Angelo Margutti

Circadian rhythm of calcitonin gene-related peptide in uncomplicated essential hypertension

Objective: To assess the existence of an altered circulating pattern of calcitonin gene-related peptide (CGRP) in hypertension. Design: The 24 h variation in plasma CGRP was measured and compared in 10 patients affected by uncomplicated essential hypertension and in nine age- and sex-matched healthy volunteers. The diurnal variations in blood pressure, atrial natriuretic peptide (ANP), plasma renin activity (PRA), plasma aldosterone and plasma cortisol were also assessed. Methods: Recumbency studies were performed under standardized, drug-free conditions beginning at 0800 h. Venous samples were drawn every 4h for 24 h and hormone levels were assessed with specific radioimmunoassays. The blood pressure was measured every 15min with a SpaceLabs 90207 monitor. Results: The mean 24-h plasma CGRP concentrations were significantly lower in the hypertensive group than in the control group. In both groups a circadian rhythm was present with the same pattern, but at a lower level in hypertension. A temporal sequence starting with the nocturnal rise in plasma CGRP concentrations and progressing with the elevations of ANP, PRA, and plasma aldosterone and cortisol was apparent in both groups. The nocturnal rise in the CGRP and ANP concentrations coincided with the blood pressure and the heart rate falls. Conclusions: Our data show that CGRP is lower than normal but maintains its circadian variability and its relationship with the diurnal variations in blood pressure and other hormones known to be active on the cardiovascular system.

Activation of the Somatotropic Axis by Testosterone in Adult Men: Evidence for a Role of Hypothalamic Growth Hormone-Releasing Hormone

Testosterone (T) is known to affect the growth hormone (GH) axis. However, the mechanisms underlying the activation of GH secretion by T still remain to be clarified. Available data in animals and humans have shown that withdrawal of somatostatin (SRIH) infusion induces a GH-releasing hormone (GHRH)-mediated rebound release of GH, and there is accumulating evidence that SRIH infusion withdrawal may be a useful test to probe the GHRH function in vivo. With the aim of investigating whether the stimulatory effect of androgens on GH release in man could be accounted for by activation of the hypothalamic GHRH tone, we evaluated the plasma GH response to SRIH withdrawal in 10 patients aged 29.6 ± 2.4 years (mean ± SEM), diagnosed with hypergonadotropic hypogonadism, before and after a 6-month replacement therapy with T enanthate (250 mg every 3 weeks, i.m.), and in 10 healthy men, aged 26.7 ± 2.8 years. To verify whether the modulation of GH secretion by T could also be mediated through changes in SRIH tone and/or pituitary releasable pool, we examined GH secretory responses to combined GHRH and L-arginine (ARG) in the same individuals. Basal plasma concentrations of GH (0.48 ± 0.11 µg/l) and IGF-I (23.79 ± 1.83 nmol/l) were significantly lower in untreated hypogonadal patients than in healthy men, and significantly increased after T replacement therapy (GH 1.13 ± 0.28 µg/l; IGF-I 28.71 ± 1.46 nmol/l). The mean ΔGH peak after SRIH withdrawal recorded in untreated hypogonadal men (2.65 ± 0.86 µg/l) was significantly (p < 0.05) lower than that observed in healthy men (6.53 ± 1.33 µg/l) and significantly increased after T replacement therapy (5.52 ± 1.25 µg/l). The GH responses to GHRH combined with ARG (a functional SRIH antagonist) were not significantly different between healthy men and untreated hypogonadal patients, and were not significantly affected by T treatment. Plasma T and estradiol (E2) levels significantly correlated with ΔGH peak after SRIH withdrawal in healthy men and in T-treated hypogonadal patients, whereas in untreated patients they did not. No significant correlation was found between GH areas under the curve after GHRH + ARG test and T and E2 plasma levels in either healthy men or in hypogonadal patients (both before and after T replacement). These findings are consistent with the view that in humans the stimulatory action of T on the GH axis appears to be mediated at the hypothalamic level primarily by promoting GHRH function.

Dermorphin inhibits spinal nociceptive flexion reflex in humans

Dermorphin (D) is a potent opiate-like peptide isolated from the skin of some species of frogs. Experimental studies in animals indicate that D has a potent antinociceptive effect, while no investigation exists about its analgesic properties in humans. Our study shows that i.v. infusion of 0.16 mg/kg D induces a marked and long-lasting increase in the threshold of nociceptive flexion reflex in healthy volunteers. This effect is also evident in a complete chronic spinal subject, showing that D depresses the nociceptive transmission mainly acting at spinal level. Naloxone, while fully antagonizing the effects of morphine and enkephalin analogue, is able to reverse only partly (ca. 50%) the depressive effect of D on nociceptive spinal reflex. This fact may suggest that D interacts with different spinal opiate receptor populations in inducing analgesia.

Growth hormone excess promotes breast cancer chemoresistance.

CONTEXT GH and IGF-I are known to promote breast carcinogenesis. Even if breast cancer (BC) incidence is not increased in female acromegalic patients, mortality is greater as compared with general population. OBJECTIVE The objective of the study was to evaluate whether GH/IGF-I excess might influence BC response to chemotherapy. DESIGN We evaluated GH and IGF-I effects on cell proliferation of a BC cell line, MCF7 cells, in the presence of doxorubicin (Doxo), frequently used in BC chemotherapy, and the possible mechanisms involved. RESULTS GH and IGF-I induce MCF7 cell growth in serum-free conditions and protect the cells from the cytotoxic effects of Doxo. GH effects are direct and not mediated by IGF-I because they are apparent also in the presence of an IGF-I receptor blocking antibody and disappear in the presence of the GH antagonist pegvisomant. The expression of the MDR1 gene, involved in resistance to chemotherapeutic drugs, was not induced by GH. In addition, c-fos transduction was reduced by Doxo, which prevented GH stimulatory effects. Pegvisomant inhibited basal and GH-induced c-fos promoter transcriptional activity. Autocrine GH action is ruled out by the lack of endogenous GH expression in this MCF7 cell strain. CONCLUSIONS These data indicate that GH can directly induce resistance to chemotherapeutic drugs with a mechanism that might involve GH-induced early gene transcription and support the hypothesis that GH excess can hamper BC treatment, possibly resulting in an increased mortality.

Circadian Rhythms of Atrial Natriuretic Peptide, Blood Pressure and Heart Rate in Normal Subjects

The occurrence and extent of a circadian rhythm in the circulating concentrations of atrial natriuretic peptide (ANP) is still a matter of controversy. In a group of hospitalized normal volunteers (6 men and 4 women, 16-76 years old), we investigated the circadian variability of ANP and its temporal relation with the circadian rhythms of blood pressure (BP) and heart rate (HR), by using a chronobiological inferential statistical method. At the end of a synchronizing period of 1 week (the diet and the daily schedule were standardized), the subjects underwent automatic BP and HR monitoring, and blood sampling for 24 h. A statistically significant mean circadian rhythm was demonstrated for ANP, BP, and HR. The mean circadian acrophase of ANP was calculated to occur around 4 a.m. BP and HR rhythms appeared to be in antiphase with ANP rhythm, i.e. the peak of BP and HR rhythms more or less coincided with a trough in ANP rhythm. ANP appears to be anticipatory in its circadian periodic rise to awakening. Therefore, postural changes cannot fully account for the diurnal variations observed.

Function of the GH/IGF-1 Axis in Healthy Middle-Aged Male Runners

In an attempt to examine the effect of prolonged physical activity on the function of the GH/IGF-1 axis during the aging process in man, we have evaluated basal and GHRH (GHRH-29: 1 microgram/kg i.v. as a bolus) stimulated GH secretion as well as basal plasma IGF-1 levels in a group of 25 healthy runners (50-60 years, mean age 55.5 +/- 0.6) and 24 age-matched relatively sedentary normal controls (mean age 55.8 +/- 0.7). The runners had a minimum distance in kilometers of 26 km/week for at least 15 years, and competed in distances ranging from 16 km to the marathon. In runners, GHRH induced an increase of GH which was significantly higher (p < 0.001) than that observed in the age-matched controls. Baseline IGF-1 levels were significantly higher (p < 0.001) in trained runners (171 +/- 8.4 micrograms/1) compared to the controls (91.1 +/- 5.5 micrograms/1). These data show that in middle-age prolonged physical activity increases the function of the GH/IGF-1 axis. To clarify the possible mechanisms underlying the GH/IGF-1 secretory pattern in the runners, the GH responses to both single and combined administration of GHRH and arginine (ARG: 30 g infused over 30 min), a GH secretagogue likely acting via inhibition of hypothalamic somatostatin release, were investigated in 6 runners (mean age 55 +/- 1.9 years) and 6 controls (mean age 55 +/- 0.9 years). ARG clearly increased the GH response to GHRH in the controls, whereas it was unable to further potentiate the GH-releasing effect of GHRH in runners, thus suggesting that the increased GH responsiveness to GHRH might be due to an exercise-related decrease in endogenous hypothalamic somatostatinergic activity.

Stimulatory Effect of Dermorphin, a New Synthetic Potent Opiate-Like Peptide, on Human Growth Hormone Secretion

Two new related heptapeptides (dermorphins) with potent central and peripheral opiate-like activity have been isolated from the skin of South American frogs, and have been chemically characterized as H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (dermorphin) and H-Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2 (Hyp6-dermorphin). The response of GH to infusion of a synthetic dermorphin (5.5 micrograms/kg/min for 30 min) was studied in 9 healthy men. Dermorphin (D) significantly increased plasma growth hormone (GH) concentrations. The GH response to D was blunted by prior administration of naloxone, suggesting that D interacts with mu-type opiate receptors. However, the evaluation of the physiological significance of D-induced GH release in humans requires further study.

Effect of pentagastrin on adrenocorticotropin hormone and thyroid-stimulating hormone release in normal subjects.

The possible role of gastrin on TSH, ACTH and cortisol secretion was evaluated by intravenous administration of pentagastrin, the carboxyl-terminal tetrapeptide of gastrin (0.5 microgram/kg b.w.) into 12 healthy subjects. Pentagastrin produced a significant rise in plasma ACTH and cortisol levels but did not alter TSH basal release. These preliminary results suggest that gastrin can influence basal activity of ACTH-cortisol axis. However, further investigation is required to determine its physiological role and mechanisms of action.

Acute administration of human galanin in normal subjects reduces the potentiating effect of pyridostigmine-induced cholinergic enhancement on release of norepinephrine and pancreatic polypeptide.

The neuropeptide galanin (GAL) is widely distributed in the central and peripheral nervous systems where it often coexists with catecholamines and acetylcholine. Recently we have reported that human GAL (hGAL) in man depresses the release of norepinephrine (NE) and the responses to both assumption of upright posture and insulin-induced hypoglycemia. To gain an insight into the action of hGAL on sympathetic nervous system activity in man, we investigated the effects of a 60-min infusion (80 pmol/kg/min) of hGAL or saline on the release of NE, epinephrine (E) and pancreatic polypeptide (PP) induced by an acetylcholinesterase inhibitor, pyridostigmine bromide (PD), in nine healthy volunteers. PD (120 mg orally) induced a significant rise in plasma concentrations of NE (1.6 +/- 0.04 vs. 1.08 +/- 0.06 nmol/l), E (0.34 +/ 0.05 vs. 0.12 +/- 0.04 nmol/l) and PP (178.06 +/- 33 vs. 37.57 +/- 7.35 pmol/l), whilst it significantly reduced heart rate (HR; 61 +/- 2 vs. 71 +/- 4 beats/min). Changes in plasma levels of PP were determined as an indirect measure of amplification of endogenous cholinergic activity produced by PD. Administration of hGAL blunted the release of NE and PP evoked by PD. The mean (+/- SEM) area under the curve produced by PD of NE (50.05 +/- 3.97 nmol/l.90 min) and PP (8,692.87 +/- 1,724 pmol/l.90 min) was significantly (p < 0.001) reduced by hGAL infusion (2.65 +/- 1.57 nmol/l.90 min and 248.1 +/- 148 pmol/l.90 min, for NE and PP, respectively). hGAL failed to affect significantly the E release evoked by PD. hGAL was able to enhance HR significantly (104 +/- 5 vs. 69 +/- 3 beats/min), and completely prevented the PD-induced slowing of HR. Both PD and hGAL did not alter supine systolic and diastolic blood pressure. We conclude that hGAL significantly reduces the release of NE and PP stimulated by PD-induced enhancement of cholinergic activity. These findings are consistent with a functional interrelationship between GAL and the cholinergic system in man, and may suggest the participation of a cholinergic pathway in the galaninergic modulation of the autonomic nervous system.

Effect of Bromocriptine on the Control of Plasma Aldosterone Diurnal Variation in Normal Supine Man

In order to investigate the role of prolactin in the control of the circadian rhythm of plasma aldosterone (PA), plasma renin activity (PRA), cortisol (PC), aldosterone and prolactin (PRL) levels were determined in samples at 4-hour intervals from 5 normal supine men over a period of 24 h under basal conditions and subsequently over a period of 24 h during suppression of prolactin release by bromocriptine (CB-154). After suppression of prolactin, statistically signific1nt circadian rhythms in PC and PA have been detected with a moderate decrease of PA concentration, while the PC level remained unalterated. PRA rhythmicity persisted with a significant shift of acrophase and remarkable reduction of plasma levels. Moreover, during CB administration a significant correlation was obtained between PA and PC, while no correlation was detected between PA and PRA. These data are consistent with the following concepts: (a) the prolactin does not play a significant role in the regulation of circadian rhythm and concentration of plasma aldosterone in normal supine men, and (b) bromocriptine induces a remarkable reduction of PRA and a variable decrease in plasma aldosterone, but it does not influence the secretion of cortisol in normal subjects.