Role of redox-active iron ions in the decomposition of S-nitrosocysteine in subcellular fractions of porcine aorta.

We recently reported that degradation of S-nitrosocysteine in homogenates of porcine aorta increased severalfold in the presence of Mg2+ ions [Kostka, P., Xu, B. & Skiles, E.H. (1999) J. Cardiovasc. Pharmacol. 33, 665-670]. The objective of the present study was to examine this in greater detail. The rate of S-nitrosocysteine degradation by aortic homogenates in the presence of Mg2+ ions exhibited differential sensitivity to chelators of iron ions. Terpyridine and diethylenetriamine penta-acetic acid (5-500 microM) caused a concentration-dependent inhibition of S-nitrosocysteine decay, whereas deferoxamine (100 microM) was ineffective. o-Phenanthroline (250 microM), a selective chelator of Fe2+ ions, potentiated the reaction at low initial concentrations of S-nitrosocysteine (< or = 15 microM) and inhibited the reaction at higher concentrations. The inhibitory effects of o-phenanthroline were related to suppression of S-nitrosocysteine decay by cysteine-mediated reduction of Fe3+. In the presence of o-phenanthroline, S-nitrosocysteine decomposition followed saturable kinetics with K0.5 = 3.8 +/- 0.3 microM and h = 1.8 +/- 0.1 (mean +/- SE, n = 4). Comparison of the rates of S-nitrosocysteine decay in different subcellular fractions showed selective association with the cytosolic fraction, as documented by copurification with lactate dehydrogenase activity. At non-limiting concentrations of S-nitrosocysteine, the rate of degradation in the cytosolic fraction was 4.1 +/- 0.3 nmol.min-1.(mg protein)-1 (n = 4). It is concluded that the cytosolic fraction of porcine aorta contains a protein factor, presumably an enzyme, capable of catalyzing heterolytic decomposition of the S-NO bond of S-nitrosocysteine in a process involving redox cycling of iron ions.

Degradation of S-nitrosocysteine in vascular tissue homogenates: role of divalent ions.

The objective of the study was to inquire about the mechanism(s) involved in the catabolism of S-nitrosothiols by vascular tissue under in vitro conditions. Incubations of S-nitrosocysteine (CYSNO) or S-nitrosoglutathione (GSNO) with homogenates isolated from porcine aortic smooth muscle resulted in only a marginal depletion of S-nitrosothiols from the reaction mixtures, which became statistically significant at relatively high concentrations of homogenate (> or =300 microg of protein/ml). Degradation of CYSNO (but not GSNO) was found to be potentiated several-fold by millimolar concentrations of either Mg2+ or Ca2+ ions. Under such conditions, the degradation of CYSNO was significantly suppressed by the removal of proteins by ultrafiltration (>80% inhibition) and eliminated completely by the alkylation of thiol groups with 1 mM N-ethylmaleimide. The potentiating effect of divalent ions on the degradation of CYSNO was insensitive to 0.1 mM neocuproine (selective chelator of Cu+ ions), although it was enhanced in the presence of 0.1 mM o-phenanthroline (selective chelator of Fe2+ ions). It is concluded that the degradation of CYSNO by tissue homogenate involves the interaction with protein-bound sulfhydryl groups, which is stimulated by Mg2+ or Ca2+ ions. The potentiating effect of o-phenanthroline suggests that the liberation of the nitrosonium moiety in such a process may be accompanied by its transfer to sulfur center(s) by transient formation of dinitrosyl-iron complexes.

Recovery of growth hormone release from suppression by exogenous insulin-like growth factor I (IGF-I): evidence for a suppressive action of free rather than bound IGF-I.

To determine the time course of recovery of GH release from insulin-like growth factor I (IGF-I) suppression, 11 healthy adults (18-29 yr) received, in randomized order, 4-h i.v. infusions of recombinant human IGF-I (rhIGF-I; 3 microg/kg-h) or saline (control) from 25.5-29.5 h of a 47.5-h fast. Serum GH was maximally suppressed within 2 h and remained suppressed for 2 h after the rhIGF-I infusion; during this 4-h period, GH concentrations were approximately 25% of control day levels [median (interquartile range), 1.2 (0.4-4.0) vs. 4.8 (2.8-7.9) microg/L; P < 0.05]. A rebound increase in GH concentrations occurred 5-7 h after the end of rhIGF-I infusion [7.6 (4.6 -11.7) vs. 4.3 (2.5-6.0) microg/L; P < 0.05]. Thereafter, serum GH concentrations were similar on both days. Total IGF-I concentrations peaked at the end of the rhIGF-I infusion (432 +/- 43 vs. 263 +/- 44 microg/L; P < 0.0001) and remained elevated 18 h after the rhIGF-I infusion (360 +/- 36 vs. 202 +/- 23 microg/L; P = 0.001). Free IGF-I concentrations were approximately 140% above control day values at the end of the infusion (2.1 +/- 0.4 vs. 0.88 +/- 0.3 microg/L; P = 0.001), but declined to baseline within 2 h after the infusion. The close temporal association between the resolution of GH suppression and the fall of free IGF-I concentrations, and the lack of any association with total IGF-I concentrations suggest that unbound (free), not protein-bound, IGF-I is the major IGF-I component responsible for this suppression. The rebound increase in GH concentrations after the end of rhIGF-I infusion is consistent with cessation of an inhibitory effect of free IGF-I on GH release.

Oral administration of growth hormone (GH) releasing peptide-mimetic MK-677 stimulates the GH/insulin-like growth factor-I axis in selected GH-deficient adults.

To determine the effect of the GH releasing peptide (GHRP)-mimetic, MK-677, on the GH/insulin-like growth factor-I (IGF-I) axis in selected GH-deficient adults, we studied nine severely GH-deficient men [peak serum GH concentration in response to insulin-induced hypoglycemia of 1.2 +/- 1.5 micrograms/L, mean +/- SD (range 0.02-4.79)], age 17-34 yr, height 168 +/- 1.5 cm, body mass index 22.6 +/- 3.3 kg/m2, who had been treated for GH deficiency with GH during childhood. In a double-blind rising-dose design, subjects received once daily oral doses of 10 or 50 mg MK-677 or placebo for 4 days over two treatment periods separated by at least 28 days. Four subjects received placebo and 10 mg/day MK-677 in a cross-over fashion in periods 1 and 2. Five subjects received 10 mg and then 50 mg/day MK-677 in a sequential, rising-dose fashion in periods 1 and 2, respectively. Blood was collected every 20 min for 24 h before treatment and at the end of each period for GH measurement using an ultrasensitive assay. The drug was generally well tolerated, with no significant changes from baseline in circulating concentrations of cortisol, PRL, and thyroid hormones. Serum IGF-i and 24-H mean GH concentrations increased in all subjects after treatment with both 10 and 50 mg/day MK-677 vs. baseline. After treatment with 10 mg MK-677, IGF-I concentrations increased 52 +/- 20% (65 +/- 6 to 99 +/- 9 micrograms/L, geometric mean +/- intrasubject SE, P < or = 0.05 vs. baseline), and 24 h mean GH concentrations increased 79 +/- 19% (0.14 +/- 0.01 to 0.26 +/- 0.02 microgram/L, P < or = 0.05 vs. baseline). Following treatment with 50 mg MK-677, IGF-I concentrations increased 79 +/- 9% (84 +/- 3 to 150 +/- 6 micrograms/L, P < or = 0.05 vs. baseline) and 24-h mean GH concentrations increased 82 +/- 29% (0.21 +/- 0.02 to 0.39 +/- 0.04 microgram/L, P < or = 0.05 vs. baseline), respectively. Serum IGF binding protein-3 concentrations increased with both 10 mg (1.2 +/- 0.1 to 1.7 +/- 0.1 micrograms/L, P < or = 0.05) and 50 mg MK-677 (1.7 +/- 0.1 to 2.2 +/- 0.2 micrograms/L, P < or = 0.05). The GH response to MK-677 was greater in subjects who were the least GH/IGF-I deficient at baseline; by linear regression analysis the increase in 24-h mean GH concentration was positively related to both baseline 24-h mean GH concentration (r = 0.81, P = 0.009) and baseline IGF-I (r = 0.79, P = 0.01) for 10 mg MK-677. IGF-I responses were not significantly related to any baseline measurement. Fasting and postprandial insulin and postprandial glucose increased significantly after MK-677 treatment, and the clinical significance of these changes will need to be assessed in longer term studies. Oral administration of such GHRP-mimetic compounds may have a role in the treatment of GH deficiency of childhood onset.

Stimulation of the growth hormone (GH)-insulin-like growth factor I axis by daily oral administration of a GH secretogogue (MK-677) in healthy elderly subjects.

Aging is associated with declining activity of the GH axis, possibly contributing to adverse body composition changes and increased incidence of cardiovascular disease. The stimulatory effects on the GH-insulin-like growth factor I (IGF-I) axis of orally administered MK-677, a GH-releasing peptide mimetic, were investigated. Thirty-two healthy subjects (15 women and 17 men, aged 64-81 yr) were enrolled in a randomized, double blind, placebo-controlled trial. They received placebo or 2, 10, or 25 mg MK-677, orally, once daily for 2 separate study periods of 14 and 28 days. At baseline and on day 14 of each study period, blood was collected every 20 min for 24 h to measure GH, PRL, and cortisol. Attributes of pulsatile GH release were assessed by 3 independent algorithms. MK-677 administration for 2 weeks increased GH concentrations in a dose-dependent manner, with 25 mg/day increasing mean 24-h GH concentration 97 +/- 23% (mean +/- SE; P < 0.05 vs. baseline). This increase was due to an enhancement of preexisting pulsatile GH secretion. GH pulse height and interpulse nadir concentrations increased significantly without significant changes in the number of pulses. With 25 mg/day MK-677 treatment, mean serum IGF-I concentrations increased into the normal range for young adults (141 +/- 21 microgram/L at baseline, 219 +/- 21 micrograms/L at 2 weeks, and 265 +/- 29 micrograms/L at 4 weeks; P < 0.05). MK-677 produced significant increases in fasting glucose (5.4 +/- 0.3 to 6.8 +/- 0.4 mmol/L at 4 weeks; P < 0.01 vs. baseline) and IGF-binding protein-3. Circulating cortisol concentrations did not change, and PRL concentrations increased 23%, but remained within the normal range. Once daily treatment of older people with oral MK-677 for up to 4 weeks enhanced pulsatile GH release, significantly increased serum GH and IGF-I concentrations, and, at a dose of 25 mg/day, restored serum IGF-I concentrations to those of young adults.