Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress.

AIMS/HYPOTHESIS: Impaired nitric oxide (NO)-dependent vasorelaxation plays a key role in the development of diabetic vascular complications. We investigated the effect of hyperglycaemia on impaired vasoreactivity and a putative role therein of the AGE precursor methylglyoxal. METHODS: The effects of high glucose and methylglyoxal on NO-dependent vasorelaxation in isolated rat mesenteric arteries from wild-type and transgenic glyoxalase (GLO)-I (also known as GLO1) rats, i.e. the enzyme detoxifying methylglyoxal, were recorded in a wire myograph. AGE formation of the major methylglyoxal-adduct 5-hydro-5-methylimidazolone (MG-H1) was detected with an antibody against MG-H1 and quantified with ultra-performance liquid chromatography (tandem) mass spectrometry. Reactive oxygen species formation was measured with a 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester probe and by immunohistochemistry with an antibody against nitrotyrosine. RESULTS: High glucose and methylglyoxal exposure of mesenteric arteries significantly reduced the efficacy of NO-dependent vasorelaxation (p < 0.05). This impairment was not observed in mesenteric arteries of GLO-I transgenic rats indicating a specific intracellular methylglyoxal effect. The diabetes-induced impaired potency (pD(2)) in mesenteric arteries of wild-type rats was significantly improved by GLO-I overexpression (p < 0.05). Methylglyoxal-modified albumin did not affect NO-dependent vasorelaxation, while under the same conditions the receptor for AGE ligand S100b did (p < 0.05). Methylglyoxal treatment of arteries increased intracellular staining of MG-H1 in endothelial cells and adventitia by fivefold accompanied by an eightfold increase in the oxidative stress marker nitrotyrosine. Antioxidant pre-incubation prevented methylglyoxal-induced impairment of vasoreactivity. CONCLUSIONS/INTERPRETATION: These data show that hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation is mediated by increased intracellular methylglyoxal levels in a pathway dependent on oxidative stress.

Mechanisms leading to increased vasodilator responses to calcitonin-gene-related peptide in mesenteric resistance arteries of early pregnant rats.

The objective of this study was to explore the mechanism responsible for the higher relaxing responses of mesenteric arteries to calcitonin-gene-related peptide (CGRP) in pregnancy. We performed myograph and ligand binding studies to determine the role of matrix metalloproteinase-2 (MMP-2) and CGRP receptor density. MMP activity was manipulated in isolated arteries by exposing them to the blocking effects of doxycycline. Vascular activity of MMP-2 was studied by gelatin zymography, and CGRP receptor density was determined by ligand binding analysis. Compared to nonpregnant rats, CGRP elicited stronger arterial relaxation in pregnant rats. The latter effect was neither accompanied by a change in relaxing responses to direct activation of adenylyl cyclase by forskolin nor by a change in the response to stimulation of G-protein-coupled adrenergic receptors by isoproterenol. Doxycycline did not affect the stronger arterial relaxation in pregnancy in spite of the observed more than threefold higher arterial MMP-2 activity. Density of binding sites for [(125)I]CGRP in arteries from pregnant rats (64 +/- 14 fmol/mg protein) and from virgin rats (54 +/- 5 fmol/mg protein) were comparable. The results of this study provide evidence for increased coupling of CGRP receptors to adenylyl cyclase in early pregnancy.

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.

Chronic hypoxia stimulates periarterial sympathetic nerve development in chicken embryo.

BACKGROUND: Epidemiological findings suggest an association between low-for-age birth weight and the risk to develop coronary heart diseases in adulthood. During pregnancy, an imbalance between fetal demands and supply may result in permanent alterations of neuroendocrine development in the fetus. We evaluated whether chronic prenatal hypoxia increases arterial sympathetic innervation. METHODS AND RESULTS: Chicken embryos were maintained from 0.3 to 0.9 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic conditions (15% O(2)). At 0.9 incubation, the degree of sympathetic innervation of the embryonic femoral artery was determined by biochemical, histological, and functional (in vitro contractile reactivity) techniques. Chronic hypoxia increased embryonic mortality (32% versus 13%), reduced body weight (21.9+/-0.4 versus 25.4+/-0.6 g), increased femoral artery norepinephrine (NE) content (78.4+/-9.4 versus 57.5+/-5.0 pg/mm vessel length), and increased the density of periarterial sympathetic nerve fibers (14.4+/-0.7 versus 12.5+/-0.6 counts/10(4) microm(2)). Arteries from hypoxic embryos were less sensitive to NE (pD(2), 5.99+/-0.04 versus 6. 21+/-0.10). In the presence of cocaine, however, differences in sensitivity were no longer present. In the embryonic heart, NE content (156.9+/-11.0 versus 108.1+/-14.7 pg/mg wet wt) was also increased after chronic hypoxia. CONCLUSIONS: In the chicken embryo, chronic moderate hypoxia leads to sympathetic hyperinnervation of the arterial system. In humans, an analogous mechanism may increase the risk for cardiovascular disease in adult life.

Remodeling of the vascular tree in hypertension: drug effects.

The primary aim of current antihypertensive therapy is to lower blood pressure through the reduction of peripheral vascular resistance. Resistance reduction is achieved primarily by interference with acutely acting pressor stimuli. However, recent research has stressed the importance of slow pressor stimuli, which act by gradually remodeling the vascular tree. Long-term remodeling is achieved by a chronic change in vessel number, vascular diameter or wall thickness, involving both physical and chemical factors. The chemical mediators belong to a group of endogenous growth-affecting factors. As Harry Struyker Boudier and colleagues explain in this review, although there are thus far no specific drugs to antagonize the effects of these factors, several therapeutically used antihypertensives influence their action. Moreover, the concept that vascular remodeling is important in hypertension offers exciting new therapeutic targets.

Chronic N(G)-nitro-L-arginine methyl ester treatment does not prevent flow-induced remodeling in mesenteric feed arteries and arcading arterioles.

Although endothelium-derived NO is an important mediator in acute flow-induced changes in arterial tone, the role of NO in chronic flow-induced changes in the resistance artery and arteriolar structure remains largely unresolved. We investigated the effects of chronic inhibition of NO synthase on arterial and arteriolar remodeling in a rat mesenteric model in which flow changes were induced. Alternating first-order mesenteric arteries were ligated to shunt blood flow through the intermittent patent arteries. Animals received no treatment (NT) or a continuous infusion of N:(G)-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg SC per day). After 2 weeks, local in vivo blood flow and in vitro arterial pressure-diameter relationships were assessed, as were the in situ diameters of arcading arterioles. Medial cross-sectional areas (CSAs) were measured histologically. In both groups of animals, blood flow was significantly increased in patent arteries and decreased in ligated arteries compared with control vessels. Nonetheless, in L-NAME-treated rats, patent artery flow was increased to a lesser extent, although control flow was not significantly reduced (0.18+/-0.05 versus 0.26+/-0.05 mL/min). In NT rats, the diameter of patent arteries was significantly larger and the diameter of ligated arteries was significantly smaller than that of control arteries. CSAs displayed the same pattern of change (11. 9+/-0.6 x 10(3), 6.1+/-0.7 x 10(3), and 8.2+/-1.0 x 10(3) microm(2) for patent, ligated, and control arteries, respectively). Arterioles in the NT collateral pathway (218+/-15 microm) had diameters similar to control arteriole diameters (201+/-15 microm) but had a significantly larger CSA (6.2+/-0.6 x 10(3) versus 4.2+/-0.4 x 10(3) microm(2)). In L-NAME-treated rats, the flow-induced changes of the diameter and CSA in patent arteries, ligated arteries, and arcading arterioles mimicked those in NT rats. Nonetheless, control feed arteries (430+/-21 versus 497+/-16 microm) and arcading arterioles (156+/-21 microm) were significantly narrower after L-NAME treatment. Thus, chronic blockade of NO oxide synthase (1) tended to reduce arterial blood flow and resulted in inward remodeling of mesenteric arteries and arterioles and (2) did not prevent arterial and arteriolar remodeling in response to imposed changes in blood flow. Endothelium-derived mediators other than NO can play a major role in flow-induced arterial remodeling.

The influence of angiotensin II-induced increase in aortic wall mass on compliance in rats in vivo.

OBJECTIVE: This study was undertaken to examine the effects of angiotensin II-induced structural changes in the aortic wall on the dynamic mechanical properties of the vessel in the rat. METHODS: Wistar rats were infused s.c. with 250 ng/kg/min angiotensin II (Ang II) for 14 days (ANG). Both ANG and control rats (CON) were equipped with an arterial catheter for measurement of arterial blood pressure. Thoracic aorta diameter, compliance coefficient (CC), and distensibility coefficient (DC) were determined non-invasively in pentobarbital-anesthetized animals using a B-mode imager attached to a vessel wall tracking system. After sacrifice, medial cross-sectional area (CSA), and elastin and collagen densities were determined by morphometry on cross-sections. Media thickness (Mt) and wall-to-lumen ratio (W/L) were subsequently calculated. RESULTS: Ang II infusion significantly increased mean arterial blood pressure in conscious rats (122 +/- 3 mmHg CON vs. 157 +/- 4 mmHg ANG). This was normalized when the rats were anesthetized, thus making it possible to determine CC and DC under isobaric conditions where the diastolic diameters were also similar. Two-week infusion of Ang II induced a significant increase in CSA from 0.48 +/- 0.02 mm2 in CON to 0.61 +/- 0.03 mm2. Mt and W/L were likewise increased, but collagen and elastin densities remained unchanged. CC and DC were not effected by this increase in aortic wall mass (CC: 0.143 +/- 0.009 CON, 0.147 +/- 0.014 mm2/kPa ANG; DC: 0.052 +/- 0.005 CON, 0.051 +/- 0.004 kPa-1 ANG). CONCLUSIONS: An increase in aortic wall mass resulting from chronic infusion of angiotensin II does not alter the dynamic compliance of the vessel under isobaric conditions.

Role of basal nitric oxide synthesis in vasoconstrictor hyporeactivity in the perfused rat hindlimb after myocardial infarction: effect of captopril.

OBJECTIVES: The contribution of vascular changes to the development of heart failure is largely unknown. In the present study, we evaluated endothelial and vascular contractile function in the rat hindlimb vascular bed after myocardial infarction (MI), including the modulatory role of basal nitric oxide (NO) production and the effects of treatment with the angiotensin converting enzyme inhibitor captopril on vascular function. METHODS: MI was induced in male Wistar rats by ligation of the left coronary artery. Acetylcholine-induced dilatations were assessed in the ex vivo perfused hindlimb at various time points. At 2 and 5 weeks post-MI, vascular contractile function in the perfused hindlimb was assessed from resistance changes induced by 35 mM and 125 mM potassium (K+) and the maximum increase in resistance (delta Rmax, 125 mM K+ and 3 mg phenylephrine). Basal NO synthesis was blocked for 2 weeks with L-nitro-arginine methylester (L-NAME) in sham and MI rats and similar contractility experiments were performed. The effect of captopril treatment from 2 to 5 weeks post-MI on vasoconstrictor responses was also tested. RESULTS: Acetylcholine-induced dilatations in the presence of 10 microM indomethacin were not different between sham and MI rats. Vasoconstrictor responses to K+ and delta Rmax were reduced at 2 weeks after MI. This reduction in vasoconstrictor ability was similar to that seen in L-NAME-treated sham rats, while chronic L-NAME treatment did not affect vasoconstrictor reactivity in MI rats. Similarly, L-NAME induced an increase in mean arterial pressure in sham rats, but not in MI rats. At 5 weeks after MI, vasoconstriction to 125 mM K+ and delta Rmax were still reduced in MI rats; this response was however partially restored after captopril treatment. CONCLUSION: The development of vascular contractile hyporeactivity in the rat hindlimb after MI may be due to reduced basal NO production. Delayed treatment with captopril improves peripheral vascular contractile function in this setting.

Peripheral blood flow and noradrenaline responsiveness: the effect of physiological hyperinsulinemia.

OBJECTIVE: Insulin seems to have vasodilator properties, but it is unclear if insulin in postprandial concentrations is a specific vasodilator of skeletal muscle resistance arterioles only or that various types of vessels are affected. The aim of the present study was to determine the direct effects and the time course of regional/local physiological hyperinsulinemia on skeletal muscle arterioles, skin blood flow and peripheral venous tone and the responsiveness of these different vascular beds to noradrenaline. METHODS: In protocol I (n = 12) we infused insulin into the brachial artery for 180 min (3.5 mU/min) and evaluated the effects on forearm (muscle) blood flow (FBF) and skin blood flow (SBF). Furthermore, noradrenaline (0.025, 0.01 and 0.4 microgram/min) was infused (i.a.) at baseline, at 90 and 180 min after the start of insulin. In protocol 2 (n = 10) the same regional forearm hyperinsulinemia was achieved, but now the local venous responsiveness to noradrenaline (1.7-55 ng/min, at baseline and at 90 and 180 min) was measured in a dorsal hand vein. In protocol 3 we evaluated the local effects of different doses of insulin (1-100 mU/min) infused directly into hand veins preconstricted with phenylephrine. RESULTS: Forearm hyperinsulinemia (approximately 50 mU/l) led to a significant increase in FBF after 180 min (median 26%, interq ranges 5-50, P < 0.05), while SBF was not altered. Forearm hyperinsulinemia did not affect the noradrenergic responsiveness in skeletal muscle or skin. Infused locally into hand veins only the highest dose of insulin (100 mU/min) caused a minor venodilation (7% [2.4-12.5], P < 0.05). CONCLUSION: Regional forearm physiological hyperinsulinemia has a vasodilator effect on resistance vessels in skeletal muscle, but is slow in onset (180 min). However, skin vasculature and peripheral veins are not affected by this hyperinsulinemia.

Regional heterogeneity of arterial structural changes.

Arterial structural changes in experimental models of hypertension and restenosis differ between vessel types and within vessels. Inspired by the diversity of short-term functional responses to vasoactive agents, hypotheses are presented with respect to the heterogeneity of structural alterations. Considered are the multifactorial nature of smooth muscle cell growth control and the possibility that vascular smooth muscle is not homogeneous but composed of different smooth muscle cell populations. These hypotheses may help explain the origin of both intervascular and intravascular heterogeneity of vascular structural responses.

Aortic wall properties in normotensive and hypertensive rats of various ages in vivo.

The distensibility of the arterial system, which is partly determined by arterial wall structure, smooth muscle tone, and actual pressure level, decreases with aging and hypertension. Our aim was to compare aortic wall properties in 3- and 6-month-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) at comparable blood pressures in vivo. During ketamine/xylazine anesthesia in rats we performed ultrasound arterial wall tracking and invasive pressure measurements to determine, at the level of the thoracic aorta, diastolic pressure, diastolic lumen area, changes in pressure and lumen area during the cardiac cycle, and indexes of compliance and distensibility. These observations were combined with histological measurements for determination of media cross-sectional area and thickness and the incremental elastic modulus under conditions as expected in situ. Anesthesia abolished the difference in diastolic pressure between SHR and WKY. Between 3 and 6 months of age in WKY, diastolic area and incremental elastic modulus increased significantly, distensibility decreased, and all other recorded variables were not modified. Between 3 and 6 months of age in SHR, diastolic area and incremental elastic modulus increased, distensibility of the aortic wall decreased, and all other mechanical and structural properties did not change significantly. At both ages, diastolic area and compliance were significantly smaller in SHR than WKY. The other mechanical and structural properties measured or calculated at comparable pressure did not differ between strains. Differences between the aorta of 3- and 6-month-old rats and between strains observed in vivo at comparable pressures can largely be attributed to differences in lumen caliber.(ABSTRACT TRUNCATED AT 250 WORDS)

Disproportional arterial hypertrophy in hypertensive mRen-2 transgenic rats.

In the present study, we investigated the role of enhanced vascular renin-angiotensin activity in vascular hypertrophy. We used transgenic (mRen-2)27 (renin TGR) rats, spontaneously hypertensive rats (SHR), and their respective normotensive control rats to study in situ pressure-diameter relationships in second-generation mesenteric arterial branches (in vivo diameter, 400 to 500 microns) over a pressure range of 0 to 200 mm Hg. We studied pressure-diameter curves under both control (Tyrode's solution) and fully relaxed (Tyrode's solution containing 100 mg/L potassium cyanide) conditions. From these curves, we determined mechanical properties at operating blood pressure. In both hypertensive strains, mesenteric arterial media cross-sectional area was increased, with a significantly (P < .05) stronger degree of hypertrophy in renin TGR rats. Arterial distensibility of relaxed vessels was decreased to an equal degree in both hypertensive strains. Under control conditions, distensibility was higher in SHR than in renin TGR rats but still significantly reduced compared with distensibility in normotensive rats. Wall tension was increased to an equal degree in both hypertensive strains, whereas circumferential wall stress was normal in SHR but significantly (P < .05) reduced in renin TGR rats. These results indicate that whereas vascular hypertrophy in SHR causes adaptive normalization of arterial wall stress, enhanced vascular renin-angiotensin activity causes vascular hypertrophy in excess of the hypertrophy associated with pressure elevation alone.

Pressure but not angiotensin II-induced increases in wall mass or tone influences static and dynamic aortic mechanics.

OBJECTIVE: To distinguish between static (due to slow changes in pressure) and dynamic (due to pressure pulsatility) components of aortic compliance over a large pressure range in vivo and to examine the effects of increased vascular mass and smooth muscle tone on these components. METHODS: Using ultrasound wall tracking, aortic lumen area-pressure curves were generated in anaesthetized rats over a broad range of pressures by altering blood volume. The compliance coefficient calculated at each mean pressure was considered the dynamic compliance at that pressure; the slope of the diastolic lumen area-pressure curve represents static compliance. Experiments were performed in control rats and rats treated with angiotensin II (ANG II) acutely (500 ng/kg per min intravenously) to modify vascular tone or chronically (250 ng/kg per min subcutaneously for 2 weeks) to modify vascular mass. RESULTS: The dynamic compliance-pressure curve approximated a parabola. Maximal dynamic compliance (0.272+/-0.026 mm2/kPa in control rats) was achieved at near-normotensive pressure (+/-105 mm Hg). The diastolic lumen area-pressure curve showed an exponential relationship within a physiological range (30-130 mm Hg). ANG II-induced increases in aortic wall mass or smooth muscle tone did not modify the relationship between static or dynamic compliance and pressure. CONCLUSIONS: These findings demonstrate that static and dynamic mechanics of the rat thoracic aorta depend differently on blood pressure. Static compliance increases slightly with pressure in a physiological range, while dynamic compliance is auto-regulated around normotensive pressures. Neither static nor dynamic compliance of the rat thoracic aorta are influenced by ANG II-induced increases in aortic wall mass or smooth muscle tone.

DNA synthesis in isolated arteries of normotensive and hypertensive rats: effects of the endothelium.

OBJECTIVES: To compare DNA synthesis in isolated arteries of normotensive and hypertensive rats and to evaluate whether removal of the endothelium affects this process. DESIGN: Carotid and renal artery segments were isolated from normotensive Wistar, Wistar-Kyoto (WKY) and Sprague-Dawley rats, and from spontaneously hypertensive rats (SHR), transgenic Sprague-Dawley rats harbouring the mouse Ren-2 gene and from WKY rats rendered hypertensive by aortic coarctation. METHODS: Artery segments were exposed in vitro to serum with or without previous gentle removal of the endothelium. Nuclear incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine was visualized by immunocytochemistry and the percentage of labelled medial nuclei was determined. RESULTS: In both types of artery, obtained from 6-week-old WKY rats and from 6-week-old SHR, removal of endothelium increased the percentage of 5-bromo-2'-deoxyuridine-labelled medial nuclei (L%). Also, in the arteries of 20-week-old Wistar rats, WKY rats and WKY rats rendered hypertensive by aortic coarctation and in vessels of 11-week-old Sprague-Dawley rats and Sprague-Dawley rats harbouring the mouse Ren-2 gene, removal of endothelium increased L%. Conversely, in the arteries of 20-week-old SHR removal of the endothelium did not alter L%. Furthermore, maximally stimulated DNA synthesis was considerably smaller in de-endothelialized arteries of adult SHR than in denuded vessels from the other strains and models. CONCLUSION: These findings confirm that the endothelium can reduce DNA synthesis in the intact rat arterial smooth muscle. This effect is not modified by hypertension, but is selectively reduced in the arteries of adult SHR.

In vivo DNA synthesis is not uniformly increased in arterial smooth muscle of young spontaneously hypertensive rats.

We compared the distribution of DNA synthesis over the arterial tree of young normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) with marginally elevated blood pressure. Six-week-old male SHR and WKY rats were therefore infused with 5-bromo-2'-deoxyuridine (BrdUrd) for 2 days and the nuclear incorporation of the thymidine analogue in the media of various arteries was determined by immunohistochemistry. In WKY rats, 2.5% of the arterial smooth muscle nuclei in elastic, muscular and resistance arteries incorporated BrdUrd. In SHR, DNA synthesis was more marked in large arteries than in resistance arteries. It was in addition significantly larger in the aorta, superior mesenteric, renal and femoral arteries of the SHR than in those of the WKY rats. However, nuclear incorporation of BrdUrd in vivo did not differ between SHR and WKY rats in aortic endothelium, carotid arterial smooth muscle, nor in mesenteric or renal resistance arteries. Between 6 and 20 weeks of age, the number of nuclear profiles per media cross-section did not increase in large arteries of WKY rats and SHR. During this period of time, however, carotid artery and thoracic aorta weight and DNA content increased. SHR large arteries gained more DNA than those of WKY rats. These data indicate that DNA synthesis is uniformly distributed over the arterial system in young WKY rats and that DNA synthesis is elevated in the smooth muscle of large arteries of 6-week-old SHR but not in their resistance arteries.

Skin blood flow disturbances in the contralateral limb in a peripheral mononeuropathy in the rat.

Electrical excitation of nociceptive afferents in an extremity has been demonstrated to increase skin blood flow in the contralateral extremity. Hence, one would expect that loose sciatic nerve ligation, which induces an experimental painful peripheral neuropathy, may also provoke a vasodilator response in the contralateral hindpaw. On the non-ligated side, such a response may involve inhibited skin vasoconstrictor activity as well as neurogenically mediated active vasodilation. We studied skin blood flow changes in the rat hindpaw consequent to contralateral loose sciatic nerve ligation. After ligation, we also investigated whether blockade of afferent input from the ligated sciatic nerve to the spinal cord, by means of lidocaine, overrules the vasodilator response in the non-ligated paw. On the non-ligated side, we assessed the vasoconstrictor response of skin microvessels to cooling of the rat abdomen as a measure of skin vasoconstrictor activity in this paw. In order to investigate the involvement of sensory and/or non-sensory nerve fibers in the non-ligated sciatic nerve on skin blood flow abnormalities in the non-ligated paw, we studied the influence of blockade of these fibers through successive capsaicin and lidocaine application. We show that loose ligation of the sciatic nerve induces a vasodilator response in the contralateral hindpaw, which is completely abolished by blockade of afferent input from the ligated sciatic nerve. From day 1 after ligation, skin vasoconstrictor activity in the non-ligated paw was reduced, as indicated by an impaired vasoconstrictor response to cooling of the rat abdomen. Besides, blockade of sensory but not of non-sensory nerve fibers on the non-ligated side attenuated the vasodilator response in this paw. The data presented here indicate that loose ligation of the rat sciatic nerve induces a vasodilator response in the contralateral hindpaw. On the non-ligated side, this vasodilator response may involve inhibition of skin vasoconstrictor activity, as well as antidromically acting sensory nerve fibers.

Effects of cyclic AMP-affecting agents on contractile reactivity of isolated mesenteric and renal resistance arteries of the rat.

1. Effects of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-affecting agents were compared in mesenteric and renal resistance arteries that had been isolated from 20 week old Wistar-Kyoto rats, chemically sympathectomized, stretched to their optimal diameter for mechanical performance and made to contract in response to 30 mM potassium. 2. In mesenteric resistance arteries, isoprenaline, dopamine, NaF, forskolin, isobutyl-methylxanthine, milrinone and dibutyryl-cyclic AMP induced relaxation. Clonidine induced further increases in tension that could be reduced by pertussis toxin and prazosin but not by yohimbine. Clonidine also reduced relaxant responses to isoprenaline. 3. In renal resistance arteries, isoprenaline and dopamine failed to induce relaxation. Compared to mesenteric resistance arteries, renal vessels were less sensitive to the relaxant effect of NaF, forskolin and isobutyl-methylxanthine. Relaxant responses to dibutyryl-cyclic AMP did not differ between the two resistance arteries. 4. Indirect evidence thus suggests that in mesenteric resistance arteries, adenylate cyclase is susceptible to pharmacological activation and inhibition and is functionally coupled to relaxation. The refractory nature of renal resistance arteries to the relaxant effects of isoprenaline and dopamine could be due primarily to absence of appropriate receptors and to a relatively low activity of adenylate cyclase.

Reduced responsiveness of rat mesenteric resistance artery smooth muscle to phenylephrine and calcium following myocardial infarction.

1. We evaluated responses of peripheral resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation in a rat model of heart failure in relation to neurohumoral changes, wall structure, receptor density and cellular calcium handling. 2. Plasma samples and third order mesenteric artery side-branches were obtained from Wistar rats after induction of left ventricular infarction (M1) or sham surgery. Vessels were denuded of endothelium, sympathectomized, depleted of neuropeptides, and mounted in a myograph for recording of isometric force development in response to calcium, agonist and high potassium. Also, the morphology of these preparations was determined. Separate vessel segments were used in radioligand binding assays with [1H]-prazosin. 3. At 1 week after MI, circulating plasma levels of adrenaline, angiotensin II, atrial natriuretic factor (ANF) and vasopressin were significantly elevated. At 5 weeks only a significant elevation of ANF persisted. 4. At 5 weeks after MI, the structure of the vessels and responsiveness to high potassium or Bay K 8466 (10(6) mol l-1) were not modified. Yet, at this stage, sensitivity to phenylephrine was increased (pD2: 6.24 +/- 0.04 vs 5.98 +/- 0.04 for controls) while maximal contractile responses to phenylephrine in the presence of 2.5 mmol l-1 calcium (2.26 +/- 0.28 vs 3.53 +/- 0.34 N m-1) and the sensitivity to calcium in the presence of phenylephrine (pD2: 2.81 +/- 0.22 vs 3.74 +/- 0.16) were reduced. Responses to the agonist in calcium-free solution and the calcium sensitivity in the presence of 125 mmol l-1 potassium or of phorbol myristate acetate (PMA, 10(-6) mol l-1) were not altered. 5. At 5 weeks after MI, the density of prazosin binding sites was not reduced (4.04 +/- 1.40 vs 2.29 +/- 0.21 fmol microgram-1 DNA in controls). 6. In conclusion, myocardial infarction leads in the rat to a reduction of contractile responses of mesenteric resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation. This seems to involve impaired agonist-stimulated calcium influx.

Impaired arterial reactivity following cytomegalovirus infection in the immunosuppressed rat.

1. Cytomegalovirus (CMV) is a major pathogen in immunocompromised individuals and may participate in the pathogenesis of atherosclerosis in the general population. We evaluated whether CMV-infection alters the function of arterial smooth muscle. 2. Blood pressure (BP) and arterial reactivity were recorded in immunosuppressed rats that had been infected with CMV (10(5) plaque forming units i.p.). Furthermore, the reactivity of isolated arteries was compared between CMV-infected rats and rats injected with bacterial endotoxin (LPS). 3. Initially resting BP and heart rate (HR) were not modified in CMV-infected rats, but baroreflex control of HR was impaired. By the eighth day post-CMV, BP dropped precipitously and could no longer be raised by phenylephrine (PHE). 4. In mesenteric resistance arteries, isolated at this stage from CMV-infected rats, contractile responses to nerve stimulation, noradrenaline, PHE and 5-hydroxytryptamine (5-HT) were virtually absent while those to high potassium and vasopressin (AVP) were not modified. In aortae of CMV-infected rats, responses to 5-HT and AVP were impaired while those to PHE or potassium were hardly affected. Reduced contractile responses could not be restored by NG-nitro-L-arginine methyl ester (L-NAME). 5. Continuous treatment of CMV-infected rats with prazosin (0.1 mg kg-1 day-1) prevented blood pressure lowering and resistance artery changes. 6. Observations in arteries of LPS-treated rats (5-10 mg kg-1, i.p.) differed markedly from those in vessels of CMV-infected animals. The contractile reactivity of their mesenteric resistance arteries was not altered while in their aortae, responses to PHE, 5-HT and AVP were reduced. With the exception of the AVP responses, this was more pronounced in the presence of 1-arginine and reversed by L-NAME. 7. These findings indicate that CMV-infection results in a reduction of resistance artery reactivity and hypotonia. This seems not to involve cytokine-mediated induction of NO synthase in the vascular wall but may be due to alterations of excitation-contraction coupling in arterial smooth muscle in response to increased sympathetic nervous input.

Effects of a phorbol ester and staurosporine on electro- and pharmacomechanical coupling in a resistance artery.

We examined the role of protein kinase-C in contractile responses of small arteries of the rat by stimulating and inhibiting protein kinase-C with phorbol myristate acetate and staurosporine, respectively. The experiments were performed in isolated mesenteric resistance arteries that had been sympathectomized and mounted for recording of isometric force development. Phorbol myristate acetate (i) at concentrations lower than 3 nM increased sensitivity for the contractile effect of potassium, but not for the effect of noradrenaline or BAY-K8644, (ii) at concentrations higher than 30 nM increased the sensitivity of depolarized vessels to extracellular calcium and (iii) at concentrations higher than 30 nM induced a contractile effect that depended on the presence of extracellular calcium and that was reduced by the calcium antagonist felodipine. Neither the phorbol ester nor staurosporine affected contractile responses to caffeine in calcium-free solution. Staurosporine (10 nM) reduced the response of resistance arteries to potassium but not to noradrenaline. These results are in agreement with direct observations by others that protein kinase-C plays a role in the activation of voltage-operated calcium channels. Protein kinase-C could participate in this way in electro-mechanical coupling in resistance arterial smooth muscle and, when strongly activated, sensitize the contractile apparatus to calcium.