Altered pituitary growth hormone (GH) regulation in streptozotocin-diabetic rats: a combined defect of hypothalamic somatostatin and GH-releasing factor

Diabetes mellitus in the rat is associated with loss of pulsatile GH secretion. An interplay between hypothalamic GH-releasing factor (GRF) and inhibitory factor [somatostatin (SRIF)] secretion is thought to account for episodic pituitary GH release. An increase in SRIF tone/action or a decrease in GRF release/response in diabetic rats could account for the suppressed GH levels. Pituitaries from streptozotocin-diabetic rats contained less GH than controls (15.9 +/- 2.5 vs. 29.5 +/- 4.6 micrograms/mg; P less than 0.05) despite normal somatotrope representation, as demonstrated using immunofluorescence studies. Basal GH secretion from monolayer culture of dispersed anterior pituitary (AP) cells from diabetic rats was proportionately decreased (150 +/- 10 vs. 103 +/- 10 ng/10(5) cells; P less than 0.005). GRF (10(-11)-10(-8) M)-induced release of GH from AP cells was decreased in diabetic rats (maximum response to 10(-8) M GRF, 401 +/- 60 vs. 618 +/- 41 ng/10(5) cells; P less than 0.01); however, sensitivity to GRF was unchanged (EC50, 79 +/- 41 vs. 128 +/- 67 pM). By contrast, SRIF (10(-7)-10(-10)-induced inhibition of GRF (10(-8) M)-mediated GH release was impaired in AP cells of diabetic rats compared to that in controls (IC50, 112 +/- 33 vs. 55 +/- 31 pM; P less than 0.05) associated with a decrease in AP plasma membrane SRIF receptor concentration (63.4 +/- 15.6 vs. 160.3 +/- 13.7 fmol/mg protein; P less than 0.05), with no change in affinity. These findings are consistent with chronic exposure to increased hypothalamic SRIF influence. GH synthesis has been shown to be independent of SRIF regulation; however, insulin-like growth factor-I and GRF inhibit and stimulate GH synthesis, respectively. In diabetic rats insulin-like growth factor-I levels were decreased, appropriate to low GH status, in serum (290 +/- 66 vs. 1662 +/- 92 ng/ml; P less than 0.001) and hypothalamus (6.8 +/- 1.0 vs. 13.0 +/- 0.4 pg/mg wet wt; P less than 0.001) and, thus, did not seem to account for the low AP GH content. Hypothalamic GRF content in diabetic rats (1.11 +/- 0.10 ng/hypothalamus) did not differ from that in controls (1.16 +/- 0.17 ng/hypothalamus). GRF mRNA levels, however, were reduced by 80% in diabetic rats compared to controls. Taken together these data support a combined role for decreased hypothalamic GRF and increased SRIF in mediating alterations of GH synthesis and secretion in streptozotocin-induced diabetes.     

Abnormal vascular responses in human chronic cardiac failure are both endothelium dependent and endothelium independent

Objective—To study underlying vascular responses in chronic heart failure in patients without ACE inhibitor treatment, and to compare them with age matched controls.
Design—Forearm blood flow was studied using venous occlusion plethysmography in patients with chronic heart failure (n = 12) and matched controls (n = 13), after infusion of L-NMMA (a nitric oxide synthase inhibitor), glyceryl trinitrate (an endothelium independent vasodilator), and serotonin (an endothelium dependent vasodilator).
Results—L-NMMA produced significant vasoconstriction in normal subjects (forearm blood flow reduced by 24%), but not in patients (6%; difference between groups p < 0.03). The vasodilator responses to glyceryl trinitrate were impaired in patients (p < 0.02). In normal controls, serotonin produced initial dilatation, followed by vasoconstriction at high doses. In patients, no vasodilator responses were observed, only late vasoconstriction (p < 0.03).
Conclusions—The vascular responses of patients are confirmed as being abnormal. The lack of response to L-NMMA suggests that nitric oxide does not contribute to basal vascular tone in patients with chronic heart failure. The responses to glyceryl trinitrate and to serotonin suggest that there is both smooth muscle and endothelial dysfunction in patients with chronic heart failure.

Keywords: heart failure;  endothelium;  nitrates;  serotonin     

Effect of somatostatin withdrawal and growth hormone (GH)-releasing factor on GH release in vitro: amount available for release after disinhibition

The secretion of GH is strikingly episodic. We have suggested that the timing of the episodic bursts of GH secretion is set by somatostatin (SRIF) withdrawal, whereas the magnitude of the bursts is determined by the amount of GH-releasing factor (GRF) impinging on the somatotrophs before and during SRIF withdrawal. We have now used an in vitro model of perifused rat pars distalis cells to examine the interaction of SRIF and GRF on GH release and, in particular, to examine the effect of GRF on the magnitude of the burst of GH release that follows SRIF withdrawal. After 30 min of perifusion with SRIF (10(-9) M), there follows an immediate but small burst of GH release. The burst of GH release following concurrent perifusion with SRIF plus GRF (10(-10) M) is increased, with a 7.5- to 9.5-fold increase in the peak secretion rate. When GRF is maintained after the withdrawal of SRIF, the peak secretion rate is not different from that seen after simple withdrawal of both SRIF and GRF, but the duration of the burst is increased. These data demonstrate that the presence of GRF during SRIF perifusion, while not altering basal release, does strikingly increase the post-SRIF release of GH. We propose that a similar relation applies in vivo, where SRIF withdrawal sets the timing of the episodic bursts of GH release, whereas GRF determines the magnitude.     

Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone.

Augmentation of vertebrate growth by growth hormone (GH) is primarily due to its regulation of insulin-like growth factor I (IGF I) and IGF II levels. To characterize the effect of GH on the levels of IGF I and IGF II mRNA in a teleost, 10 micrograms of bovine GH (bGH) per g of body weight was administered to juvenile rainbow trout (Oncorhynchus mykiss) through i.p. injection. The levels of IGF I and IGF II mRNA were determined simultaneously, by using RNase protection assays, in the liver, pyloric ceca, kidney, and gill at 0, 1, 3, 6, 12, 24, 48, and 72 hr after injection. In the liver, IGF I mRNA levels were significantly elevated at 6 and 12 hr (approximately 2- to 3-fold, P < or = 0.01), while IGF II mRNA levels were significantly elevated at 3 and 6 hr (approximately 3-fold, P < or = 0.01). In the pyloric ceca, IGF II mRNA levels were significantly elevated at 12, 24, and 48 hr (approximately 3-fold, P < or = 0.01), while IGF I mRNA was below the limits of assay accuracy. GH-dependent IGF mRNA appearance was not detected in the gill and kidney. Serum bGH levels, determined by using a radioimmunoassay, were significantly elevated at 3 and 6 hr (P < 0.005). In primary hepatocyte culture, IGF I and IGF II mRNA levels increased in a bGH dose-dependent fashion, with ED50 values of approximately 45 and approximately 6 ng of bGH per ml, respectively. The GH-dependent appearance of IGF II mRNA in the liver and pyloric ceca suggests important roles for this peptide hormone exclusive of IGF I.     

Bradykinin induced dilatation of human epicardial and resistance coronary arteries in vivo: effect of inhibition of nitric oxide synthesis

Objective—To clarify whether endothelium derived nitric oxide contributes to exogenous bradykinin induced dilatation of human epicardial and resistance coronary arteries in vivo.
Design—Quantitative coronary angiography and Doppler flow velocity measurements were used to determine the effects of the nitric oxide synthesis inhibitor, N^G-monomethyl-L-arginine (L-NMMA), on bradykinin induced dilatation of the epicardial and resistance coronary arteries.
Setting—Hiroshima University Hospital.
Patients—20 patients (16 men and four women, mean (SD) age 56 (9) years) with angiographically normal smooth epicardial coronary arteries.
Interventions—Serial infusions of bradykinin (0.5, 1.5, and 2.5 µg/min) were given into the left coronary ostium before and after L-NMMA infusion (60 µmol/min).
Main outcome measures—Epicardial coronary diameter, coronary blood flow, and coronary vascular resistance.
Results—Bradykinin-induced epicardial coronary vasodilatation after L-NMMA (dilatation by 2.5 µg/min, 3.8(1.4)% in the proximal and 5.9(1.8)% in the distal segments, mean (SEM)) was less (p < 0.001, respectively) than before L-NMMA (11.7(2.5)% and 15.1(2.0)%, respectively). In contrast, L-NMMA did not affect the bradykinin induced increase in coronary blood flow and decrease in coronary vascular resistance.
Conclusions—Endothelium derived nitric oxide contributes to bradykinin induced dilatation of epicardial coronary arteries, but may be less important in coronary resistance vasodilatation.

Keywords: bradykinin;  nitric oxide;  coronary artery;  coronary blood flow     

Inhibition of hypothalamic somatostatin release by beta-adrenergic antagonists

A number of in vivo studies suggest that hypothalamic somatostatin (SRIF) tone is stimulated by the beta-adrenergic system. Employing dispersed adult male rat hypothalamic cells, we studied the effects of beta-adrenergic antagonists on the release of hypothalamic SRIF. Propranolol, at concentrations of 1-100 microM, had no detectable effect on basal SRIF release, but caused dose-dependent inhibition of SRIF release stimulated by ouabain. Two other beta-adrenergic antagonists, labetolol and metoprolol, also caused inhibition of ouabain-stimulated SRIF release. The alpha 2-agonist clonidine was without effect on SRIF release under basal or stimulated conditions. GH secretion from monolayers of dispersed rat anterior pituitary cells was also examined. Propranolol (1-100 microM) had no significant effect on basal GH secretion or GH secretion stimulated by rat GRF. In conclusion, 1) beta-adrenergic antagonists caused inhibition of stimulated SRIF release; 2) clonidine had no detectable effect on SRIF release; and 3) propranolol did not affect GH secretion in vitro. These findings support the hypothesis that beta-adrenergic antagonists augment GH responsivity by inhibiting hypothalamic SRIF release.     

A hypothalamic hormone-somatostatin--from endocrinology to neurophysiology]

The densest distribution of somatostatin (SRIF) neuron perikarya is localized in the hypothalamic periventricular nucleus (Pe) close to the third ventricle, from which many fibers are projected to the median eminence. The release of SRIF in the neurohemal organ into the anterior pituitary modulates GH secretion from pituitary somatotrophs. When SRIF input from the hypothalamus to rat anterior pituitary is reduced by either neurosurgery or SRIF antiserum iv injection, the responsiveness of the pituitaries to human GH releasing factor (hGRF) in an in vitro perifusion system is markedly attenuated. Moreover, SRIF pretreatment facilitates the GH release response of dispersed anterior pituitary cells to hGRF. The long lasting SRIF effect to sensitize somatotrophs appears to take place beyond cAMP formation or as an unknown distal effect. These findings indicate that SRIF neurons in the Pe play a role in maintaining the pituitary responsiveness to GRF in addition to the original action to inhibit GH secretion. Neuronal networks between Pe-SRIF neurons, and intra- and extrahypothalamic nuclei are identified by Pe stimulation test on GRF-GH secretion. In addition to the physiological role in maintaining pituitary responsiveness, Pe SRIF neurons have a wide influence on specific SRIF receptor binding in various brain regions as well as in the anterior pituitary. Shortly after lesioning the Pe neurons, there is a continuous increase in plasma GH level with a transient increase in specific binding of 125I-Tyr 11-SRIF-14 to the anterior pituitary. Furthermore, there is a similar but a little longer increase in binding of the radioligand to some brain areas such as the cerebral cortex, hippocampus, and amygdala nuclei. However, neuronal connections between the SRIF neurons and nuclei which are up-regulated by the lesioning have not been fully proven. When the labeled ligand is infused into the lateral ventricle, it is rapidly and widely distributed in many periventricular structures in the lateral and third ventricles. These findings suggest that SRIF produced in the Pe neurons is transported to other brain areas via cerebrospinal fluid in addition to neuronal connections for modulating the activity of neurons which have SRIF receptors. Thus, hypothalamic Pe SRIF neurons have dualistic roles for controlling anterior pituitary function and modulating CNS neuron activity.     

The interrelationship between the effects of somatostatin and human pancreatic growth hormone-releasing factor on growth hormone release by cultured pituitary tumor cells from patients with acromegaly

The effects of somatostatin (SRIF) and human pancreatic tumor GRF on GH release by cultured pituitary tumor cells obtained during transsphenoidal operation from 15 acromegalic patients were investigated. In a study of the sensitivity of pathological GH release to SRIF, 1-10 nM SRIF induced maximal inhibition of hormone release in 3 consecutive tumors. In 12 of 15 tumor cell cultures, 10 nM SRIF produced statistically significant inhibition of basal GH release by 39 +/- 3% (mean +/- SEM). In 2 of the 3 other tumors, SRIF inhibited GRF-stimulated GH release, while this was not investigated in the third tumor. A dose-response study of the effect of GRF on GH release by cultured pituitary tumor cells showed that doses of 0.1, 1, 10, and 100 nM induced similar maximal (35%) stimulation of hormone secretion. In four of five consecutive tumor cell suspensions, 1 and 10 nM GRF induced statistically significant GH stimulation by 18-300%. Preincubation of the tumor cells with 5 nM dexamethasone greatly increased the sensitivity and the maximal stimulation in response to GRF and made one tumor cell suspension, which did not react to GRF initially, sensitive to GRF. In the tumors of four patients, the interrelationship between the effects of SRIF and GRF on GH release were also studied. SRIF (10 nM) inhibited the stimulatory effects of GRF on GH release virtually completely. In conclusion, GH release by in vitro cell cultures of GH-secreting pituitary adenomas was inhibited by SRIF and stimulated by GRF. The interaction of GRF and SRIF on GH release by these pituitary tumor cells was similar to that in normal rat GH cells, as SRIF virtually completely overcame the GRF-induced GH release.     

Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormone-releasing factor in the rat

Possible inhibitory effects of somatostatin (SRIF) on GRF were studied by assessing spontaneous GH secretion and GRF content and release in adult male rats depleted of hypothalamic SRIF by anterolateral hypothalamic deafferentation (AHD) or electrolytic lesions in the medial preoptic area (MPO). Plasma GH levels were measured 7 days postoperatively every 20 min in conscious animals with indwelling iv cannulae. Median eminence SRIF was markedly reduced 8 days postoperatively in both AHD and MPO rats, as determined by immunohistochemistry and RIA (P less than 0.01). Although GRF immunoreactivity in the median eminence of AHD and MPO animals appeared well preserved immunocytochemically, hypothalamic GRF content by RIA was significantly decreased at 8 days (P less than 0.01). Spontaneous GH secretion was pulsatile in sham-operated animals. In contrast, basal GH levels in AHD and MPO animals were markedly elevated (P less than 0.01), and secretory pulses were absent. Intravenous injection of specific anti-GRF serum into MPO animals decreased the elevated plasma GH levels (P less than 0.01), indicating increased hypothalamic GRF secretion. GRF release from hypothalamic median eminence-arcuate nucleus complexes in vitro was significantly greater in AHD and MPO animals than in control animals 4 and 8 days postoperatively in response to 30 mM K+ (P less than 0.01), but not under basal conditions. These results suggest that hypothalamic medial preoptic area somatostatinergic neurons play a tonic inhibitory role in the regulation of GRF release and that GH hypersecretion observed after MPO and AHD is attributable to changes in both SRIF and GRF.     

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)     

Intracellular calcium concentration and growth hormone secretion in individual somatotropes: effects of growth hormone-releasing factor and somatostatin

The cytosolic free calcium concentration and cumulative GH release were measured simultaneously in normal pituitary cells. This was made possible by a novel combination of fluorescence microscopy using the calcium indicator fura-2 and a reverse hemolytic plaque assay. GRF (10 nM) rapidly increased the intracellular free calcium concentration ([ Ca2+]i) from a basal level of 234 +/- 17 nM (mean +/- SE) to a peak value of 480 +/- 61 nM 1 min after stimulation. This GRF-induced calcium rise was totally abolished in calcium-free medium or in the presence of calcium channel blockers cobalt chloride (2 mM) and verapamil (100 microM). When somatostatin (SRIF; 1 nM) was added after basal recordings, cytosolic calcium decreased to 96 +/- 23 nM in identified somatotropes. [Ca2+]i returned to baseline upon the removal of SRIF inhibition. This rebound was higher when a sequential treatment of SRIF followed by GRF was applied. Exposing cells to a combination of GRF (10 nM) plus SRIF (1 nM) resulted in a decrease in [Ca2+]i identical to that caused by SRIF treatment alone. Despite the 10-fold excess of GRF, SRIF not only inhibited hormone secretion, but also totally overcame the GRF-induced rise of [Ca2+]i. In summary, stimulation by GRF increases cytosolic calcium in normal somatotropes. This increase is proposed to be due to the influx of calcium through membrane ion channels. In contrast, SRIF decreases [Ca2+]i. This might explain the cAMP-independent effects of this peptide. The effect of SRIF dominates over that of GRF with respect to both changes in [Ca2+]i and hormone release. Changes in the GH secretory rate are, therefore, accompanied by parallel changes in [Ca2+]i, both of which are primarily regulated by SRIF.     

Cysteamine-induced enhancement of growth hormone-releasing factor (GRF) immunoreactivity in arcuate neurons: morphological evidence for putative somatostatin/GRF interactions within hypothalamus

The episodic secretion of GH is regulated through the reciprocal release into the hypophyseal portal circulation of two hypothalamic peptides, SRIF and GRF. Recent physiological evidence suggests that, in addition to exerting their well documented opposite actions at the level of the anterior pituitary, SRIF and GRF may interact within the central nervous system to modulate GH secretion. The aim of the present study was to provide morphological evidence to support the concept that SRIF exerts an influence on the GRF-containing neuronal system within the hypothalamus. To accomplish this, we used the thiol agent cysteamine (CSH; 300 mg/kg sc) to manipulate endogenous hypothalamic SRIF and examined its effects on the immunoreactivity and mRNA content of GRF-containing arcuate (ARC) neurons, using immunohistochemistry and semi-quantitative in situ hybridization, respectively. Hypothalamic SRIF content and pulsatile GH secretion were also monitored. CSH treatment reduced hypothalamic immunoreactive SRIF concentrations (to 60% of vehicle-injected control values) and severely suppressed the spontaneous surges of GH release (mean GH peak amplitude: 24.2 +/- 2.7 vs. 168.0 +/- 27.8 ng/ml in H2O-injected controls; P less than 0.001). CSH treatment also resulted in a striking increase in the number and labeling density of GRF-immunoreactive cells detected in the ARC nucleus. Cell counts revealed an overall 126% increase, over controls, in the mean number of detectable GRF-positive ARC neurons. This increase (P less than 0.01) was apparent over all rostrocaudal levels of the ARC nucleus, but was most pronounced within the caudal tier. Administration of CSH also produced a massive qualitative increase in the intensity and extent of staining of GRF-positive terminal elements in the external zone of the median eminence. Conversely, CSH treatment resulted in a decline in GRF mRNA-associated signal in cells of the ARC nucleus, with this decline being most evident in the caudal third of the nucleus. The finding of an increase in GRF immunoreactivity in ARC neurons in association with a decrease in plasma GH, as well as with a decline in relative GRF mRNA content, suggests that CSH leads to an overall decrease in GRF release from the median eminence. These results provide morphological evidence to support the concept of a SRIF-mediated central influence on the GRF-containing ARC neuronal system. Such central regulation of GRF by SRIF may be an important mechanism in the physiological control of pulsatile GH secretion.     

Cyclic GMP release by acute enhanced external counterpulsation

BACKGROUND: Enhanced external counterpulsation (EECP) is a noninvasive, pneumatic technique that provides favorable effects in patients with coronary artery disease and heart failure. The mechanisms by which EECP exerts its beneficial effects remain poorly understood. Cyclic GMP (cGMP) regulates vascular smooth muscle tone that may improve arterial function. We investigated the effect of a single session of EECP on plasma and platelet cGMP in asymptomatic subjects with cardiovascular risk factors (HCVR) and in patients with coronary artery disease (CAD). METHODS: Fifty-five subjects were included (25 HCVR and 30 CAD) and randomized into two groups to receive either sham (control) or active EECP during 1 h. Plasma and platelet cGMP were measured immediately before and after EECP by radioimmunoassay. RESULTS: One hour of EECP increased cGMP plasma concentration by 52% +/- 66% (SD) (P < .001) and platelet content by 19% +/- 28% (P < .01). The increase in plasma cGMP was particularly marked in CAD patients receiving active EECP (P < .01), mainly in those with low LDL-cholesterol. Platelets, inhibition of nitric oxide synthesis by N(G)-monomethyl-l-arginine (L-NMMA) reduced cGMP by 23% +/- 31% (P < .001), whereas presence of superoxide dismutase and inhibition of phosphodiesterase-5 increased cGMP by 46% +/- 49% and 70% +/- 77%, respectively (P < .001). In all of the cases EECP increased additional platelet cGMP content, which suggests nitric oxide synthase activation. CONCLUSIONS: Acute external counterpulsation showed that very early treatment increases the cGMP production that may participate in the mechanism by which EECP exerts its clinical benefit. Analysis of the modulation of platelet cGMP content suggests that EECP activated the nitric oxide-dependent pathways.     

Thyrotrophin-releasing hormone-induced growth hormone (GH) secretion in anaesthetized chickens: inhibition by GH-releasing factor at central sites.

Intracerebroventricular (i.c.v.) administration of GH-releasing factor (GRF) (at 1 or 10 micrograms) to anaesthetized immature (6- to 8-weeks-old) or adult (greater than 24-weeks-old) domestic fowl had no effect on basal GH concentrations in peripheral plasma, but suppressed (after 20 min) the acute GH response to exogenous (i.v.) thyrotrophin-releasing hormone (TRH) (1 micrograms/kg). The i.c.v. injection of GRF also reduced the content of somatostatin (SRIF) and dopamine (DA) in the hypothalamus, while increasing the concentration of the DA metabolite 3,4-dihydroxyphenyl acetic acid (DOPAC) and the DOPAC/DA ratio. The release of SRIF from hypothalamic tissue was stimulated in vitro by 100 nmol GRF/l. The inhibitory effect of i.c.v. GRF on TRH-induced GH secretion was blocked when it was simultaneously injected i.c.v. with SRIF antiserum. These results demonstrate central effects of GRF on avian hypothalamic function and suggest an inhibitory role for this peptide in GH regulation, possibly mediated through increased SRIF secretion.     

Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite

OBJECTIVES: This study examined the effects of endotoxin on cardiac contractility in human myocardium. BACKGROUND: In animal myocardium, endotoxin and cytokine treatment led to enhanced inducible nitric oxide synthase (iNOS) expression and contractile dysfunction. Effects in human myocardium are unknown. METHODS: Left ventricular myocardial preparations from failing (n = 18) and nonfailing (n = 5) human hearts were incubated for 6 and 12 h in tyrode solution or in tyrode plus lipopolysaccharides (LPS), with LPS plus N(G)-mono-methyl-L-arginine (L-NMMA), with LPS plus hemoglobin or with LPS plus the superoxide scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). Force of contraction in response to isoprenaline (0.001 to 3 micromol/liter) was determined in electrically stimulated muscle preparations. The iNOS mRNA expression was examined by in situ hybridization and by polymerase chain reaction. The cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay. RESULTS: Isoprenaline concentration dependently increased force of contraction. Six and 12 hours of LPS treatment of failing myocardium decreased maximum inotropic response to isoprenaline by 54% (p = 0.009) and by 69% (p = 0.0023), respectively. In nonfailing myocardium, 12 h of LPS treatment decreased maximum inotropic effect of isoprenaline by 66% (p < 0.001). The LPS effects were attenuated by L-NMMA, hemoglobin and also Tiron. The iNOS mRNA was expressed in all LPS-treated preparations but also in most control myocardial preparations. In situ hybridization revealed iNOS expression within cardiac myocytes. There was no increase in myocardial cGMP content in response to endotoxin. CONCLUSIONS: Endotoxin exposure of human myocardium leads to a depression of cardiac contractility, which is mediated by enhanced iNOS activity and release of nitric oxide (NO). Consecutive reaction of NO with superoxide and formation of peroxynitrite may contribute to the decrease in force of contraction.     

Magnesium causes nitric oxide independent coronary artery vasodilation in humans

OBJECTIVE—To determine how magnesium affects human coronary arteries and whether endothelium derived nitric oxide (EDNO) is involved in the coronary arterial response to magnesium.
DESIGN—Quantitative coronary angiography and Doppler flow velocity measurements were used to determine the effects of the nitric oxide synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA) on magnesium induced dilation of the epicardial and resistance coronary arteries.
SETTING—Hiroshima University Hospital a tertiary cardiology centre.
PATIENTS—17 patients with angiographically normal coronary arteries.
INTERVENTIONS—Magnesium sulfate (MgSO₄) (0.02 mmol/min and 0.2 mmol/min) was infused for two minutes into the left coronary ostium before and after intracoronary infusion of L-NMMA.
MAIN OUTCOME MEASURES—Diameter of the proximal and distal segments of the epicardial coronary arteries and coronary blood flow.
RESULTS—At a dose of 0.02 mmol/min, MgSO₄ did not affect the coronary arteries. At a dose of 0.2 mmol/min, MgSO₄ caused coronary artery dilation (mean (SEM) proximal diameter 3.00 (0.09) to 3.11 (0.09) mm; distal 1.64 (0.06) to 1.77 (0.07) mm) and increased coronary blood flow (79.3 (7.5) to 101.4 (9.9) ml/min, p < 0.001 v baseline for all). MgSO₄ increased the changes in these parameters after the infusion of L-NMMA (p < 0.001 v baseline).
CONCLUSIONS—Magnesium dilates both the epicardial and resistance coronary arteries in humans. Furthermore, the coronary arterial response to magnesium is dose dependent and independent of EDNO.


Keywords: coronary artery; coronary blood flow; magnesium sulfate; nitric oxide     

Characterization of the glucose-dependent release of growth hormone-releasing factor and somatostatin from superfused rat hypothalami

The glucose-dependent secretion of the neuropeptides, growth hormone-releasing factor (GRF) and somatostatin (SRIF), by hypothalamic fragments was studied in vitro using a superfusion system. After equilibration of mediobasal hypothalami in HEPES-buffered Krebs-Ringer solution containing 5.5 mM glucose, glucose levels in the superfusion medium were altered. Lowering the glucose concentration in the medium from 5.5 to 2.7 or 1.1 mM provoked a rapid increase in GRF and SRIF release in a concentration and Ca2+-dependent manner. At 1.1 mM glucose, neuropeptide secretion was elevated 3- to 4-fold. The increase of GRF and SRIF release induced by low glucose was transient since stimulated neuropeptide secretion declined to basal levels in the continued presence of low glucose. Furthermore, after reequilibration in 5.5 mM glucose, no second stimulation of neuropeptide release could be induced by reduced glucose. Intracellular glucopenia induced by addition of 2-deoxy-D-glucose (16.5 mM) to the superfusion medium containing 5.5 mM glucose, also evoked increases in GRF and SRIF release. The sensitivity of GRF and SRIF neurons to glucose was absent in the postnatal period until day 9 after birth and then gradually increased. The parallel increases of GRF and SRIF release in response to low glucose observed in the present in vitro study, together with the suppression of plasma GH levels occurring in hypoglycemia in the rat, suggest that, in this condition, the inhibition of GH release induced by elevated SRIF levels predominates whereas the increase of GRF release might serve to attenuate this effect of SRIF.     

Effects of rat growth hormone (rGH)-releasing factor and somatostatin on the release and synthesis of rGH in dispersed pituitary cells

The effects of rat hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) on the release and biosynthesis of rat GH were studied by RIA and quantitative immunoprecipitation using monolayer cultures of rat anterior pituitary cells. In kinetic studies, GRF stimulation of GH release appeared at the first sampling time (20-min incubation) and the effect began to diminish after 2-h incubation with GRF. On the other hand, total (cell plus medium) content of GH significantly increased only after 24-h incubation. To examine the GH-synthesizing effect of GRF more directly, newly synthesized GH labeled by [35S]methionine during incubation with GRF was quantified by immunoprecipitation. The amount of immunoprecipitable GH increased significantly and specifically (compared with the total amount of labeled proteins) also only after 24-h incubation. When GH pools were labeled with [35S]methionine under different schedules, the basal release of newly synthesized GH, which was labeled for 1 h immediately before chase incubation was lower during the first 15 min than stored GH which had been labeled earlier. Basal newly synthesized GH secretion exceeded stored GH secretion after 30 min. GRF stimulated the release of GH from both pools but the stimulation of stored GH was greater. In this system, SRIF suppressed both the basal and stimulated release of GH but did not modify GH biosynthesis under either condition. Newly synthesized GH showed significant degradation during 24-h incubation; neither GRF nor SRIF affected the rate of GH degradation during the same incubation period. These results indicate that 1) GRF stimulates both release and synthesis of GH; 2) these two effects have different kinetics and different sensitivities to SRIF; and 3) GRF stimulates the release of GH from heterogeneous pools disproportionally.     

L-arginine stimulates cyclic guanosine 3',5'-monophosphate formation in rat islets of Langerhans and RINm5F insulinoma cells: evidence for L-arginine:nitric oxide synthase.

L-Arginine (L-Arg) is metabolized by nitric oxide synthase to the reactive intermediate nitric oxide. Since nitric oxide stimulates guanylyl cyclase and cGMP synthesis, L-Arg effects on cGMP accumulation in isolated pancreatic islets of the rat and RINm5F insulinoma cells were determined. Both L-Arg and glucose stimulation increased islet cGMP levels, and glucose potentiated the response to L-Arg alone. A competitive inhibitor of L-Arg metabolism to nitric oxide, NG-monomethyl-L-arginine, reduced glucose- and L-Arg-stimulated insulin release and glucose-induced increases in cGMP; however, basal insulin release was slightly increased. D-Arg and L-ornithine did not affect islet cGMP levels, although insulin release was stimulated. RINm5F cell cGMP levels and insulin release increased in response to L-Arg in a concentration- and time-related manner, whereas glucose and L-histidine were without effect. 8-Bromo-cGMP also slightly increased RINm5F cell insulin release. Sodium nitroprusside as a source of nitric oxide increased RINm5F cell cGMP production. Methylene blue and LY83583, inhibitors of soluble guanylyl cyclase activation, reduced RINm5F cell cGMP levels in the presence and absence of L-Arg; LY83583 also reduced glucose-stimulated cGMP levels in islets. Insulin release by glucose and L-Arg was also inhibited by methylene blue and LY83583 in islets. We conclude that glucose and L-Arg stimulate guanylyl cyclase activity and cGMP formation in beta-cells at least in part through metabolism to the reactive intermediate nitric oxide. However, neither nitric oxide nor cGMP synthesis is obligatory for insulin secretion.