The Protective Effect of Propofol Against Ischemia-Reperfusion Injury in the Interlobar Arteries: Reduction of Abnormal Cx43 Expression as a Possible Mechanism.

BACKGROUND/AIMS: This experimental study aims to observe whether the protective effect of propofol against renal ischemia-reperfusion injury (IRI) in the rat interlobar artery occurs through altered expression of the gap junction protein connexin 43 (Cx43). METHODS: This study randomly divided male Sprague Dawley (SD) rats into an untreated control group, a sham-operated control group (sham group), an ischemia-reperfusion group (IR group), a propofol group (propofol+IR group) and a fat emulsion group (Intralipid group). The ischemia/reperfusion model was prepared through resection of the right kidney and noninvasive arterial occlusion of the left kidney. Forty-five minutes after renal ischemia-reperfusion, an automatic biochemical analyzer was employed to measure blood urea nitrogen (BUN) and serum creatinine (SCr); changes in renal tissue pathology were observed using hematoxylin and eosin (HE) staining, and the vasomotor activity of the interlobar artery was detected using a pressure mechanogram technique. The protein expression of Cx43 in renal artery cross-sections was determined through western blotting. RESULTS: The experimental study confirmed that the BUN and SCr of rats markedly increased after ischemia-reperfusion injury; additionally, we observed some coagulation necrosis and shedding of cells, some solidification of nuclear chromatin, degeneration of cytoplasmic vacuoles, high renal interstitial vascular congestion and obvious inflammatory cell infiltration, characterized by focal hemorrhages. Furthermore, the contraction activity of the renal interlobar artery greatly decreased, and the tension of the arteries in the renal lobe increased remarkably. After the gap junction blocking agents 2-APB and Gap27 were applied, the systolic velocity of blood vessels and the vascular contraction rate both decreased. In addition, the expression of Cx43 in kidney tissues increased markedly. The damage was more severe after 24 h of ischemic reperfusion than after only 4 h. However, after pretreatment with propofol, regardless of whether ischemia-reperfusion was applied for 4 h or 24 h, the previously increased expression of Cx43 decreased obviously, and all forms of renal damage were reversed. CONCLUSION: Our research suggests new ways for propofol to relieve ischemia-reperfusion injury by decreasing the abnormal expression of the gap junction protein Cx43. This study reveals a novel mechanism for the action of propofol against IRI, and we hope this finding will lead to new treatments for IRI.

Interlobular hyaline arteriopathy reflects severe arteriolopathy in renal allografts.

AIM: The present study was performed to examine the clinicopathological significance of hyaline deposits in the smooth muscle of the interlobular artery (interlobular hyaline arteriopathy [IHA]) in renal allografts. METHODS: Tissue specimens that included the interlobular artery from biopsies performed from January 2012 to December 2015, as well as specimens from biopsies performed ≥1 year after living kidney transplantation were analyzed. Biopsies of recipients with new-onset diabetes mellitus after transplantation were excluded, as well as those of recipients who had undergone transplantation because of diabetic nephropathy. Arteriolopathy was evaluated using the aah score determined by the Banff 2007 classification. RESULTS: In total, 51 specimens with IHA lesions were identified among 381 biopsies obtained from 243 recipients performed ≥1 year after kidney transplantation. Among these 51 biopsies, 18 specimens had a score of aah3, 29 had a score of aah2, and four had a score of aah1. The incidence of IHA lesions was 3.6% at ≥1 to <4 years, 18.5% at ≥4 to <8 years, and 54.1% at ≥8 years. Older kidney grafts exhibited more IHA lesions. Among the biopsy specimens obtained ≥8 years after transplantation, no significant differences in the recipient or donor age, duration after transplantation, or prevalence of hypertension were observed between the IHA and non-IHA groups. The aah scores were significantly higher in the IHA group ≥8 years after transplantation as determined by the mean score test (P < 0.01). CONCLUSION: IHA in renal allografts is associated with severe arteriolopathy.

Intranuclear expression of progesterone receptors in smooth muscle cells of renovascular fibromuscular dysplasia: a pilot study.

BACKGROUND: The pathogenesis of fibromuscular dysplasia (FMD) remains poorly understood. Yet, understanding this mechanism has taken on new urgency after recent evidence indicating that FMD is not as rare as previously thought. We speculated that hormonal receptors in the walls of dysplastic renal arteries were implicated in the pathogenesis of FMD. METHODS: We undertook a pilot prospective case-control study comparing histologic findings from renal arteries that were surgically removed in 2 patient groups. The case group included 6 samples from FMD patients who underwent surgery for stenosis or aneurysm caused by FMD. The control group included 3 FMD-free patients who underwent nephrectomy for nonvascular causes. Surgical specimens were sent to the histology laboratory. FMD was defined preoperatively using conventional radiologic criteria and was confirmed by histologic examination. RESULTS: Immunohistochemical staining detected intense progesterone receptor expression in the nuclei of smooth muscle cells in FMD patients. No progesterone receptor expression was found in the FMD-free patients. Estrogen receptor expression was not noted in the 2 groups. CONCLUSIONS: This preliminary finding may suggest that progesterone plays a key role in the pathogenesis of FMD and opens the fields of genetic and therapeutic approaches.

Renal myogenic constriction protects the kidney from age-related hypertensive renal damage in the Fawn-Hooded rat.

INTRODUCTION: Intact myogenic constriction plays a role in renal blood flow autoregulation and protection against pressure-related (renal) injury. However, to what extent alterations in renal artery myogenic constriction are involved in development of renal damage during aging is unknown. Therefore, we studied two strains of fawn-hooded rats, which differ in expression of hypertension and chronic renal failure. METHODS: Ten-week-old fawn-hooded hypertensive (FHH) and fawn-hooded low blood pressure (FHL) rats were followed for SBP and proteinuria for 1 year. At 52 weeks of age, the kidney was removed and studied for focal glomerulosclerosis (FGS) and glomerular cross-sectional area, and myogenic constriction of isolated small renal arteries in a vessel perfusion set up. Renal myogenic constriction and FGS were additionally determined in 10-week-old fawn-hooded rats. RESULTS: At young age, fawn-hooded rats did not differ in SBP, FGS, and urinary protein excretion, but renal artery myogenic constriction already was significantly smaller (∼50%) in FHH compared with FHL rats. Aging in fawn-hooded rats was associated with increase in SBP and urinary protein excretion and loss of renal artery myogenic constriction. These changes occurred in both fawn-hooded strains, although that in FHH rats the onset of hypertension occurred earlier and the increase in proteinuria by far exceeded (>4 times) that observed in FHL rats, and came along with 5.5 times increase in FGS and 1.3 times increase in glomerular cross-sectional area and significantly less preserved renal artery myogenic constriction in FHH rats. CONCLUSION: Better preservation of renal myogenic constriction protects the kidney from age-related hypertensive renal damage in the fawn-hooded rat.

Atherosclerotic plaque components characterization and macrophage infiltration identification by intravascular ultrasound elastography based on b-mode analysis: validation in vivo.

Intravascular ultrasound elastography (IVUSE) is a promising imaging technique for early investigation of vulnerable plaques. Compared to radiofrequency signal processing, digital B-mode analysis is simple and of higher portability. However, rare studies have been reported validating the latter technique in vivo. In this study, we developed an IVUSE computer software system involving semi-automatic border delineation and block-matching algorithm and validated the system in vivo. Seven minipigs were fed with atherogenic diet for 40 weeks. For each pig, the endothelium of one side of the renal arteries was denuded at the fifth week. With cross-correlation analysis, Lagrangian strain was calculated from two intravascular ultrasound images acquired in situ. Sixty regions of interests were selected from 35 elastograms matched well with the corresponding histological slices. Plaque types within these regions were classified as fibrous, fibro-fatty or fatty on Masson's trichrome and Oil-red O staining. Macrophage infiltration was also evaluated with immunohistology. Comparison between the mean strain value of the region of interest and the histological results revealed significant differences in strain values among different plaque types and non-diseased artery walls. The extent of macrophage infiltration was found to be correlated positively with strain values. For identification of fibro-fatty and fibrous plaques and macrophage infiltration, the system showed high sensitivity (93, 96 and 92%, respectively) and specificity (89, 76 and 66%, respectively), as revealed by receiver operating characteristic analysis. Our IVUSE system based on B-mode analysis is capable of characterizing fibrous and fibro-fatty plaques and macrophage intensity, thus holds potential for identifying vulnerable plaque.

Aqueous garlic extracts prevent oxidative stress and vascular remodeling in an experimental model of metabolic syndrome.

The organosulfur profile and the effect on oxidative stress and vascular remodeling in fructose-fed rats (FFR) were evaluated in Fuego INTA and Morado INTA garlic cultivars. Wistar rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. During the last 6 weeks of a 12 week period of the corresponding diet, a subgroup of control and FFR received an aqueous extract of Fuego INTA and Morado INTA. Fuego INTA showed higher levels of total thiosulfinates, allicin, and pungency than Morado INTA. FFR showed an increase of systolic blood pressure, aortic NAD(P)H oxidase activity, plasma thiobarbituric acid reactive substances, and vascular remodeling that was significantly reduced after both garlic administrations. The beneficial effect was slightly higher when Fuego INTA was administered. Both aqueous garlic extracts prevent oxidative stress and vascular remodeling in rats with metabolic syndrome, suggesting the existence of slight differences among cultivars.

Function of connexins in the renal circulation.

Connexins form intercellular channels that span two plasma membranes and directly couple the cytoplasm of adjacent cells. This morphological contact enables the exchange of ions, second messengers, and metabolites, which act to regulate several biological functions. This review focuses on the significance of connexins in the renal circulation. Cells of the renal vasculature are coupled and express connexins in a vessel and cell-specific pattern. This finding indicates that renal connexins likely play an important role in renal autoregulatory mechanisms (Bayliss effect, tubuloglomerular feedback) and in the control of vasomotor responses. The described coupling of endothelial and vascular smooth muscle cells in the afferent arterioles may also contribute to the communication of neighboring nephrons, called 'nephron coupling.' Furthermore, deletion of the Cx40 and Cx43 genes results in an altered functional behavior of the renin-producing cells, suggesting involvement of these connexin isoforms in the regulation of renin secretion and synthesis. In addition, this review discusses the role of renal connexin expression in the pathogenesis of hypertension or diabetes.

Adenosine2A receptor vasodilation of rat preglomerular microvessels is mediated by EETs that activate the cAMP/PKA pathway.

Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine A2A receptors (A2AR) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G(salpha), adenylyl cyclase, PKA, and Ca2+-activated K+ (KCa) channel activity, to the vasoactive responses to A2AR activation by a selective A2A agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 microm) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 microM), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 microM) and found that both were inhibited by approximately 70% (P<0.05), whereas the response to SNP (10 microM) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 microM), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 microM), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 microM) with PGMV resulted in an increase in cAMP levels (P<0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of KCa channel activity. Thus in rat PGMV activation of A2AR is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in Gsalpha protein activation.

Effect of nebivolol on cardiovascular changes associated with a rat model of insulin-resistance.

Nebivolol is a vasodilator that combines beta-adrenergic blocking activity with a relaxant effect on vascular smooth muscle cells (VSMC) mediated by the endothelial nitric oxide (NO) pathway. FFR provide a model of dietary-induced insulin-resistance syndrome, which has been used to study the pathophysiological mechanisms associated with this syndrome. Our main objective was to examine the effect of long-term administration of nebivolol on metabolic and cardiovascular variables in fructose-fed rats (FFR), a model in which an altered bioavailability of NO has been already described. Male Wistar rats were randomly assigned to 4 groups (n = 8 each): I. Control (C); II. Control + nebivolol (C+N): 1 mg/kg(-1) x day(-1) in drinking water during the last 4 weeks. III. FFR: rats receiving fructose in drinking water as a 10% (w/v) solution during 8 weeks, and IV. FFR+N: idem II plus III. During the 8 weeks experimental period, variations in systolic blood pressure (SBP), glucose tolerance test (GTT) and plasma thiobarbituric acid-reactive substances (TBARS) were assessed. At the end of this experimental period, rats were killed and heart and kidneys were excised for calculation of relative heart weight (RHW) and histological evaluation of lumen to media ratio (L/M) in renal arteries. Rats from FFR group increased their SBP and RHW, showed glucose intolerance and an increment in lipid peroxidation. Moreover, FFR showed vascular remodeling in renal arteries evidenced by changes in L/M. Although the metabolic changes were not reverted by the administration of nebivolol, this drug successfully decreased SBP, TBARS levels and reverted structural changes such as cardiac hypertrophy and renal arterial remodeling. Data demonstrate that nebivolol administration could participate in the reversion of cardiovascular structural changes associated with the insulin-resistance syndrome.

Age-related changes of elements in renal arteries of Thai and Japanese and the relationships among elements.

To examine whether there were differences between races in regard to age-related changes of elements and the relationships among elements in the arteries, the authors investigated both the renal arteries of Thai and Japanese. The Thai subjects consisted of 27 men and 11 women, ranging in age from 27 to 88 yr, whereas the Japanese subjects consisted of 19 men and 26 women, ranging in age from 61 to 99 yr. After the ordinary dissections at Chiang Mai University and Nara Medical University were completed, the bilateral renal arteries were resected and the element contents were determined by inductively coupled plasma-atomic emission spectrometry. In the Thai, a slight accumulation of calcium and phosphorus occurred in the fifties, but thereafter hardly increased. In contrast, in the Japanese, an accumulation of calcium and phosphorus began to occur in the seventies and increased markedly in the eighties. The result revealed that a higher accumulation of calcium and phosphorus occurred in the renal arteries of the Japanese in old age compared with those of the Thai. Regarding the relationships among elements, extremely or very significant direct correlations were found among the contents of calcium, phosphorus, magnesium, and sodium in both the renal arteries of the Thai and Japanese, except for magnesium and sodium contents in the renal arteries of the Thai.

Rapid and simple determination of epoxyeicosatrienoic acids in rabbit renal artery by reversed-phase HPLC with fluorescence detection.

A liquid chromatographic method with fluorescence detection coupled with a solid-phase extraction was applied to the rapid determination of epoxyeicosatrienoic acids (EETs) in the rabbit renal artery. The EETs were extracted with an acetonitrile from renal artery homogenate and concentrated by a solid-phase extraction method. The concentrated EETs were reacted directly with a 6, 7-dimethoxy-1-methyl-2 (1H)-quinoxalinone-3-propionyl-carboxylic acid (DMEQ) hydrazide and separated by a reversed-phase HPLC with eluting a combination of a step-wise and a gradient of a mixture of methanol and water. The content of EETs in the renal arteries was significantly greater in the 0.5% cholesterol fed rabbits than in control rabbits. It is suggested that hyperchlesterolemia increases the production of EETs in the rabbit renal artery.

Rapamycin attenuates vascular wall inflammation and progenitor cell promoters after angioplasty.

Rapamycin combines antiproliferative and antiinflammatory properties and reduces neointima formation after angioplasty in patients. Its effect on transcriptional programs governing neointima formation has not yet been investigated. Here, we systematically analyzed the effect of rapamycin on gene expression during neointima formation in a human organ culture model. After angioplasty, renal artery segments were cultured for 21 or 56 days in absence or presence of 100 ng/ml rapamycin. Gene expression analysis of 2312 genes revealed 264 regulated genes with a peak alteration after 21 days. Many of those were associated with recruitment of blood cells and inflammatory reactions of the vessel wall. Likewise, chemokines and cytokines such as M-CSF, IL-1beta, IL-8, beta-thromboglobulin, and EMAP-II were found up-regulated in response to vessel injury. Markers indicative for a facilitated recruitment and stimulation of hematopoetic progenitor cells (HPC), including BST-1 and SDF-1, were also induced. In this setting, rapamycin suppressed the coordinated proadhesive and proinflammatory gene expression pattern next to down-regulation of genes related to metabolism, proliferation, and apoptosis. Our study shows that mechanical injury leads to induction of a proinflammatory, proadhesive gene expression pattern in the vessel wall even in absence of leukocytes. These molecular events could provide a basis for the recruitment of leukocytes and HPC. By inhibiting the expression of such genes, rapamycin may lead to a reduced recruitment of leukocytes and HPC after vascular injury, an effect that may play a decisive role for its effectiveness in reducing restenosis.

Interstitial fluid analysis for assessment of organ function.

Evaluation methods are required for non-heart-beating donor (NHBD) kidneys to ensure the success of transplantation. In this study, the microdialysis technique was employed for the ex-vivo assessment of hypothermically preserved NHBD kidney function. Microdialysis probes were placed in the renal cortex of 2 h warm ischaemic porcine kidneys to monitor interstitial pyruvate dynamics during hypothermic machine perfusion with perfusate containing 29.4 mM fructose-1,6-diphosphate (FDP). The presence of exogenous FDP in the perfusate induced no changes in the renal flow rate and vascular resistance, renal artery effluent biochemistry, or pyruvate concentration relative to untreated control kidneys. Significant increases in pyruvate production (P < 0.05), however, were observed after 12 h of perfusion in the interstitial fluid of FDP-treated kidneys relative to control kidneys. After 24 h of perfusion, interstitial fluid concentrations of pyruvate were 149.1 +/- 58.4 vs. 55.6 +/- 17.9 micro M (P < 0.05) in the FDP and control group, respectively. The microdialysis probe collected the interstitial fluid directly from the cellular sites of metabolic and synthetic activity, where perfusate dilution was minimal. Consequently, the biochemical changes induced by the organ metabolic activity were detected only at the interstitial level, in the microdialysates. Interstitial fluid pyruvate may be a good indicator of kidney function. The addition of FDP to the perfusion solution during ischaemic kidney preservation resulted in enhanced pyruvate production in the extracellular space, indirectly reflecting an increase in anaerobic ATP production. The pyruvate will be transformed during organ reperfusion into acetyl Co-A enzyme allowing an immediate start of aerobic metabolism. This in turn can increase the amount of ATP available to the cells and may help prevent reperfusion injury upon transplantation.

Expression of A20 in the vessel wall of rat-kidney allografts correlates with protection from transplant arteriosclerosis.

BACKGROUND: Chronic rejection with development of transplant arteriosclerosis is the major culprit involved in loss of kidney allografts. The allografts' fate was thought to depend on the intensity of the host immune responses and the potency of immunosuppressive regimens. Recent data suggests that grafts contribute to their own survival by way of up-regulation of "cytoprotective" genes. METHODS: We analyzed the expression of four cytoprotective genes, A20, Bcl-2, Bcl-x(L) and heme oxygenase (HO)-1, in three rat renal allograft models of chronic rejection: Fisher 344-Lewis (F344/Lew), Dark Agouti-Brown Norway (DA/BN), and DA-Wistar-Furth (WF). We chose these genes for their known anti-inflammatory and anti-apoptotic function in endothelial cells (EC) and the atheroprotective function of A20 in smooth muscle cells (SMC). RESULTS: Twenty-eight and 9 weeks following transplantation, F344/Lew and DA/BN transplants had stable graft function. Histopathologic analysis showed moderate tissue damage, minimal cellular infiltrates, and preserved vascular integrity correlating with high expression of A20 in SMC. Conversely, impaired allograft function in the DA/WF combination with substantial transplant arteriosclerosis was noted in 60% of the grafts correlating with absent or decreased A20 expression in EC and SMC. In all combinations, expression of HO-1, Bcl-2, and Bcl-x(L) colocalized with infiltrating cells and was not informative on the graft status. CONCLUSIONS: We demonstrate for the first time a strict correlation between A20 expression in the vessel and the absence of transplant arteriosclerosis in rat kidney-allograft models. This data is similar to data obtained in human kidney allografts and suggests that A20 may represent a novel therapeutic target for the prevention of chronic allograft rejection.

Renal arterial 20-hydroxyeicosatetraenoic acid levels: regulation by cyclooxygenase.

20-HETE, a potent vasoconstrictor, is generated by cytochrome P-450 omega-hydroxylases and is the principal eicosanoid produced by preglomerular microvessels. It is released from preglomerular microvessels by ANG II and is subject to metabolism by cyclooxygenase (COX). Because low-salt (LS) intake stimulates the renin-angiotensin system and induces renal cortical COX-2 expression, we examined 20-HETE release from renal arteries (interlobar and arcuate and interlobular arteries) obtained from 6- to 7-wk-old male Sprague-Dawley rats fed either normal salt (0.4% NaCl) or LS (0.05% NaCl) diets for 10 days. With normal salt intake, the levels of 20-HETE recovered were similar in arcuate and interlobular arteries and interlobar arteries: 30.1 +/- 8.5 vs. 24.6 +/- 5.3 ng. mg protein(-1). 30 min(-1), respectively. An LS diet increased 20-HETE levels in the incubate of either arcuate and interlobular or interlobar renal arteries only when COX was inhibited. Addition of indomethacin (10 microM) to the incubate of arteries obtained from rats fed an LS diet resulted in a two- to threefold increase in 20-HETE release from arcuate and interlobular arteries, from 39.1 +/- 13.2 to 101.8 +/- 42.6 ng. mg protein(-1). 30 min(-1) (P < 0.03), and interlobar arteries, from 31.7 +/- 15.1 to 61.9 +/- 29.4 ng. mg protein(-1). 30 min(-1) (P < 0.05) compared with release of 20-HETE when COX was not inhibited. An LS diet enhanced vascular expression of cytochrome P-4504A and COX-2 in arcuate and interlobular arteries; COX-1 was unaffected. Metabolism of 20-HETE by COX is proposed to represent an important regulatory mechanism in setting preglomerular microvascular tone.

Sex-specific acute effect of estrogen on endothelium-derived contracting factor in the renal artery of hypertensive Dahl rats.

OBJECTIVE: To determine whether estrogen rapidly affects endothelium-derived contracting factor (EDCF) in the renal artery of hypertensive Dahl rats, and whether factors other than nitric oxide (NO) contribute to the effect of estrogen. DESIGN: Acute effects of estrogen on the acetylcholine-induced vasomotor responses and on prostaglandin H2/thromboxane A2 mimetic, U46619,-induced contraction were examined in isolated arterial rings. METHODS AND RESULTS: Dahl salt-sensitive male and female rats were fed an 8% NaCl diet for 4 weeks. The blood pressure increased more rapidly and to a greater extent in males than in females. Renal arterial rings were prepared for isometric tension recording. 17beta-Estradiol, but not the biologically less active stereoisomer, 17alpha-estradiol, improved the relaxation response to acetylcholine in renal arteries from females. Estrogen also rapidly decreased the contraction evoked by acetylcholine (10(-6) to approximately 10(-4) mol/l) in renal arteries from females and it was effective at a physiological concentration (10(-9) mol/l) in the presence of Nomega-nitro-l-arginine methyl ester (an NO synthase inhibitor). The estrogen receptor antagonist, ICI 182,780, abolished the effect of estrogen, whereas the cytochrome P450 inhibitor, miconazole, had no effect. The contraction induced by U46619 was also suppressed by estrogen, without any contribution from NO. Estrogen had no effect on either relaxation or contraction responses in renal arteries from males. CONCLUSION: 17beta-Estradiol antagonizes increases in vascular tone in hypertensive females by enhancing NO-dependent relaxation, and by suppressing EDCF-mediated mechanisms in an NO-independent manner.

P2X receptor immunoreactivity in different arteries from the femoral, pulmonary, cerebral, coronary and renal circulations.

The expression of the seven P2X receptor subunits (P2X1-7) in the rat vascular system was determined using subtype-selective antibodies. Arteries of different sizes (from arterioles to conduit vessels) from a range of vascular beds were used to give an overview of receptor expression. P2X1 receptor immunoreactivity was detected in the smooth muscle layer of arteries. The relative level of P2X1 receptor immunoreactivity was dependent on the size of the artery and the vascular bed; expression was highest in small and medium arteries. P2X4 receptors were detected in all arteries; once again, the relative level of expression was dependent on the size of the artery and the vascular bed. P2X5 receptor immunoreactivity was barely detectable in most arteries studied. P2X7 receptor immunoreactivity was generally punctate and associated with the outer adventitial layer. Immunoreactivity for P2X2, P2X3 and P2X6 receptors was not detected in arteries. These results demonstrate that arteries express multiple P2X receptor subunits and that there is a heterogeneity in the level of expression. The properties of artery P2X receptors correspond to homomeric P2X1 receptors, and the function of P2X4 and P2X5 receptor subunits in arteries is unclear.

Paradoxical effects of temperature on vascular tone.

Temperature may have significant influence on vascular tone in such cases as organ preservation, coronary bypass surgery, and extracorporeal circulation. The aim of this research was to study the direct effect of temperature variation on vascular tone in an attempt to elucidate the mechanisms involved. In a first series of experiments, the isometric tension of two different vessels (rat thoracic aorta and pig renal branch artery) was studied at different temperatures. To study the role of calcium in this response, a second series of experiments was performed. In this the vessels were incubated with the intracellular chelator BAPTA/AM. Further experiments were performed to test the effect of cold storage. Our results show that changes in temperature lead to different results in pig renal artery and rat aorta. A decrease in temperature induced a highly reproducible relaxation in rat aorta, whereas pig renal artery presented cooling-induced contraction. Moreover, whereas calcium depletion failed to inhibit cooling-induced relaxation in rat aorta, it did not provoke cooling-induced contraction in pig renal artery. Similar responses were obtained with cold storage and calcium depletion. We intend to demonstrate that, just as the effect of temperature variation on pig renal artery is due to a metabolic mechanism, its effect on rat aorta may be due to structural factors. This hypothesis is supported by the result of histological studies which demonstrate a higher proportion of elastin fibres in rat aorta than in pig renal artery.

Regulation of renal artery smooth muscle tone by alpha1-adrenoceptors: role of voltage-gated calcium channels and intracellular calcium stores.

The ischemia induced vasospasm of the renal arterial blood vessels mediated by alpha1-adrenoceptors is of importance for the loss of kidney function. This is based on reduced perfusion of the kidney cortex occurring in kidney transplant and organ preserving surgery. The present study considered the intracellular mechanism of the norepinephrine (NE) induced renal artery vasospasm by using swine renal artery smooth muscle ring. Norepinephrine and phenylephrine (PE) induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 10 nM (n = 7) and 10 nM (n = 4), and an EC50 of 0.3 microM and 1 microM, respectively. The receptor was identified as alpha1A-subtype. The contraction was completely inhibited by verapamil (IC50 = 1.51 microM; n = 11) and diltiazem (IC50 = 9.49 microM; n = 8) and 85% by nifedipine (IC50 = 0.13 microM; n = 21). Blockade of the intracellular inositol- 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store by thapsigargin (1 microM, n = 7) or suppression of Ca2+ release from the intracellular Ca2+-sensitive Ca2+ store by ryanodine (100 microM, n = 4) inhibited the PE induced contraction by 39.5% and 47.6%, respectively. The results suggest a key role of voltage-dependent Ca2+ channels and intracellular Ca2+ stores in the alpha1A-adrenoceptor induced contraction of the renal artery.

Lysophosphatidylcholine triggers intracellular calcium release and activation of non-selective cation channels in renal arterial smooth muscle cells.

The effects of a lipid component of oxidized low-density lipoproteins (ox-LDL), L-alpha-palmitoyl-lysophosphatidylcholine (LPC), on membrane currents of isolated canine renal artery smooth muscle cells (RASMC) were examined using the whole-cell configuration of the patch-clamp technique. In RASMC exposed to nominally Ca2+-free solutions and dialyzed with 0.1 mM EGTA and 140 mM K+, superfusion with LPC (10 microM) elicited spontaneous transient outward currents (STOCs) and/or spontaneous transient inward currents (STICs), followed by the activation of a large voltage-independent current with a reversal potential (Er) close to 0 mV. Buffering intracellular Ca2+ with 10 mM BAPTA prevented the appearance of STOCs and STICs, but not the activation of the voltage-independent current. Er of the LPC-induced voltage-independent current exhibited sensitivity to changes in [K+]o and [Na+]o in a manner consistent with a non-selective cation current (I(NSC)) and was blocked by gadolinium (Gd3+; 10 microM). Shifts in Er of the LPC-induced I(NSC) in response to changes in [Ca2+]o were used to estimate a relative Ca2+ to Na+ permeability ratio (P(Ca)/P(Na)) of 1.67. These results suggest that LPC causes abnormal sarcoplasmic reticulum Ca2+ regulation, leading to the appearance of STOCs and STICs, and the activation of I(NSC) in vascular smooth muscle cells. These effects may explain the ability of ox-LDLs to elevate [Ca2+]i in vascular smooth muscle and inhibit endothelium-dependent relaxation.