Insulin-mediated venodilation is impaired in young, healthy carriers of the 825T allele of the G-protein beta3 subunit gene (GNB3).

OBJECTIVE: A C825T polymorphism has been identified for the gene encoding the G-protein beta 3 subunit ( GNB3 ). The 825T allele is associated with hypertension and obesity, which in turn are closely linked with resistance to the metabolic and vascular effects of insulin. We hypothesized that venodilation in response to insulin would be impaired in GNB3 825T-allele carriers. Because vasodilatory properties of insulin are mainly mediated by nitric oxide, we also investigated the influence of the T-786C polymorphism of the gene for endothelial nitric oxide synthase ( NOS3 ) on insulin-mediated venous responses. METHODS: We used the linear variable transducer technique to compare dorsal hand vein compliance in 31 young, healthy men ( GNB3 C825T: 15 CC, 14 CT, and 2 TT; NOS3 T-786C: 14 TT, 13 TC, and 4 CC). Individual dose-response curves to phenylephrine (3.2-10,000 ng/min) were established, and veins were preconstricted by a constant infusion of phenylephrine at the individual dose needed to procure 70% of maximal constriction. Then insulin was infused (50-250,000 microU/min), and the changes in venous diameter were recorded. RESULTS: Venous response to insulin was biphasic, with venoconstriction at low doses being followed by venodilation at higher doses. Insulin dose-response curves of GNB3 825T-allele carriers were significantly shifted to the right (ANOVA, P < .001, versus CC). NOS3 T-786C-allele carrier status had no influence on insulin-induced vascular responses ( P = .60 for TC/CC versus TT). CONCLUSION: This study is the first to show the influence of a genetic polymorphism on insulin-mediated venodilation in men in vivo. Further studies are needed to determine whether these results translate to other vascular beds, and possible gender-specific differences remain to be investigated.

Clonidine lowers blood pressure by reducing vascular resistance and cardiac output in young, healthy males.

PURPOSE: Clonidine is a classical sympatholytic drug that is widely used for the treatment of hypertension. Experimental and clinical studies suggest that Clonidine may activate baroreflex. The aim of this study was to determine the hemodynamic response to Clonidine under physiological conditions and to test the hypothesis that Clonidine would reduce cardiac output and blood pressure resulting in an increase in total peripheral resistance. METHODS: Clonidine's hemodynamic effect was evaluated in 28 young, healthy subjects after a single i.v. dose of 1 microg x kg(- 1). Impedance cardiography, systolic time intervals and pulse wave analysis were used to characterize myocardial and vascular function. RESULTS: Clonidine lowered blood pressure, heart rate, left ventricular ejection time, and pulse wave velocity and increased pre-ejection period. Stroke volume and cardiac output decreased gradually over the investigation time of 240 min. Central systolic blood pressure (SBP) was lowered to a larger extent than peripheral SBP. Total peripheral resistance was characterized by an immediate fall of short duration followed by a continuous rise above baseline after 120 min. Placebo did not have any significant effect on hemodynamic parameters. CONCLUSIONS: Clonidine's blood pressure lowering effect is mediated by both an immediate decrease in vascular resistance and a prolonged decrease in cardiac output, and Clonidine lowers central SBP more than peripheral SBP.

Laser Doppler imager (LDI) scanner and intradermal injection for in vivo pharmacology in human skin microcirculation: responses to acetylcholine, endothelin-1 and their repeatability.

AIMS: The purpose of this study was to evaluate the repeatability of forearm skin blood flow responses to intradermal injections of acetylcholine (ACh) and endothelin-1 (ET-1) using a double injection technique (DIT) and a laser Doppler imager (LDI) scanner in the human skin microcirculation. METHODS: We used a laser Doppler imager (Moor LDI V3.01) to continuously monitor the change in skin blood flow during intradermal administration of physiological saline (0.9% NaCl), acetylcholine (ACh 10(-7), 10(-8), 10(-9) M) and endothelin-1 (ET-1 10(-14), 10(-16), 10(-18) M) in 10 healthy male subjects. Subjects were examined on 3 different days for assessment of interday and interobserver repeatability. Injections of either drug were randomly placed on different sites of the forearm. Laser Doppler images were collected before and after injection at 2.5 min intervals for 30 min. Data were analysed after the completion of each experiment using Moor Software V.3.01. Results are expressed as changes from baseline in arbitrary perfusion units (PU). RESULTS: ACh caused a significant vasodilation (P < 0.0001 anova, mean +/- SE: 766 +/- 152 PU, ACh 10(-9) M; 1868 +/- 360 PU, ACh 10(-8) M; 4188 +/- 848 PU, ACh 10(-7) M; mean of days 1 and 2, n = 10), and ET-1 induced a significant vasoconstrictive response (P < 0.0001 anova, -421 +/- 83 PU, ET-1 10(-18) M; -553 +/- 66 PU, ET-1 10(-16) M; -936 +/- 90 PU, ET-1 10(-14) M; mean of days 1 and 2, n = 10). There was no difference on the response to either drug on repeated days. Bland-Altman analyses showed a close agreement of responses between days with repeatability coefficients of 1625.4 PU for ACh, and 386.0 PU for ET-1 (95% CI: ACh, -1438 to 1747 PU, ET-1, -399 to 358 PU) and between observers with repeatability coefficients of 1057.2 PU for ACh and 255.8 PU for ET-1 (95% CI: ACh, -1024 to 1048 PU, ET-1, -252 to 249 PU). The variability between these responses was independent of average flux values for both ACh and ET-1. There was a significant correlation between responses measured in the same site, in the same individual on two different days by the same observer (ACh, r = 0.94, P < 0.0001; ET-1, r = 0.90, P < 0.0006), and between responses measured by two different observers (ACh, r = 0.94, P < 0.0001; ET-1, r = 0.91, P < 0.0003). CONCLUSION: We have shown that interday and intraobserver responses to intradermal injections of ET-1 and ACh, assessed using the DIT in combination with an LDI scanner, exhibited good reproducibility and may be a useful tool for studying the skin microcirculation in vivo.

Effects of systemic endothelin A receptor antagonism in various vascular beds in men: in vivo interactions of the major blood pressure-regulating systems and associations with the GNB3 C825T polymorphism.

OBJECTIVE: We used the orally available endothelin A (ETA) receptor antagonist darusentan to characterize interactions between the major blood pressure-regulating systems in healthy men. Mediators of the endothelin system, the sympathetic nervous system, and the renin-angiotensin system act via G protein-coupled receptors with a possible involvement of the G-protein beta3 subunit (GNB3) C825T polymorphism. We studied the influence of this polymorphism on the responses to ETA antagonism in the presence of endothelin 1 (ET-1), norepinephrine (NA), and angiotensin II (ANGII). METHODS: Thirty-seven individuals were included in a randomized, double-blind, placebo-controlled, crossover trial with 100 mg darusentan. Systemic hemodynamics and plasma ET-1, NA, and ANGII concentrations were assessed. Local studies were performed in the dorsal hand veins (n=18) and skin microcirculation (n=12), respectively. RESULTS: Darusentan lowered systolic and diastolic blood pressure ( P <.001 versus placebo) without any differences according to genotype (mean maximum Delta systolic blood pressure, -7 +/- 2 mmHg for CT/TT versus -5 +/- 3 mmHg for CC, P=.37; mean maximum Delta diastolic blood pressure, -3 +/- 2 mmHg for CT/TT versus -4 +/- 2 mmHg for CC, P=.96). Venoconstriction to ET-1 and NA was not affected by ET A blockade in either group; however, carriers of the 825T allele demonstrated a markedly enhanced venoconstriction to ET-1 and NA (median effective concentration [ED50] for ET-1 after darusentan [placebo]: 2.5 +/- 0.2 pmol/min for CT/TT [2.7 +/- 0.3 pmol/min], P=.42; 3.9 +/- 0.6 pmol/min for CC [4.6 +/- 0.3 pmol/min], P=.42; P=.046 [P=.0005] for CT/TT versus CC) (ED50 for NA after darusentan [placebo]: 5.2 +/- 1.2 ng/min for CT/TT [7.3 +/- 1.2 ng/min], P=.20; 32.9 +/- 7.1 ng/min for CC [19.7 +/- 5.5 ng/min], P=.75; P=.0008 [P=.026] for CT/TT versus CC). Darusentan dilated veins at baseline in CC homozygous subjects (+0.21 +/- 0.05 mm, P=.004 versus placebo). Systemic ET A antagonism inhibited constriction to ET-1 and also to NA and ANGII in the skin microcirculation without differences according to genotype (ET-1, P=.017 for all individuals versus placebo; NA, P=.0005; and ANGII, P=.002). CONCLUSION: GNB3 C825T allele carrier status did not influence systemic hemodynamic or local vascular responses to ET A blockade with darusentan in young, healthy men. However, it determined venoconstriction to exogenous ET-1 and NA. Darusentan markedly inhibited not only ET-1-induced but also NA-induced and ANGII-induced vasoconstriction in the skin microcirculation. In contrast, it had no effects on either ET-1-mediated or NA-mediated venoconstriction, indicating that, in the presence of high local ET-1 concentrations, constrictive endothelin B receptors may be of greater importance in the venous vasculature than has been recognized so far.

Endothelin-B-receptor-selective antagonist inhibits endothelin-1 induced potentiation on the vasoconstriction to noradrenaline and angiotensin II.

OBJECTIVE: Endothelin-A-receptor-antagonists inhibit angiotensin II- and noradrenaline-induced vasoconstriction. Whether functional constrictive endothelin-B-receptors play a role in the endothelin-1-mediated potentiation of vasoconstriction to angiotensin II and noradrenaline is thus far unknown. METHODS: We studied the effects of noradrenaline and angiotensin II (10 mol/l) in the presence of exogenous endothelin-1 (10 mol/l) with and without selective endothelin-B-receptor-blockade by BQ-788 (10 mol/l) and dual receptor blockade with BQ-788 and the endothelin-A-selective antagonist BQ-123 (10 mol/l) in 14 healthy male volunteers (aged 20-28). Studies were performed in the human skin microcirculation under in vivo conditions using laser-Doppler flowmetry and double injection technique. The area under the time-response curve of all doses was calculated. RESULTS: Endothelin-1 potentiated the effects of angiotensin II and noradrenaline (-944 +/- 139 perfusion units (PU), P < 0.01; -926 +/- 117 PU, P < 0.05, respectively). In the presence of BQ-788, the potentiating effect of endothelin-1 was significantly blunted (-624 +/- 132 PU, P < 0.01; -549 +/- 136 PU, P < 0.01, respectively). In the presence of BQ-123 and BQ-788 the vasoconstriction was fully inhibited (431 +/- 108 PU, P < 0.001 and 421 +/- 86 PU, P < 0.001, respectively). CONCLUSIONS: These data suggest that functional vasoconstrictive endothelin-B receptors on vascular smooth muscle cells may contribute to the potentiating effects of high local concentrations of endothelin-1 on the vasoconstriction to noradrenaline and angiotensin II in human microcirculation.

The I1-imidazoline agonist moxonidine decreases sympathetic tone under physical and mental stress.

AIMS: Moxonidine is an I1-imidazoline receptor agonist that reduces blood pressure by inhibition of central sympathetic activity. The effects of the drug under physical and mental stress have not been studied in detail. METHODS: We investigated the effects of 0.4 mg moxonidine orally on sympathetic activity, blood pressure and heart rate in a double-blind, placebo-controlled crossover study in 12 healthy volunteers. The subjects underwent physical exercise test using bicycle ergometry and a mental stress test using an adaptive reaction test device. Potential association of parameters with the GNB3 C825T polymorphism was also assessed. RESULTS: Under resting conditions, moxonidine decreased plasma noradrenaline (NA: -66.1 +/- 12 pg ml(-1); P < 0.01 vs placebo) and adrenaline (A: -18.8 +/- 6 pg ml(-1); P < 0.05 vs placebo). Physical exercise evoked a significant increase in plasma NA and A (NA: 760 +/- 98 pg ml(-1); A: 97 +/- 9 pg ml(-1); P < 0.001 vs baseline), which was significantly reduced after pretreatment with moxonidine (NA: 627 +/- 68 pg ml(-1); P < 0.05 vs placebo; A: 42.8 +/- 4 pg ml(-1); P < 0.01 vs placebo). Maximal physical exercise capacity was not limited by moxonidine (NS). During the mental stress test, increases in NA (placebo: 146 +/- 24 pg ml(-1), moxonidine: 84 +/- 26 pg ml(-1); P < 0.01 vs placebo) and A (placebo: 22.8 +/- 9 pg ml(-1), moxonidine: 8.0 +/- 8 pg ml(-1); P < 0.01 vs placebo) were significantly reduced after pretreatment with moxonidine. Increases in blood pressure during mental stress were significantly lower after pretreatment with moxonidine (P < 0.05 vs placebo). There was no association of the response to moxonidine with GNB3 genotypes (NS). CONCLUSIONS: Moxonidine decreases total sympathetic tone under basal conditions as well as during physical exercise and mental stress without limiting absolute exercise capacity. Thus, moxonidine appears suitable for the treatment of patients with high SNS activity and hypertension induced by physical or mental stress. As the drug does not reduce exercise capacity, it may be considered as an alternative to beta-adrenoceptor blockers in selected patients.

Left ventricular ejection time: a potential determinant of pulse wave velocity in young, healthy males.

OBJECTIVE: Pulse wave velocity (PWV) is a classic marker of vascular stiffness. Recent studies showed that heart rate is an important determinant of PWV. The purpose of this study was to evaluate the role of myocardial function in determining PWV under resting conditions and under adrenergic stimulation. DESIGN AND METHODS: Hemodynamic parameters were investigated under resting conditions in 102 young, healthy males and under stimulation of either beta- or alpha(2)-adrenoceptors in six young, healthy males. PWV was determined from pressure tracing over the carotid and femoral artery. Central hemodynamics were assessed by impedance cardiography and systolic time intervals. Simple (r) and multiple (beta) regression analyses were used to assess the relationships between PWV and hemodynamic parameters. RESULTS: Under resting conditions, PWV was correlated to age (beta = 0.259, P = 0.0052), diastolic blood pressure (beta = 0.279, P = 0.0072) and left ventricular ejection time (beta = -0.314, P = 0.0277). Under alpha(2)-adrenergic stimulation PWV was only correlated to diastolic blood pressure (DBP) (beta = 0.806, P = 0.0020). Under beta-adrenergic stimulation PWV was only correlated to left ventricular ejection time index (r = -0.52, P = 0.0325). CONCLUSIONS: Left ventricular ejection time may be an important determinant of pulse wave velocity under resting and adrenergic conditions in young, healthy males. Further studies are needed to evaluate this relationship in other populations including females and patients with cardiovascular disease.

Venous response to nitroglycerin is enhanced in young, healthy carriers of the 825T allele of the G protein beta3 subunit gene (GNB3).

OBJECTIVE: The ultimate mode of action by which nitroglycerin elicits vasodilation remains elusive. Animal studies point to an involvement of pertussis toxin-sensitive G proteins. The 825T allele of the GNB3 C825T polymorphism in the gene encoding the G protein beta3 subunit is associated with enhanced signal transduction via pertussis toxin-sensitive pathways in vitro. We hypothesized that G proteins have a role in nitroglycerin-mediated vasodilation and that carriers of the 825T allele would exhibit a stronger response to nitroglycerin. METHODS: We used the linear variable transducer technique to compare dorsal hand vein compliance in 28 young, healthy men with and without the T allele (n = 15 CC, n = 8 CT, and n = 5 TT). After individual dose-response curves to phenylephrine had been established, veins were preconstricted to 70% of the maximal phenylephrine-induced constriction. Nitroglycerin was then infused in ascending doses (0.02-2000 ng/min), and the vasodilatory response was measured. RESULTS: The vasodilatory response to nitroglycerin was significantly greater in carriers of the 825T allele. The maximal response to nitroglycerin was 102% +/- 6% venodilation in the CT/TT group and 78% +/- 5% in the CC control group (P =.0045) (mean difference, -24% +/- 8%; 95% confidence interval, 8%-40%). Comparison of the nitroglycerin dose-response curves by ANOVA confirmed an enhanced nitroglycerin-induced venodilation in 825T-allele carriers (P <.0001). CONCLUSION: Our results suggest that the GNB3 C825T polymorphism determines venous response to nitroglycerin and that G proteins may be involved in the signal transduction pathway.

Metabolic and haemodynamic effects of oral glucose loading in young healthy men carrying the 825T-allele of the G protein beta3 subunit.

BACKGROUND: A C825T polymorphism was recently identified in the gene encoding the beta3 subunit of heterotrimeric G-proteins (GNB3). The T-allele is significantly associated with essential hypertension and obesity. In order to further explore a possible pathogenetic link between the T-allele and impaired glucose tolerance we studied metabolic and haemodynamic responses to oral glucose loading in young, healthy subjects with and without the 825T-allele. METHODS: Twelve subjects with and 10 without the 825T-allele were investigated at rest and following glucose ingestion (75 g). Blood glucose, serum insulin and haemodynamics were determined prior to and over 2 hours following glucose ingestion. We non-invasively measured stroke volume (SV, by impedance-cardiography), blood pressure (BP), heart rate (HR), and systolic-time-intervals. Cardiac output (CO) was calculated from HR and SV. Total peripheral resistance was calculated from CO and BP. Metabolic and haemodynamic changes were quantified by maximal responses and by calculation of areas under the concentration time profile (AUC). Significances of differences between subjects with and without the T-allele were determined by unpaired two-tailed t-tests. A p < 0.05 was considered statistically significant. RESULTS: Metabolic and haemodynamic parameters at baseline were very similar between both groups. The presence of the T-allele did not alter the response of any metabolic or haemodynamic parameter to glucose loading. CONCLUSIONS: In conclusion, this study does not support the hypothesis that the C825T polymorphism may serve as a genetic marker of early impaired glucose tolerance.

Terlipressin plus hydroxyethyl starch infusion: an effective treatment for hepatorenal syndrome.

Ornipressin is a vasopressin analogue that can cause potent splanchnic vasoconstriction. It has been shown that, in combination with albumin infusion, ornipressin is able to reverse hepatorenal syndrome. However, its clinical use is limited by possible severe ischaemic complications. In our case, a 47-year-old man received a right hemihepatectomy for cholangiocellular carcinoma. On post-operative day three, he developed hepatorenal syndrome with ascites, peripheral oedema and oliguria (250-500 ml/day). Serum creatinine was increased to 3.5 mg/dl. The patient was treated with terlipressin, another vasopressin analogue with fewer side effects than ornipressin, (1 mg every 4 h intravenously) and hydroxyethyl starch (500 ml/day). Urine output increased to 3000 ml/day, serum creatinine decreased to normal range within 4 days and ascites and oedema disappeared. We hereby report the first case of successful treatment of hepatorenal syndrome with terlipressin and hydroxyethyl starch, which appears to be a safe and effective treatment.

Effect of the C825T polymorphism of the G protein beta 3 subunit on the systolic blood pressure-lowering effect of clonidine in young, healthy male subjects.

The T allele of the C825T polymorphism in the gene encoding the G-protein beta 3 subunit (GNB3) is associated with hypertension. An enhanced signal transduction in response to alpha(2)-adrenergic receptor stimulation has been shown in carriers of the T allele in vitro. We hypothesized that T allele carriers would show an enhanced antihypertensive response to stimulation of central alpha(2)-adrenergic receptors by clonidine. We compared the response to intravenous clonidine in 30 young, healthy male subjects with and without the T allele (15 CC, 10 CT, and 5 TT). Clonidine lowered blood pressure and total peripheral resistance, lengthened the duration of electromechanical systole (QS(2)c), and slowed down pulse wave velocity. Carriers of the T allele showed significantly greater reductions in systolic blood pressure (P =.009; mean change +/- SEM: CC, -8.9 +/- 0.5; CT and TT, -10.6 +/- 0.4) and total peripheral resistance (P <.0001; mean change +/- SEM: CC, 40 +/- 17; CT and TT, -48 +/- 14) and more marked lengthening of QS(2)c (P =.002; mean change +/- SEM: CC, 2.2 +/- 0.5; CT and TT, 4.7 +/- 0.6) and slowing of pulse wave velocity (P =.012; mean change +/- SEM: CC, -0.25 +/- 0.02; CT and TT, -0.33 +/- 0.03). The results of this study suggest that the 825T allele may be a relevant pharmacogenetic marker in the use of centrally acting sympatholytic drugs.

Augmentation index is associated with cardiovascular risk.

OBJECTIVES: Augmentation index is a parameter measured by pulse wave analysis (PWA) and is used as a surrogate measure of arterial stiffness. The aim of this study was to assess whether augmentation index is associated with cardiovascular risk, as well as to evaluate whether the determinants of augmentation index are different in patients with cardiovascular disease compared to healthy subjects. DESIGN AND METHODS: We related augmentation index to risk scores in 216 subjects with or without a cardiovascular disease. Subjects without cardiovascular disease were classified according to the 'coronary risk chart' of the European Society of Cardiology (ESC), and those with cardiovascular disease were classified using the SMART (Second Manifestations of ARTerial disease) score and the EPOZ (Epidemiological Prevention study Of Zoetermeer) function. Augmentation index was derived by PWA using carotid applanation tonometry. Augmentation index was also correlated to age, blood pressure, heart rate, smoking history, cholesterol, height, body mass index and gender in subjects categorized as healthy or with cardiovascular disease. RESULTS: Augmentation index significantly increased with increasing risk scores (P < 0.0001) and was significantly correlated to cardiovascular risk (ESC: P < 0.0001; SMART: P < 0.0001; EPOZ: P < 0.0001). In subjects with and without cardiovascular disease, augmentation index was correlated with diastolic blood pressure, heart rate, height and gender. Age was found to be significantly correlated with augmentation index only in healthy subjects but not in those with atherosclerotic disease. CONCLUSIONS: Our findings suggest that augmentation index may be a useful marker of cardiovascular risk. Further studies are required to investigate the relationship between age and augmentation index in subjects with atherosclerotic disease.

Enhanced vasoconstriction to endothelin-1, angiotensin II and noradrenaline in carriers of the GNB3 825T allele in the skin microcirculation.

Hypertension is associated with enhanced peripheral vascular resistance, which may be mediated by enhanced vasoconstriction. The impact of the recently detected G-protein beta3-subunit gene C825T polymorphism on the response to the major pressor mediators has been studied in vivo in the human microcirculation. We assessed the effects of endothelin-1 (ET-1), angiotensin II (AT), endothelin-antagonists (BQ-123 and BQ-788) and noradrenaline (NA, each 10-16-10-8 mol) on vasoconstriction in the human skin microcirculation in vivo in 25 healthy male volunteers (13 with CC genotype, 12 TC/TT genotype) using laser Doppler flowmetry. The effects of endothelium-derived vasodilation on NA-induced effects were studied using the NO-synthase inhibitor l-nitro-monomethyl-arginine (L-NMMA) and the alpha2-adrenoceptor-antagonist yohimbine (YO). ET-1, AT and NA caused a dose-dependent vasoconstriction (P < 0.001). In carriers of the 825T allele the response to ET-1, AT and NA was significantly enhanced leading to a shift to the left of the dose-response curve of up to two log units (ET-1: P < 0.001 vs. CC; AT: P < 0.01 vs. CC; NA: P < 0.05 vs. CC). After pretreatment with L-NMMA or YO, NA induced vasoconstriction was no longer different between subjects with the CC- and CT/TT genotypes. However, following combined pretreatment with both L-NMMA and YO, vasoconstriction to NA was significantly potentiated in carriers of the T-allele. Vasodilatation to an ETA-antagonist (BQ-123) was more pronounced in the CT/TT genotype, while ETB-antagonism (BQ-788) led to a more pronounced vasoconstriction in the CT/TT genotype (not significant vs. CC). Healthy, normotensive carriers of the 825T-allele have enhanced vasoconstriction to ET-1, AT and NA in the skin microcirculation. This enhanced vasoconstriction appears to be partially antagonized by an enhanced release of endothelium derived vasodilators mediated by the stimulation of endothelial alpha2-adrenoceptors. The GNB3 C825T polymorphism is potentially an attractive pharmacogenetic marker to predict hormone-mediated responses in humans.