Positive effects of aggressive vasodilator treatment of well-treated essential hypertensive patients.

Increased systemic vascular resistance and coronary microvascular dysfunction are well-documented in essential hypertension (EH). We investigated the effect of additional vasodilating treatment on coronary and peripheral resistance circulation in EH patients with high systemic vascular resistance index (SVRI) despite well-treated blood pressure (BP). We enroled patients on stable antihypertensive treatment that were given intensified vasodilating therapy (ACE inhibitor, angiotensin II receptor blocker or calcium channel blocker). Before and following 6 months of intensified therapy, coronary resting and maximal artery flow were measured by transthoracic Doppler echocardiography to calculate coronary flow reserve (CFR) and minimum vascular resistance (C-Rmin). Cardiac output was estimated by inert gas rebreathing to calculate SVRI. Maximal forearm blood flow was determined by venous occlusion plethysmography to calculate minimum vascular resistance (F-Rmin). Patients were assigned into two groups: high-SVRI and low-SVRI subgroups, based on a median split at baseline. Following additional treatment SVRI decreased more in the high-SVRI group than in the low-SVRI group (14.4 vs -2.2%: P=0.003), despite similar baseline ambulatory BP (132/81 mm Hg) and BP reduction (6.5 and 4.6%: P=0.19). F-Rmin remained unchanged (6.5 vs -2.0%: P=0.30), while C-Rmin decreased by 22 and 24% (P=0.80) and CFR increased by 23 and 17% (P=0.16). Thus, intensified vasodilating therapy improved SVRI more in patients with high SVRI than in those with low SVRI. Regardless of SVRI status, the treatment improved cardiac but not forearm dilatation capacity. The substantial improvement of the hypertensive cardiac microvascular dysfunction was not related to the reduction in SVRI.

Effect of cystamine on blood pressure and vascular characteristics in spontaneously hypertensive rats.

BACKGROUND: Tissue transglutaminase (t-TG) has been implicated in small artery remodelling. The aim of this study was to determine if cystamine, an inhibitor of t-TG, could reduce blood pressure in spontaneously hypertensive rats (SHR) and if so to what extent this is mediated through small arteries. METHODS: In vitro inhibition of t-TG, with cystamine, was studied in organ culture and wire myograph setups in small mesenteric arteries obtained from SHR. In vivo treatment with cystamine (80 mg/kg/day) or amlodipine (10 mg/kg/day) was performed with osmotic pumps in adult SHR, and hemodynamic parameters determined with telemetry. Plasma concentrations of cystamine were determined with a liquid chromatography setup. Small arteries were harvested following administration of cystamine, and structural as well as functional characteristics were determined. RESULTS: SHR small arteries showed inward remodelling following in vitro activation. Administration of cystamine caused attenuation of the inward remodelling induced by activation. In vivo administration of cystamine caused a 9 ± 2 mm Hg reduction in blood pressure, but with no detectable alterations in small artery structure. CONCLUSION: t-TG is potentially involved in vascular remodelling of SHR small arteries and results support a possible role for t-TG in blood pressure control.

Orthostatic hypotensive effect of antipsychotic drugs in Wistar rats by in vivo and in vitro studies of alpha1-adrenoceptor function.

RATIONALE: Many antipsychotics cause orthostatic hypotension possibly due to antagonist action on resistance vessel alpha1A-adrenoceptors (alpha1A-AR). OBJECTIVE: We have tested this possibility by determining in Wistar rats how the orthostatic hypotensive effect of several antipsychotic drugs compares with their affinity for adrenoceptors in mesenteric small arteries (MSA with mainly alpha1A-AR) and aorta (mainly alpha1D-AR). MATERIALS AND METHODS: Using a tilt setup, orthostatic hypotension was measured in anaesthetized rats for prazosin and the antipsychotics haloperidol, sertindole, risperidone, clozapine, ziprasidone, domperidone, olanzapine, and aripiprazole. For in vitro studies, segments of MSA and aorta were mounted on a wire myograph for isometric tension recording. Cumulative concentration-response curves were constructed to phenylephrine (PE) in the absence and presence of the drugs. Apparent affinity (pA2) was calculated by Schild analysis. RESULTS: Prazosin antagonized tilt-induced and PE responses in both studies (threshold 4 ng/ml, pA2 9.52 MSA, 10.1 aorta). The rank order of the potency of the antipsychotics in the tilt experiments correlated (r2 = 0.69, P = 0.01) with the pA2-values in MSA: Risperidone and sertindole had the highest potency in the tilt test (threshold 159 and 97 ng/ml) and the highest apparent affinity in MSA (pA2 8.92 and 8.78), in contrast with aripiprazole and domperidone, which had the lowest in each case (threshold 4.1 and 3.0 microg/ml, pA2 7.17 and 6.99). In aorta, the pA2 values did not correlate with the in vivo potencies; in particular, sertindole had no functional affinity in aorta. CONCLUSION: We conclude that the orthostatic hypotensive effect in rats of the antipsychotic drugs investigated is mediated through alpha1A-ARs.

Forearm plethysmography in the assessment of vascular tone and resistance vasculature design: new methodological insights.

AIM: High peripheral resistance and structural alteration in resistance arteries are central phenomena in essential hypertension and have been widely examined by forearm venous occlusion plethysmography; at rest for studying vascular tone, and during reactive hyperaemia for studying vascular structure. This work concerns the influence of venous pressure on hyperaemic vascular resistance (Rmin), the reproducibility of hyperaemic and resting vascular resistances (Rrest) and the relation between forearm and total peripheral vascular resistance (TPR). METHODS: In four healthy subjects, intravenous and intra-arterial blood pressures were measured simultaneously with plethysmographic recordings of hyperaemic and resting forearm blood flows. Reproducibility was examined in 15 young and 14 middle-aged healthy subjects and in 21 untreated hypertensive patients. RESULTS: Rmin remained low in the first recorded cardiac cycle, but rose in the second, even though corrected for the venous pressure rise, suggesting vascular tone recovery along with venous congestion. Between-day reproducibility of Rmin was high in middle-aged normotensive (8.7%) and hypertensive subjects (10.6%), but Rmin fell significantly between successive days in the young subjects. Rrest correlated with TPR, but required up to 40 min to reach steady state and showed high day-to-day variation in young (21.8%) and hypertensive subjects (16.2%). CONCLUSIONS: During hyperaemia, vascular resistance should be measured in the first cardiac cycle following venous occlusion to minimize influences of venous pressure rise and possible tone recovery. Rrest seems to reflect TPR. About 20 subjects may be needed to detect 15% changes between days in Rrest, fewer when concerning Rmin and TPR.

Combination of Ca2+ -activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery.

BACKGROUND AND PURPOSE: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation. EXPERIMENTAL APPROACH: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured. KEY RESULTS: A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ -activated K channels abolished acetylcholine (10 microM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 microM), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA. CONCLUSIONS AND IMPLICATIONS: Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.

Involvement of guanylyl cyclase, protein kinase A and Na+ K+ ATPase in relaxations of bovine isolated bronchioles induced by GEA 3175, an NO donor.

The present study was designed to investigate the role of the sodium potassium adenosine triphosphatase (the Na(+)K(+) ATPase) in relaxation of bovine isolated bronchioles by a new NO donor, GEA 3175 (3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulphonyl]amino]-)hydroxide)). Bronchioles were mounted in a wire myograph for isometric tension recordings and contracted with 5-hydroxytryptamine (5-HT) or a K(+) rich solution. Concentration-dependent relaxations evoked by GEA 3175 were inhibited by ouabain or K(+) free solution. The guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 microM) and ouabain (10 nM) reduced GEA 3175-evoked relaxations to the same extent without any additive effect. Iberiotoxin (10 nM), an inhibitor of large conductance Ca(2+)-activated K(+) channels inhibited GEA 3175-evoked relaxations to the same extent as ouabain. Combining ouabain and iberiotoxin completely abolished GEA 3175 relaxation. An inhibitor of protein kinase G (PKG), Rp-beta-phenyl-1,N(2)-etheno-8-bromo-guanosine-3'-5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPs), slightly reduced GEA 3175-induced relaxations. An inhibitor of cyclic AMP-dependent kinase (PKA), Rp-adenosine-3'-5'-cyclic phosphorothioate (Rp-cAMPs), inhibited the GEA 3175-induced relaxations to the same extent as ouabain. Inhibition of both PKG and PKA abolished GEA 3175 relaxation. The study provides evidence that the NO donor GEA 3175 causes guanylyl cyclase-dependent relaxations, taking place through cyclic GMP and cyclic AMP-dependent protein kinases followed by opening of large conductance Ca(2+)-activated K(+) channels and activation of smooth muscle Na(+)K(+) ATPase.

Structure of renal afferent arterioles in the pathogenesis of hypertension.

Renal vascular resistance is increased in essential hypertension, as in genetic models of hypertension. Here we review the evidence that this is at least in part due to structural changes in the afferent arterioles. Rat studies show that the renal afferent arteriole is structurally narrowed in young and adult spontaneously hypertensive rats (SHR). Furthermore, in the second generation of crossbred SHRs/normotensive rats (SHR/WKY F(2)-hybrids), a narrowed afferent arteriole lumen diameter at 7 weeks is a predictor of later development of high blood pressure. The reduced lumen diameter of resistance vessels is accompanied by a decrease in media cross-sectional area in SHR and could therefore be due to inhibited growth. Evidence from a primate model of hypertension has shown a negative correlation between left ventricular hypertrophy and afferent arteriole diameter, but apparently no relation to blood pressure. In SHR, the antihypertensive effect of angiotensin converting enzyme (ACE) inhibitors is mediated through renal vascular mechanisms, while ACE inhibitors (like AT(1) antagonists) have a more persistent effect on blood pressure after treatment withdrawal compared with other antihypertensive drugs. Taken together, the evidence suggests that structural narrowing of the renal afferent arteriole could be an important link in the pathogenesis of primary hypertension, at least in the SHR.

Force-independent expression of c-fos mRNA by endothelin-1 in rat intact small mesenteric arteries.

AIM: Wall stress-independent signalling pathways were studied for endothelin-1 (ET-1)-induced c-fos expression in rat intact mesenteric small arteries. METHODS: Arteries were kept unmounted in Krebs buffer, equilibrated for 1 h and stimulated with vasoactive substances for 15-60 min. The c-fos mRNA expression was determined by real-time polymerase chain reaction. RESULTS: Stimulation with fetal bovine serum (FBS), phorbol 12-myristate 13-acetate (PMA) and ET-1 caused about a doubling of c-fos mRNA. The ET-1-induced c-fos expression was steady (15-60 min) and was inhibited by the inhibitor of the ET(A) receptor, BQ-123. Platelet-derived growth factor-B, angiotensin II and U46619 did not cause increased c-fos mRNA levels. The broad specificity inhibitor staurosporine inhibited the response to ET-1, but inhibitors of Rho-A kinase and phosphatidylinositol 3-kinase had no effect. However, inhibitors to tyrosine kinases, the MAP kinases [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase, p38], and to conventional protein kinase C showed no inhibition. Consistent with these findings, ET-1 did not cause activation of ERK1/2, a finding also seen in vessels held under pressure. In contrast, ET-1-induced c-fos expression was inhibited by the calcium chelator BAPTA, suggesting a role for intracellular calcium. This possibility was supported by the finding that raising the extracellular K(+) concentration caused increased expression of c-fos in a concentration-dependent manner. CONCLUSION: The results suggest that in the absence of wall stress, ET-1 is able to induce increased expression of c-fos independent of traditional growth pathways, such as MAP kinase. The mechanism appears to be calcium-dependent.

Influence of nitric oxide synthase and adrenergic inhibition on adenosine-induced myocardial hyperemia.

BACKGROUND: Myocardial perfusion during adenosine-induced hyperemia is used both in clinical diagnosis of coronary heart disease and for scientific investigations of the myocardial microcirculation. The objective of this study was to clarify whether adenosine-induced hyperemia is dependent on endothelial NO production or is influenced by adrenergic mechanisms. METHODS AND RESULTS: In 12 healthy men, myocardial perfusion was measured with PET in 2 protocols performed in random order, each including 3 perfusion measurements. First, perfusion was measured at rest. Second, either saline or the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 4 mg/kg) was infused, and perfusion during adenosine-induced hyperemia was determined. Last, in both protocols, the alpha-receptor blocker phentolamine was infused, and perfusion during adenosine-induced hyperemia was determined again. Resting perfusion was similar in the 2 protocols (0.69+/-0.14 and 0.66+/-0.18 mL. min(-1). g(-1)). L-NAME increased mean arterial blood pressure by 12+/-7 mm Hg (P<0.01) and reduced heart rate by 16+/-7 bpm (P<0.01). Adenosine-induced hyperemia (1.90+/-0.33 mL. min(-1). g(-1)) was attenuated by L-NAME (1.50+/-0.55 mL. min(-1). g(-1), P<0.01). The addition of phentolamine had no effect on the adenosine-induced hyperemia (2.10+/-0.34 mL. min(-1). g(-1), P=NS). In the presence of L-NAME, however, when the adenosine response was attenuated, phentolamine was able to increase hyperemic perfusion (2.05+/-0.44 mL. min(-1). g(-1), P<0.05). CONCLUSIONS: Inhibition of endogenous NO synthesis attenuates myocardial perfusion during adenosine-induced hyperemia, indicating that coronary vasodilation by adenosine is partly endothelium dependent. alpha-Adrenergic blockade has no effect on adenosine-induced hyperemia unless NO synthesis is inhibited.

Initial and sustained phases of myogenic response of rat mesenteric small arteries.

A possible role for a metabolite of cytochrome P-450 omega-hydroxylase in the initial and sustained phases of the myogenic response in cannulated rat mesenteric small arteries was studied. With slight preconstriction (norepinephrine and neuropeptide Y), pressure was raised from 60 to 100 mmHg, and both initial (within 2 min) and sustained phases (at 10 min) of the myogenic response were quantified. The myogenic response was fully inhibited by D600 (methoxyverapamil). Ketoconazole and 17-octadecanoic acid did not affect the initial phase but inhibited the sustained phase. In contrast, miconazole did not affect either phase. Charybdotoxin and iberiotoxin potentiated the initial phase but eliminated the sustained phase. Apamin, glibenclamide, 4-aminopyridine, and barium had no effect on either phase. The results demonstrate different mechanisms for the initial and sustained phases of the myogenic response of rat mesenteric small arteries. Only the sustained phase appears mediated through a cytochrome P-450 omega-hydroxylase metabolite and calcium-activated K+ channels. However, both phases of the response are dependent on calcium influx through voltage-dependent calcium channels.

Reduced medication and normalization of vascular structure, but continued hypertension in renovascular patients after revascularization.

OBJECTIVE: The effect of invasive treatment of renal artery stenosis on the use of antihypertensive medication, blood pressure, and morphology and function of resistance arteries was investigated in 14 renovascular hypertensive patients before and after treatment compared to normotensive controls. METHODS: Use of antihypertensive medication was calculated as defined daily doses (DDD). Resistance arteries were taken from gluteal subcutaneous biopsies and analyzed in a myograph. RESULTS: Prior to invasive treatment, blood pressure of the patients was elevated compared to normotensive controls. Six months after technically successful invasive treatment, patients were still hypertensive compared to time-matched controls. The use of antihypertensive medication was reduced from 4.4+/-0.7 DDD before invasive treatment to 3.0+/-0.6 DDD 6 months after treatment. Vascular structure of resistance arteries, expressed as media/lumen ratio (media thickness to diameter), was greater in patients before invasive treatment (10.7+/-1.0%) compared to normotensive controls (7.9+/-0.8%). Media/lumen ratio of resistance arteries was reduced to that of the controls 6 months after invasive treatment despite the remaining hypertension. The functional studies showed no difference in response to acetylcholine, adenosine, noradrenaline or angiotensin II between patients and controls before or after treatment. CONCLUSIONS: This study shows that hypertension and increased media/lumen ratio of resistance arteries prevail in renovascular hypertensive patients despite antihypertensive medication and that invasive treatment is of benefit as regards use of antihypertensive medication. The study provides the novel information that the remaining hypertension is not due to uncorrected media/lumen ratio of the resistance arteries.

Smooth muscle cell changes during flow-related remodeling of rat mesenteric resistance arteries.

To obtain information on the molecular and cellular mechanisms of flow-induced arterial remodeling, we analyzed the morphology and smooth muscle cell (SMC) characteristics in rat mesenteric resistance arteries after interventions that decreased and increased flow. Juvenile male Wistar Kyoto rats were subjected to surgery that, compared with control arteries, provided arteries with chronic low flow and chronic high flow. Low flow resulted in a decreased passive lumen diameter, hypotrophy of the artery wall, and both loss and decreased size of SMCs. Time course studies, with intervention length ranging from 2 to 32 days of altered blood flow, showed that the narrowing of the lumen diameter in low-flow arteries appeared within 2 days and that an early dedifferentiation of SMC phenotype was indicated by markedly reduced levels of desmin mRNA. High flow resulted in an increased passive lumen diameter and in hypertrophy of the artery wall. The hypertrophy resulted from SMC proliferation because SMC number, measured by the 3D-dissector technique, was increased and immunohistochemical assessment of proliferating cell nuclear antigen also showed an increase. The widening of high-flow arteries required 16 days to become established, at which time desmin mRNA was reduced. This time was also required to establish changed wall mass in both low-flow and high-flow arteries. Apoptotic cells detected by TdT-mediated dUTP-biotin nick end labeling staining were mainly located in the medial layer, and evaluation of DNA fragmentation indicated that increased apoptosis occurred in both low flow and high flow. This study shows for the first time direct evidence that reduced and elevated blood flow in resistance arteries produce, respectively, decrease and increase in SMC number, with dedifferentiation of the SMCs in both cases.

Vasodilatation, not hypotension, improves resistance vessel design during treatment of essential hypertension: a literature survey.

Correction of structural abnormalities in resistance arteries of patients with essential hypertension is a potential treatment goal, in addition to blood pressure reduction. However, available evidence from human as well as from animal studies indicates that antihypertensive therapy is not always accompanied by normalization of resistance vessel structure, despite normalization of blood pressure. Thus, blood pressure is not the only factor determining resistance vessel structure, and experimental studies show that several factors could play a role, including shear stress and hormonal stimulation. To date, there has been no systematic review of the many published papers which have studied the structural effects of antihypertensive therapy, and it is not known which conditions are best able to normalize resistance vessel structure. We have therefore made a survey of the available literature. The survey shows that change in blood pressure in indeed a poor indicator of change in resistance vessel structure. However, it is a remarkably consistent finding that normalization of resistance vessel structure is obtained with therapeutic regimens which reduce blood pressure by vasodilation rather than by lowering cardiac output Thus, to the extent that normalization of resistance vessel structure is deemed a goal of antihypertensive treatment, the survey points towards the importance of considering not only the treatment effect on blood pressure, but also the haemodynamic effects within patients with essential hypertension.

Morphology of renal afferent arterioles and glomeruli, heart weight, and blood pressure in primates.

In a Caribbean outbred population of African green monkeys (Cercopithecus aethiops), 5 to 10% of feral adults have elevated blood pressure (BP). We have investigated whether the increased pressure is associated with abnormal renal afferent arteriole structure or glomerular number. In seven young adult (aged 7 to 13 years) male monkeys with consistently high BP (mean BP, 111 mm Hg; ketamine anesthesia) and seven controls (mean BP, 81 mm Hg), the morphology of the renal vasculature has been analyzed in three cortical zones. In each animal, the left kidney vasculature was fixed while relaxed and at known intravascular pressure, and afferent arteriolar diameter and media cross-sectional area were estimated. The right kidney was perfusion-fixed and prepared for unbiased stereologic estimation of glomerular number and size. No difference was found in afferent arteriole lumen diameter or media cross-sectional area, or in glomerular number or size, between the high BP group and controls. There was no difference in heart weight between the two groups, but there was a negative correlation between left ventricle heart weight and afferent arteriole diameter (controls: r = -0.81, P = .025; all animals: r = -0.70, P = .005, slope about 3.5% reduction in lumen diameter for 10% increase in heart weight). The results suggest that cardiac mass and renal afferent arteriole structure may be controlled by a common mechanism unrelated to BP measured in anesthesia. However, the lack of conscious measurements prevents conclusions as to whether this mechanism involves ambulatory BP.

Location of resistance arteries.

Thickening and narrowing of resistance arteries must, by definition, be key elements in the control of the cardiovascular system. However, the precise location of resistance arteries is difficult to establish. This is due to technical problems related to the small size of the vessels, to the measurement conditions disturbing the hemodynamics, and to the status of the animals while the measurements are being made. Furthermore, due to large data heterogeneity, previous studies do not give unequivocal information concerning the pressure profile in the vascular system, or the level of arterial diameter responsible for blood flow. Finally, and importantly, there is little evidence regarding the conscious state, which is thus a major limitation to understanding the mechanisms of blood distribution and the pathogenesis for disease processes such as genetic hypertension. This review first summarizes briefly the techniques which are available for identifying resistance arteries and the inherent technical limitations which are involved. The review then provides a critical assessment of the available data, both as regards measurement of local blood pressures and as regards control of peripheral resistance. The evidence suggests that, at least as regards rats and other small animals, feed arteries as well as more distal microvessels contribute to the maintenance and regulation of blood flow and resistance. Evidence from larger animals is however lacking, and it is thus unclear if resistance function should be based on arterial diameter or anatomic location. Furthermore, evidence concerning man is not available. We therefore conclude the review with suggestions for future research in this area.

Angiotensin II stimulates extracellular signal-regulated kinase activity in intact pressurized rat mesenteric resistance arteries.

The activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was assessed in isolated rat mesenteric resistance arteries (200-micrometer diameter) in a pressure myograph and stimulated for 5 minutes by angiotensin II (Ang II, 0.1 micromol/L) with a pressure of 70 mm Hg. ERK1/2 activity was measured by using an in-gel assay, and ERK1/2 phosphorylation was measured by Western blot analysis with use of a phospho-specific ERK1/2 antibody. Ang II (0.1 micromol/L) induced contraction (28% of phenylephrine contraction, 10 micromol/L). ERK kinase inhibitor PD98059 (10 micromol/L) attenuated this contraction by 36% but not that to phenylephrine or K(+) (60 mmol/L). In unpressurized arteries, Ang II increased ERK1/2 activity by 26%, and pressure (70 mm Hg) itself increased ERK1/2 activity by 72%. Ang II and pressure together acted synergistically, increasing ERK1/2 activity by 264%. Thus, in pressurized vessels, Ang II (0.1 micromol/L) increased ERK1/2 activity by 112%, calculated as [(364/172)-1]x100, which was confirmed by a measured 72% increase in ERK1/2 phosphorylation. Ang II type 1 receptor blockade by candesartan (10 micromol/L) abolished the Ang II-induced increase in ERK1/2 activity, but Ang II type 2 receptor blockade (PD123319, 10 micromol/L) did not. The Ang II-induced increase in ERK1/2 activity was inhibited by protein kinase C inhibitors Ro-31-8220 (1 micromol/L) and Go-6976 (300 nmol/L) and tyrosine kinase inhibitors genistein (1 micromol/L, general) and herbimycin A (1 micromol/L, c-Src family). The present findings show for the first time in intact resistance arteries that ERK1/2 activation is rapidly regulated by Ang II, is synergistic with pressure, and is involved in contraction. The ERK1/2 signaling pathway apparently includes upstream protein kinase C and c-Src.

Effect of mitogens on growth and contractile responses of rat small arteries: In vitro studies.

Rat mesenteric and epigastric small arteries were cultured to investigate influences of mitogens on contractility, proliferation and protein synthesis. Wistar rat arteries were cultured in serum-free Dulbecco's Modified Eagle Medium, first, for 24 h to equilibrate and then for a further 24-48 h either in the absence or presence of test substances: angiotensin II (AII), 1 microM; AII, 1 microM + platelet derived growth factor BB-chain (PDGF-BB), 1 ng mL-1; PDGF-BB, 1 ng mL-1; PDGF-BB, 30 ng mL-1. No mechanical stress was applied. Viability was assessed by myography, protein synthesis by 6-h incorporation of 35S-methionine and proliferation by both 48-h 3H-thymidine-incorporation and immunohistochemical analysis using the thymidine analogue 5-bromo-2'-deoxyuridine. After 3 days in culture, the contractile responses of arteries to phenylephrine, serotonin, AII and PDGF-BB were preserved. Stimulation with PDGF-BB (30 ng mL-1) increased protein synthesis 1.5- (mesenteric) and 1. 9-fold (epigastric). Similarly, stimulation with PDGF-BB (30 ng mL-1) increased 3H-thymidine incorporation of unstimulated arteries 3.4- (mesenteric) and 2.8-fold (epigastric). The other treatments affected neither protein synthesis nor proliferation. Immunohistochemical analysis showed that the proliferation was occurring primarily in the adventitia and that the levels of apoptosis were unaltered by culture. The effects of AII and PDGF-BB on remodelling did not correlate with their contractile effects: epigastric arteries responded strongly to AII and PDGF-BB, while mesenteric arteries responded weakly. The results suggest that organ culture conditions which preserve contractile function may not be sufficient to preserve trophic mechanisms.

Cellular hypertrophy in subcutaneous small arteries of patients with renovascular hypertension.

Structural alterations of small arteries in patients with essential hypertension are characterized by inward eutrophic remodeling. However, small arteries in patients with secondary hypertension, as well as in experimental models of hypertension with high circulating renin, are characterized by inward hypertrophic remodeling, which is characterized by smooth muscle cell hypertrophy in animal models. The aim of our study was to determine whether remodeling of subcutaneous small arteries in patients with secondary forms of hypertension is associated with smooth muscle cell hypertrophy and/or alterations in the elastic modulus of the vessel wall. Fifteen patients with renovascular hypertension, 9 with primary aldosteronism, and 13 with essential hypertension and 9 normotensive subjects were included in the study. A biopsy of subcutaneous fat was taken from all subjects. Small arteries were dissected, and morphology was determined on a micromyograph. Unbiased estimates of cell volume and number were made in fixed material. From the resting tension-internal circumference relation of the small arteries, the incremental elastic modulus was calculated and plotted as a function of wall stress. Blood pressure was greater in patients with essential hypertension, renovascular hypertension, or primary aldosteronism than in normotensive subjects, but no significant difference was observed among the 3 groups of hypertensive patients. The media/lumen ratio, the medial cross-sectional area, and the smooth muscle cell volume were significantly greater in patients with renovascular hypertension than in normotensive subjects and patients with essential hypertension. No difference in cell number or in the elastic properties was observed among the 4 groups of subjects. In conclusion, our data demonstrate for the first time that a pronounced activation of the renin-angiotensin-aldosterone system is associated with vascular smooth muscle cell hypertrophy in human hypertension in a manner similar to that found in animal models.

Neuropeptide Y regulates intracellular calcium through different signalling pathways linked to a Y(1)-receptor in rat mesenteric small arteries.

Simultaneous measurements of intracellular calcium concentration ([Ca(2+)](i)) and tension were performed to clarify whether the mechanisms which cause the neuropeptide Y (NPY)-elicited contraction and potentiation of noradrenaline contractions, and the NPY inhibition of forskolin responses are linked to a single or different NPY receptor(s) in rat mesenteric small arteries. In resting arteries, NPY moderately elevated [Ca(2+)](i) and tension. These effects were antagonized by the selective Y(1) receptor antagonist, (R)-N(2)-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]-D-argininea mide (BIBP 3226) (apparent pK(B) values of 8.54+/-0.25 and 8.27+/-0.17, respectively). NPY (0.1 microM) caused a near 3 fold increase in sensitivity to noradrenaline but did not significantly modify the tension-[Ca(2+)](i) relationship for this agonist. BIBP 3226 competitively antagonized the contractile response to NPY in arteries submaximally preconstricted with noradrenaline (pA(2) 7.87+/-0.20). In arteries activated by vasopressin, the adenylyl cyclase activator forskolin (3 microM) induced a maximum relaxation and a return of [Ca(2+)](i) to resting levels. NPY completely inhibited these effects. The contractile responses to NPY in arteries maximally relaxed with either sodium nitroprusside (SNP) or nifedipine were not significantly higher than those evoked by the peptide at resting tension, in contrast to the contractions to NPY in forskolin-relaxed arteries. BIBP 3226 competitively antagonized the contraction to NPY in forskolin-relaxed arteries with a pA(2) of 7.92+/-0.29. Electrical field stimulation (EFS) at 8-32 Hz caused large contractions in arteries relaxed with either forskolin or noradrenaline in the presence of phentolamine. These responses to EFS were inhibited by BIBP 3226. Similar EFS in resting, non-activated arteries did not produce any response. The present results suggest that different intracellular pathways are linked to a single NPY Y(1) receptor in intact rat mesenteric small arteries, and provide little support for involvement of other postjunctional NPY receptors in the contractile responses to NPY. Neurally released NPY also seems to act through Y(1) receptors, and may serve primarily as an inhibitor of vasodilatation.

Nitric oxide, prostanoid and non-NO, non-prostanoid involvement in acetylcholine relaxation of isolated human small arteries.

The main purpose of the study was to clarify to which extent nitric oxide (NO) contributes to acetylcholine (ACh) induced relaxation of human subcutaneous small arteries. Arterial segments were mounted in myographs for recording of isometric tension, NO concentration and smooth muscle membrane potential. In noradrenaline-contracted arteries, ACh induced endothelium-dependent relaxations. The NO synthase inhibitor, N(G)-nitro-L-arginine (L-NOARG) had a small significant effect on the concentration-response curves for ACh, and in the presence of L-NOARG, indomethacin only caused a small additional rightward shift in the ACh relaxation. The NO scavenger, oxyhaemoglobin attenuated relaxations for ACh and for the NO donor S-nitroso-N-acetylpenicillamine (SNAP). Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), and inhibition of protein kinase G with beta-phenyl-1, N2-etheno-8-bromoguanosine- 3', 5'- cyclic monophosphorothioate, Rp-isomer, slightly attenuated ACh relaxation, but abolished SNAP induced relaxation. ACh induced relaxation without increases in the free NO concentration. In contrast, for equivalent relaxation, SNAP increased the NO concentration 32+/-8 nM. ACh hyperpolarized the arterial smooth muscle cells with 11.4+/-1.3 mV and 10.5+/-1.3 mV in the absence and presence of L-NOARG, respectively. SNAP only elicited a hyperpolarization of 1.6+/-0.9 mV. In the presence of indomethacin and L-NOARG, ACh relaxation was almost unaffected by lipoxygenase inhibition with nordihydroguaiaretic acid, or cytochrome P450 inhibition with 17-octadecynoic acid or econazole. ACh relaxation was strongly reduced by the combination of charybdotoxin and apamin, but small increments in the extracellular potassium concentration induced no relaxations. The study demonstrates that the NO/L-arginine pathway is present in human subcutaneous small arteries and to a limited extent is involved in ACh induced relaxation. The study also suggests a small contribution of arachidonic acid metabolites. However, ACh relaxation is mainly dependent on a non-NO, non-prostanoid endothelium dependent hyperpolarization. British Journal of Pharmacology (2000) 129, 184 - 192