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Obstructive sleep apnoea (OSA) is an independent risk factor of hypertension and cardiovascular diseases. Recurrent episodes of upper airways collapse during sleep causing blood oxygen desaturation, hypercapnia, and micro-arousals, are known to activate the sympathetic nervous system (SNS). However, whether changes in the renin-angiotensin-aldosterone system and endothelial activation also occur remains contentious. Based on routine use of drug-induced sleep endoscopy (DISE) for the work-up of OSA patients in our centre, we designed a prospective study to investigate the haemodynamic and humoral changes occurring during the apnoeic episodes reproduced in vivo in the course of DISE. Specifically, plasma aldosterone concentration and renin activity, C-terminal fragment of proendothelin-1, as a marker of endothelial damage, and free plasma catecholamines, will be measured at fixed times during DISE. The activity of catechol-O-methyltransferase (COMT), a key catecholamine-inactivating enzyme that has been scantly investigated thus far owing to the lack of commercially available kits, will be also determined by a newly developed high performance liquid chromatography method, which is herein described. The aim of this study is to provide novel information on the haemodynamic, hormonal, and SNS changes, and also on COMT activity modification concomitantly occurring during apnoea, thus contributing substantively to the understanding of the pathophysiology of OSA.

Aims/hypothesis. To verify whether individual susceptibility to diabetic nephropathy resides in an intrinsic difference in Protein Kinase C (PKC) activity. Methods. We compared the effect of different glucose concentrations on PKC activity, PKC isoform expression and diacylglycerol (DAG) content in cultured fibroblasts from 14 Type I diabetic patients who developed nephropathy with those in cells from 14 patients without nephropathy. We recruited 14 normal subjects as control patients. Forearm skin fibroblasts were cultured in either normal (5 mmol/l) or high (20 mmol/l) glucose concentrations. Results. In normal glucose, in situ PKC activity was higher in Type 1 patients with nephropathy (10.1 +/- 1.4 pmol/min/mg protein; p<0.01) than in those without (6.8 +/- 0.8) and the normal control subjects (6.3 +/- 0.5). This difference was due to increased concentrations of PKC(x isoform in the membrane fraction of fibroblasts from patients with nephropathy. DAG content was also higher in cells from Type 1 patients with nephropathy. Incubation in high glucose concentration caused a further increase in PKC activity and DAG content in quiescent fibroblasts from patients with diabetic nephropathy, with no significant changes in cells from diabetic patients without nephropathy and normal control subjects. Conclusion/interpretation. Differences in PKC activation could contribute to the individual susceptibility to renal damage in Type 1 diabetic patients.

We have recently shown that insulin attenuates angiotensin II-induced intracellular Ca2+ mobilization in human skin fibroblasts from normotensive subjects. This study was designed to investigate the effects of angiotensin II and the interactions between insulin and angiotensin II on intracellular Ca2+ mobilization in skin fibroblasts from patients with essential hypertension. Fibroblasts were obtained from 9 normotensives and is hypertensives. Spectrofluorophotometric free Ca2+ measurement was performed in monolayers of 24-hour serum-deprived cells. Resting intracellular Ca2+ level and angiotensin II-stimulated intracellular Ca2+ peak were higher in fibroblasts from hypertensives compared with those from normotensives. The effect of acute insulin exposure was evaluated in fibroblasts from hypertensives subdivided on the basis of insulin sensitivity. In insulin-sensitive hypertensives, insulin significantly blunted the effects of angiotensin TS on intracellular Ca2+ response, whereas in insulin-resistant patients, insulin did not modify intracellular Ca2+ response to angiotensin II. Pertussis toxin, a G(i alpha)-inhibitor, reduced angiotensin II-stimulated Ca2+ peak in insulin-sensitive but not in insulin-resistant hypertensives. In conclusion, the effects of angiotensin Il on intracellular Ca2+ mobilization are more pronounced in fibroblasts from hypertensives compared with those from normotensives, and the inhibitory effect of insulin is blunted in insulin-resistant hypertensives by a G(i alpha) pertussis toxin-sensitive abnormality.

Background. Angiotensin II stimulates synthesis and deposition of collagen and might contribute to the vascular and cardiac dysfunction associated with arterial hypertension. Insulin attenuates angiotensin II-induced responses of intracellular Ca2+ concentration ([Ca2+]) in many cell types but this effect is less in insulin-resistant states. The mechanisms of the interaction between insulin and angiotensin II are still not known. Objective. To characterize the effects of angiotensin II on intracellular [Ca2+] and the effects of insulin on the angiotensin II-induced response of intracellular [Ca2+] in human skin fibroblasts. Methods. Spectrofluorophotometric measurements of intracellular [Ca2+] in monolayers of cultured human skin fibroblasts from 15 normotensive patients were performed using Fura-2 at 510 nm emission with excitation wavelengths of 340 and 380 nm. Results. Basal intracellular [Ca2+] in quiescent (24 h serum-deprived) human fibroblasts was 75 ± 3 nmol/l (n = 20). Administration of angiotensin II elevated intracellular [Ca2+] dose-dependently with a concentration for half-maximal effect of 20 nmol/l. Administration of 100 nmol/l angiotensin II stimulated a rapid and transient increase in intracellular [Ca2+] (from 75 ± 3 to 130 ± 2 nmol/l, n = 20). Removal of extracellular calcium did not change peak intracellular [Ca2+], but it did reduce the time to recovery of [Ca2+] (from 64 ± 4 to 48 ± 2 s, n = 10, P < 0.01), suggesting that an angiotensin II-induced transmembrane calcium influx had occurred. This hypothesis was confirmed by quenching studies with manganese. The angiotensin II-induced changes in intracellular [Ca2+] were completely blocked by administration of 100 nmol/l of the angiotensin II type 1 receptor inhibitor losartan but not by administration of 100 nmol/l of the angiotensin II type 2 receptor blocker CGP42112A. Acute (20 min) exposure to 100 nmol/l insulin did not alter basal intracellular [Ca2+] in quiescent fibroblasts, but signifi