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Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were used to study whether protons in the immediate protein environment around CuA in cytochrome c oxidase are susceptible to solvent exchange. The enzyme was incubated in buffered D2O under resting or turnover conditions for 90 min and then frozen to quench the hydrogen/deuterium-exchange process. ENDOR spectra of the deuterated sample were essentially identical to those of control samples. The ESEEM spectra, however, provided a clear indication of the introduction of deuterium into the CuA environment following incubation in buffered D2O. The extent of deuterium incorporation was not affected by enzyme turnover. An analysis of the ESEEM data indicated that water is in reasonably close proximity to the CuA site, but not in the immediate coordination sphere of the metal(s). We estimate a minimum distance of 5.4 A between the CuA center and the protein/water interface. This relatively short surface separation distance is consistent with the role of CuA as the immediate oxidant of cytochrome c in the cytochrome oxidase (Hill, B. C. (1991) J. Biol. Chem. 266, 2219-2226).

The influence of somatostatin (a bolus injection of 250 micrograms i.v. followed by 6 micrograms/min for 40 min) on the plasma cyclic AMP, insulin, glucose and non-esterified fatty acid (NEFA) responses to i.v. adrenaline (0.07 micrograms/kg body-weight/min for 20 min) and the plasma cyclic AMP, insulin and glucose responses to a bolus injection of i.v. glucagon (0.01 mg/kg BW) were studied in normal subjects. Somatostatin suppressed the insulin response to glucagon and inhibited the insulin rebound observed on termination of adrenaline infusion. The plasma glucose response to glucagon was exaggerated by somatostatin, reflecting insulin deficiency. Neither the plasma glucose or plasma non-esterified fatty acid responses to adrenaline were influenced by somatostatin. Adrenaline produced a three-fold and glucagon a twenty-fold rise in plasma cyclic AMP and 15 min. This was not influenced by concurrent somatostatin infusion, indicating that somatostatin is not a universal inhibitor of hormone stimulated adenylate cyclase activity. This is supported by the failure of somatostatin to inhibit the metabolic actions of glucagon and adrenaline thought to be mediated by cyclic AMP.

Five diabetic and 14 non-diabetic uraemic patients on long-term haemodialysis were studied during twenty-one 24 h periods including 5 to 7 h of haemodialysis against glucose-free acetate buffered dialysis fluid. Half-hourly blood samples were collected for hormonal and metabolite analysis. In addition, blood samples were analyzed in 40 experiments covering the haemodialysis and a pre-dialysis period. Before dialysis, plasma growth hormone levels were high and fluctuating, but almost always fell to low normal values within the first 2 h of haemodialysis. In the diabetic uraemic patients, the occasional severe hypoglycaemic episodes occurring during haemodialysis did not provoke growth hormone release, and hypoglycaemic reactions were not encountered. Intravenous acetate infusion studies resulted in plasma concentrations ranging from 1.3 to 2.7 mmol, i.e. about 60 per cent of the levels reached during haemodialysis and in suppression of growth hormone secretion. It is suggested that the fall in growth hormone levels and the lack of hypoglycaemic symptoms during haemodialysis is due to the use of acetate as a fuel in brain.

Injection of somatostatin, the hypothalamic growth-hormone-inhibiting factor, lowered basal plasma-insulin levels. In intravenous glucose-tolerance tests in five normal subjects, glucose-induced insulin release was markedly suppressed and the glucose-disappearance rate was lowered after somatostatin. Perfusion experiments on the isolated canine pancreas showed that somatostatin acts directly on the beta cells, causing suppression of the initial and the late sustained insulin release phase.

Growth hormone secretion in uremic patients is completely suppressed immediately after initiation of and during hemodialysis using acetate dialysate. The present study demonstrates that growth hormone secretion stimulated by arginine is also inhibited by hemodialysis. Acetate infusion experiments proved that the high blood acetate level obtained during dialysis is at least partly responsible for this growth hormone suppression by unknown mechanisms.