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The activity of many vasomotor signaling pathways strongly depends on extracellular/intracellular pH. Nitric oxide (NO) is one of the most important vasodilators produced by the endothelium. In this review, we present evidence that in most vascular beds of mature mammalian organisms metabolic or respiratory acidosis increases functional endothelial NO-synthase (eNOS) activity, despite the observation that direct effects of low pH on eNOS enzymatic activity are inhibitory. This can be explained by the fact that acidosis increases the activity of signaling pathways that positively regulate eNOS activity. The role of NO in the regulation of vascular tone is greater in early postnatal ontogenesis compared to adulthood. Importantly, in early postnatal ontogenesis acidosis also augments functional eNOS activity and its contribution to the regulation of arterial contractility. Therefore, the effect of acidosis on total peripheral resistance in neonates may be stronger than in adults and can be one of the reasons for an undesirable decrease in blood pressure during neonatal asphyxia. The latter, however, should be proven in future studies. [ABSTRACT FROM AUTHOR]

Cyanotoxins (CTs) are a large and diverse group of toxins produced by the peculiar photosynthetic prokaryotes of the domain Cyanoprokaryota. Toxin-producing aquatic cyanoprokaryotes can develop in mass, causing "water blooms" or "cyanoblooms," which may lead to environmental disaster—water poisoning, extinction of aquatic life, and even to human death. CT studies on single cells and cells in culture are an important stage of toxicological studies with increasing impact for their further use for scientific and clinical purposes, and for policies of environmental protection. The higher cost of animal use and continuous resistance to the use of animals for scientific and toxicological studies lead to a progressive increase of cell lines use. This review aims to present (1) the important results of the effects of CT on human and animal cell lines, (2) the methods and concentrations used to obtain these results, (3) the studied cell lines and their tissues of origin, and (4) the intracellular targets of CT. CTs reviewed are presented in alphabetical order as follows: aeruginosins, anatoxins, BMAA (β-N-methylamino-L-alanine), cylindrospermopsins, depsipeptides, lipopolysaccharides, lyngbyatoxins, microcystins, nodularins, cyanobacterial retinoids, and saxitoxins. The presence of all these data in a review allows in one look to advance the research on CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines for future toxicological, pharmacological, and physiological studies. [ABSTRACT FROM AUTHOR]

Background and Purpose: The vasomotor role of K2P potassium channels during early postnatal development has never been investigated. We tested the hypothesis that TASK‐1 channel (K2P family member) contribution to arterial vascular tone and BP is higher in the early postnatal period than in adulthood. Experimental Approach We studied 10‐ to 15‐day‐old ("young") and 2‐ to 3‐month‐old ("adult") male rats performing digital PCR (dPCR) (using endothelium‐intact saphenous arteries), isometric myography, sharp microelectrode technique, quantitative PCR (qPCR) and Western blotting (using endothelium‐denuded saphenous arteries), and arterial pressure measurements under urethane anaesthesia. Key Results: We found mRNA of Kcnk1–Kcnk7, Kcnk12, and Kcnk13 genes to be expressed in rat saphenous artery, and Kcnk3 (TASK‐1) and Kcnk6 (TWIK‐2) were most abundant in both age groups. The TASK‐1 channel blocker AVE1231 (1 μmol·L−1) prominently depolarized arterial smooth muscle and increased basal tone level and contractile responses to methoxamine of arteries from young rats but had almost no effect in adult rats. The level of TASK‐1 mRNA and protein expression was higher in arteries from young compared with adult rats. Importantly, intravenous administration of AVE1231 (4 mg·kg−1) had no effect on mean arterial pressure in adult rats but prominently raised it in young rats. Conclusion and Implications: We showed that TASK‐1 channels are important for negative feedback regulation of vasocontraction in young but not adult rats. The influence of TASK‐1 channels most likely contributes to low BP level at perinatal age. [ABSTRACT FROM AUTHOR]

Background: This study was undertaken to test the hypothesis that the newly synthesized curcuminoids B2BrBC and C66 supplementation will overcome hyperoxia‐induced tracheal hyperreactivity and impairment of relaxation of tracheal smooth muscle (TSM). Materials and methods: Rat pups (P5) were exposed to hyperoxia (>95% O2) or normoxia for 7 days. At P12, tracheal cylinders were used to study in vitro contractile responses induced by methacholine (10−8–10−4M) or relaxation induced by electrical field stimulation (5–60 V) in the presence/absence of B2BrBC or C66, or to study the direct relaxant effects elicited by both analogs. Results: Hyperoxia significantly increased contraction and decreased relaxation of TSM compared to normoxia controls. Presence of B2BrBC or C66 normalized both contractile and relaxant responses altered by hyperoxia. Both, curcuminoids directly induced dose‐dependent relaxation of preconstricted TSM. Supplementation of hyperoxic animals with B2BrBC or C66, significantly increased catalase activity. Lung TNF‐α was significantly increased in hyperoxia‐exposed animals. Both curcumin analogs attenuated increases in TNF‐α in hyperoxic animals. Conclusion: We show that B2BrBC and C66 provide protection against adverse contractility and relaxant effect of hyperoxia on TSM, and whole lung inflammation. Both analogs induced direct relaxation of TSM. Through restoration of catalase activity in hyperoxia, we speculate that analogs are protective against hyperoxia‐induced tracheal hyperreactivity by augmenting H2O2 catabolism. Neonatal hyperoxia induces increased tracheal contractility, attenuates tracheal relaxation, diminishes lung antioxidant capacity, and increases lung inflammation, while monocarbonyl CUR analogs were protective of these adverse effects of hyperoxia. Analogs may be promising new therapies for neonatal hyperoxic airway and lung disease. [ABSTRACT FROM AUTHOR]

Background and Purpose: BK channels play important roles in various physiological and pathophysiological processes and thus have been the target of several drug development programmes focused on creating new efficacious BK channel openers, such as the GoSlo-SR compounds. However, the effect of GoSlo-SR compounds on vascular smooth muscle has not been studied. Therefore, we tested the hypothesis that GoSlo-SR compounds dilate arteries exclusively by activating BK channels.Experimental Approach: Experiments were performed on rat Gracilis muscle, saphenous, mesenteric and tail arteries using isobaric and isometric myography, sharp microelectrodes, digital droplet PCR and the patch-clamp technique.Key Results: GoSlo-SR compounds dilated isobaric and relaxed and hyperpolarised isometric vessel preparations and their effects were abolished after (a) functionally eliminating K+ channels by pre-constriction with 50 mM KCl or (b) blocking all K+ channels known to be expressed in vascular smooth muscle. However, these effects were not blocked when BK channels were inhibited. Surprisingly, the Kv 7 channel inhibitor XE991 reduced their effects considerably, but neither Kv 1 nor Kv 2 channel blockers altered the inhibitory effects of GoSlo-SR. However, the combined blockade of BK and Kv 7 channels abolished the GoSlo-SR-induced relaxation. GoSlo-SR compounds also activated Kv 7.4 and Kv 7.5 channels expressed in HEK 293 cells.Conclusion and Implications: This study shows that GoSlo-SR compounds are effective relaxants in vascular smooth muscle and mediate their effects by a combined activation of BK and Kv 7.4/Kv 7.5 channels. Activation of Kv 1, Kv 2 or Kv 7.1 channels or other vasodilator pathways seems not to be involved. [ABSTRACT FROM AUTHOR]

Recently, medical students' scientific thinking skills have been identified as an important issue in medical education. Scientific thinking cannot be imparted in conventional lectures, but rather requires actively involving students. We modified a practical course in physiology. A study was designed to test whether the new course fosters scientific thinking without impairing the transfer of physiological knowledge. The study group consisted of 226 first-year medical students at the Medical Faculty Mannheim of Heidelberg University. Written consent to participate in the study was obtained from all participants. The group was then randomly divided into two groups (traditional vs. modified course). The subject of both courses was a laboratory experiment in skeletal muscle physiology. In the traditional course, the students addressed topics already presented in lectures. In the modified course, students dealt with the same topics as in the traditional course, but the experiment was expanded to include one issue not taught before. When working on this issue, the students were instructed in scientific thinking. All participants filled out a questionnaire with 15 multiple-choice questions addressing the physiological subject matter and four open-ended questions addressing the criteria of scientific methodology. Physiological knowledge in both groups did not differ [F(1) = 2.08, P = 0.15]. Scores in scientific thinking in the modified course were higher (mean = 4.20, SD = 1.89) than in the traditional course (mean = 2.04, SD = 1.91) with F(1) = 70.69, P < 0.001, η2 = 0.24 (large effect). Our study demonstrates that small adjustments to courses in medical education can promote scientific thinking without impairing knowledge transfer. [ABSTRACT FROM AUTHOR]

Abstract Alterations in the retinal microvessel (RMV) compartment occurring in systemic disease states such as diabetes may eventually contribute to blindness. To specifically address the pathophysiological role of the microvasculature we developed a new method for RMV bulk isolation from individual rats. The extraction procedure performed in the cold throughout takes less than one hour. Slight modifications enable isolation of brain microvessels (BMVs) for comparison. Microscopically, RMVs and BMVs consisted mainly of capillaries of good structural integrity. The endothelial cell/pericyte ratio was approximately 1.8 in RMVs and 2.7 in BMVs, well in agreement with data from intact vascular beds. Total RNA extracted from individual rats amounted to approximately 7 ng in RMVs, 50 ng in BMVs, and 155 ng in pial arteries (which were also isolated) with highly preserved integrity throughout. Measurements using microfluidic card methodology revealed segregation of RMVs, BMVs, and pial arteries in distinct clusters based on principal component analysis. In all three vascular compartments endothelial cell-specific markers were significantly enriched. Similarly, pericyte-specific markers displayed accumulation in RMVs, BMVs, and pial arteries, the latter probably reflecting the common ontogenetic origin of pericytes and smooth muscle cells. Isolation of RMVs, BMVs, and pial arteries from rats suffering from 8-weeks hyperglycemia yielded expression patterns of endothelial cell- and pericyte-specific marker genes largely comparable to those obtained in control rats. Our newly developed protocols allow for selective studies of RMVs from individual rats to characterize reactive pathways, in comparison with the ontogenetically closely related BMVs. Moreover, our protocols with inclusion of pial arteries enable comparative studies of the macro- and microvasculature from the same organ. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

The Src tyrosine kinase family contributes to the signalling mechanism mediating serotonin (5-hydroxytryptamine (5-HT))-induced vasoconstriction. These kinases were reported to influence the calcium sensitivity of the contractile apparatus. Whether Src kinases affect also the intracellular calcium concentration during constriction of intact arteries is unknown. Thus, we tested the hypothesis that constriction of arteries is associated with a Src kinase-dependent alteration of the intracellular calcium concentration. Contractility of gracilis arteries of Wistar rats was studied using isometric and isobaric myography. The intracellular calcium concentration was measured simultaneously with tension by FURA-2 fluorimetry. Inhibition of Src kinases with 10 μM PP2, 30 μM dasatinib and 100 μM AZM 475271 resulted in a strong attenuation of 5-HT-induced contractions. Vessel incubation with 10 μM PP3, an inactive analogue of PP2, had no effect. Removal of the endothelium did not alter vessel contractile responses to 5-HT nor the action of the Src-kinase inhibitor PP2. The PP2-mediated inhibition of 5-HT-induced contraction was associated with a reduced response of [Ca] to 5-HT. In particular, inhibition of Src kinases attenuates 5-HT-induced calcium influx as well as calcium release from intracellular stores. In contrast, the calcium sensitivity of the contractile apparatus and the filling state of the sarcoplasmic reticulum were not influenced by Src kinases during 5-HT-induced contractions. We conclude that Src kinase activation is a powerful mechanism to produce vasoconstriction of small skeletal muscle arteries of rats. This effect is endothelium-independent. The data further suggest that the action of c-Src kinases is associated with a change in the intracellular calcium concentration that involves Ca entry and Ca release pathways. [ABSTRACT FROM AUTHOR]

Stretch-induced depolarizations of cardiomyocytes, which are related to activity of mechano-gated cation channels (MGCs), can lead to serious arrhythmias. However, signaling pathways leading to activation of mechano-gated channels by stretch remain almost unexplored. Using standard sharp microelectrodes, the present study addresses the hypothesis that tumor necrosis factor-alpha (TNF-α) modulates stretch-induced electrophysiological abnormalities in rat atrial myocardium by a mechanism involving nitric oxide (NO)-dependent pathways. TNF-α (50 ng/ml) produced a marked prolongation of action potential, subsequently transforming into humplike depolarizations and, finally, leading to occurrence of arrhythmias. These effects developed slowly during 25 min of TNF-α application. Similar electrical effects were induced by stretching the preparations. A blocker of MGCs, Gd 3+ (40 μM), completely abolished action potential (AP) prolongations and electrical abnormalities caused by TNF-α or stretch. Further, a donor of exogenous NO, S-nitroso- N-acetylpenicillamine SNAP (300 μM), evoked the same electrical abnormalities as TNF-α and tissue stretch. Both TNF-α and stretch failed to produce their typical effects after pretreatment of the preparations with the NO-synthase inhibitor L-N G-nitroarginine methyl ester (L-NAME) (100 μM). Thus, the present study shows (i) that TNF-α and the NO-donor SNAP evoke MGC-mediated electrical abnormalities in rat atrial myocardium in the absence of stretch that is very similar to stretch-evoked electrical events and (ii) that the TNF-α-induced electrical abnormalities are mediated by NO synthase. In conclusion, our data suggest that NO is an endogenous modulator of MGCs and mediates proarrhythmic effects of TNF-α in mammalian organism. [ABSTRACT FROM AUTHOR]

Aims Functional remodelling takes place permanently in the circulatory system. Whether this process also affects the anti-contractile effect of the endothelium during vasoconstrictor action is unknown. Therefore, the hypothesis was tested that the impact of the anti-contractile effect of the endothelium on agonist-induced contractions changes during early postnatal development. Methods and results We studied isometric contractions in saphenous arteries of young (1–2 weeks) and adult (2–3 months) rats. Real-time PCR and western blot were performed to evaluate the levels of mRNA expression and protein phosphorylation, respectively. In young but not in adult rats, methoxamine-induced contractions of endothelium-intact vessels exhibited a lower sensitivity compared with endothelium-denuded vessels. The endothelial influence on methoxamine-induced contractions in arteries of young rats was completely blocked by inhibition of endothelial NO-synthase (eNOS) and guanylate cyclase. NO-donor-induced vessel relaxations were not different in young and adult rats. The expression level of eNOS mRNA was prominently higher in arteries from young compared with adult rats. eNOS inhibition alone induced tonic contractions of endothelium-intact arteries from young but not from adult animals that were associated with corresponding changes in phosphorylation of the myosin regulatory light chains, the regulatory subunit of smooth muscle cell myosin light chain phosphatase, and vasodilator-stimulated phosphoprotein, the latter two being considered to be good markers of NO/sGC/PKG pathway activity. Conclusion We demonstrated that agonist-induced contractions in arteries of young rats are attenuated by the endothelium possessing an active NO-pathway. The active NO-pathway is due to a constitutive eNOS activity that disappears with age. [ABSTRACT FROM AUTHOR]

Abstract: In the gut, dopamine is released by enteric neurons and modulates motility of small intestine smooth muscle cells. Here, we systematically analyzed the dopamine-induced effects on the longitudinal smooth muscle of different sections of the rat isolated small intestine. We found that exogenous dopamine had biphasic effects and could lead to both an early contraction and a late relaxation, depending on the region of small intestine. Thus, dopamine-induced early contractions were commonly observed in the duodenum, but less frequently in the jejunum, and rarely in the ileum. The amplitudes of these early contractions showed a striking regional dependence (duodenum>jejunum>ileum) and were significantly blocked by SCH23390 and raclopride. Conversely, dopamine-induced late relaxations were regularly obtained in the ileum and in the jejunum, but less frequently in the duodenum. Interestingly, the amplitudes of these relaxations showed an inverse regional dependence (ileum>jejunum>duodenum), and were insensitive to dopamine receptor antagonists. Rather, they were significantly inhibited by propranolol and prazosin. We conclude that dopamine exerts differential effects on smooth muscle motility in different regions within the rat small intestine. In proximal parts, dopamine predominantly causes D1 and D2 dopamine receptor-dependent contraction, whereas it leads to α and β adrenoceptor-dependent relaxation in more distal parts. [Copyright &y& Elsevier]

Nitric oxide (NO) is a powerful vasodilator in different vascular beds and NO-donors are widely used in clinical practice. Early data suggested that NO and NO-donors activate vascular smooth muscle high-conductance, calcium-activated potassium channels (BK channels). There exist two hypotheses explaining the effect of NO and NO-donors on BK channels—one stating that protein kinase G (PKG) mediates the effect of NO, and the other one stating that NO acts directly on the channel. Thus, the degree of contribution of PKG to the NO-induced activation of the BK channel is still not completely clear. This study tested the hypothesis that the sodium nitroprusside (SNP)-induced activation of vascular smooth muscle BK channels is fully mediated by PKG. This hypothesis was investigated using the patch-clamp technique and freshly isolated smooth muscle cells from rat tail artery. In whole-cell experiments, SNP considerably increased the outward current compared with the addition of the bath solution. SNP did not alter the current in the presence of iberiotoxin, the specific blocker of BK channels, during co-application with hydroxocobalamin, an NO-scavenger, and in the presence of Rp-8-Br-PET-cGMPS, the specific PKG-inhibitor. In inside-out patches, the activity of BK channels was increased by SNP, SNAP, and DEA-NO. However, these effects did not differ from the effect of the application of drug-free bath solution. Furthermore, a similar increase in single BK channel activity was induced by Rp-8-Br-PET-cGMPS, Rp-8-Br-PET-cGMPS together with SNP, hydroxocobalamin, and hydroxocobalamin together with SNP or DEA-NO. Finally, the activity of excised BK channels did not change in the absence of any application but was considerably increased by PKG compared with the addition of drug-free bath solution. These results suggest that NO released from NO-donors stimulates the BK current only through activation of PKG. [ABSTRACT FROM AUTHOR]

Maturation of the cardiovascular system is associated with crucial structural and functional remodeling. Thickening of the arterial wall, maturation of the sympathetic innervation, and switching of the mechanisms of arterial contraction from calcium-independent to calcium-dependent occur during postnatal development. All these processes promote an almost doubling of blood pressure from the moment of birth to reaching adulthood. This review focuses on the developmental alterations of potassium channels functioning as key smooth muscle membrane potential determinants and, consequently, vascular tone regulators. We present evidence that the pattern of potassium channel contribution to vascular control changes from Kir2, Kv1, Kv7 and TASK-1 channels to BKCa channels with maturation. The differences in the contribution of potassium channels to vasomotor tone at different stages of postnatal life should be considered in treatment strategies of cardiovascular diseases associated with potassium channel malfunction. [ABSTRACT FROM AUTHOR]

It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current-voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current-voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.

Bone morphogenetic protein (BMP)-9, a member of the TGFβ-family of cytokines, is believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients' samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The study of the mechanisms of regulation of vascular tone is an urgent task of modern science, since diseases of the cardiovascular system remain the main cause of reduction in the quality of life and mortality of the population. Myography (isometric and isobaric) of isolated blood vessels is one of the most physiologically relevant approaches to study the function of cells in the vessel wall. On the one hand, cell-cell interactions as well as mechanical stretch of the vessel wall remain preserved in myography studies, in contrast to studies on isolated cells, e.g., cell culture. On the other hand, in vitro studies in isolated vessels allow control of numerous parameters that are difficult to control in vivo . The aim of this review was to 1) discuss the specifics of experimental design and interpretation of data obtained by myography and 2) highlight the importance of the combined use of myography with various complementary techniques necessary for a deep understanding of vascular physiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schubert, Gaynullina, Shvetsova and Tarasova.)

Aim: TASK-1 channels are established regulators of pulmonary artery tone but their contribution to the regulation of vascular tone in systemic arteries is poorly understood. We tested the hypothesis that TASK-1 channel functional impact differs among systemic vascular beds, that this is associated with differences in their expression and may increase with alkalization of the extracellular environment. Therefore, we evaluated the expression level of TASK-1 channels and their vasomotor role in mesenteric and renal arteries. Methods: Pulmonary, mesenteric and renal arteries from male Wistar rats were used for TASK-1 channel mRNA (qPCR) and protein content (Western blotting) measurements. The functional role of TASK-1 channels was studied by wire myography using the TASK-1 channel blocker AVE1231. In some experiments, the endothelium was removed with a rat whisker. Results: Expression levels of both mRNA and protein of the TASK-1 channel pore-forming subunit were highest in pulmonary arteries, lowest in mesenteric arteries and had an intermediate value in renal arteries. Blockade of TASK-1 channels by 1 µM AVE1231 increased U46619-induced contractile responses of pulmonary arteries but did not affect basal tone and contractile responses to methoxamine of mesenteric and renal arteries at physiological extracellular pH (pHo = 7.41). At alkaline extracellular pH = 7.75 (increase of NaHCO 3 to 52 mM) AVE1231 evoked the development of basal tone and increased contractile responses to low concentrations of methoxamine in renal but not mesenteric arteries. This effect was independent of the endothelium. Conclusion: In the rat systemic circulation, TASK-1 channels are abundant in renal arteries and have an anticontractile function under conditions of extracellular alkalosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shvetsova, Lazarenko, Gaynullina, Tarasova and Schubert.)

Although the vasoactive properties of carbon monoxide (CO) have been extensively studied, the mechanism by which CO mediates vasodilation is not completely understood. Through-out published studies on CO mediated vasodilation there is inconsistency on the type of K + -channels that are activated by CO releasing molecules (CORMs). Since the vasorelaxation properties of enzyme triggered CORMs (ET-CORMs) have not been studied thus far, we first assessed if ET-CORMs can mediate vasodilation of small mesenteric arteries and subsequently addressed the role of soluble guanylate cyclase (sGC) and that of K-channels herein. To this end, 3 different types of ET-CORMs that either contain acetate ( rac - 1 and rac - 4 ) or pivalate ( rac - 8 ) as ester functionality, were tested ex vivo on methoxamine pre-contracted small rat mesenteric arteries in a myograph setting. Pre-contracted mesenteric arteries strongly dilated upon treatment with both types of acetate containing ET-CORMs ( rac - 1 and rac - 4 ), while treatment with the pivalate containing ET-CORM ( rac - 8 ) resulted in no vasodilation. Pre-treatment of mesenteric arteries with the sGC inhibitor ODQ abolished rac - 4 mediated vasodilation, similar as for the known sGC activator SNP. Likewise, rac - 4 mediated vasodilation did not occur in KCL pretreated mesenteric arteries. Although mesenteric arteries abundantly expressed a variety of K + -channels only Kv7 channels were found to be of functional relevance for rac - 4 mediated vasodilation. In conclusion the current results identified Kv7 channels as the main channel by which rac - 4 mediates vasodilation. In keeping with the central role of Kv7 in the control of vascular tone and peripheral resistance these promising ex-vivo data warrant further in vivo studies, particularly in models of primary hypertension or cardiac diseases, to assess the potential use of ET-CORMs in these diseases., Competing Interests: PW was employed by Sanofi Aventis Deutschland GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Krause, Schmalz, Wohlfart, Yard and Schubert.)

Background: Voltage-gated potassium (Kv) channels, especially Kv7 channels, are major potassium channels identified in vascular smooth muscle cells with a great, albeit differential functional impact in various vessels. Vascular smooth muscle Kv7 channels always coexist with other K channels, in particular with BK channels. BK channels differ in the extent to which they influence vascular contractility. Whether this difference also causes the variability in the functional impact of Kv7 channels is unknown. Therefore, this study addressed the hypothesis that the functional impact of Kv7 channels depends on BK channels., Experimental Approach: Experiments were performed on young and adult rat gracilis and saphenous arteries using real-time PCR as well as pressure and wire myography., Key Results: Several subfamily members of Kv7 (KCNQ) and BK channels were expressed in saphenous and gracilis arteries: the highest expression was observed for BKα, BKβ1 and KCNQ4. Arterial contractility was assessed with methoxamine-induced contractions and pressure-induced myogenic responses. In vessels of adult rats, inhibition of Kv7 channels or BK channels by XE991 or IBTX, respectively enhanced arterial contractility to a similar degree, whereas activation of Kv7 channels or BK channels by retigabine or NS19504, respectively reduced arterial contractility to a similar degree. Further, IBTX increased both the contractile effect of XE991 and the anticontractile effect of retigabine, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991. In vessels of young rats, inhibition of Kv7 channels by XE991 enhanced arterial contractility much stronger than inhibition of BK channels by IBTX, whereas activation of Kv7 by retigabine reduced arterial contractility to a greater extent than activation of BK channels by NS19504. Further, IBTX increased the anticontractile effect of retigabine but not the contractile effect of XE991, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991., Conclusion: Kv7 and BK channels are expressed in young and adult rat arteries and function as negative feedback modulators in the regulation of contractility of these arteries. Importantly, BK channels govern the extent of functional impact of Kv7 channels. This effect depends on the relationship between the functional activities of BK and Kv7 channels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Ma, Gaynullina, Schmidt, Mladenov and Schubert.)

Perivascular adipose tissue (PVAT) is implicated in the pathophysiology of cardiovascular disease, especially in obese individuals in which the quantity of renal and visceral PVAT is markedly increased. The control of arterial tone by PVAT has emerged as a relatively new field of experimental hypertension research. The discovery of this prototype of vasoregulation has been mostly inferred from data obtained using wire myography. Currently, there is a major discussion on distinguishing between biological vs. technical replicates in biomedical studies, which resulted in numerous guidelines being published on planning studies and publishing data by societies, journals, and associations. Experimental study designs are determined depending on how the experimentator distinguishes between biological vs. technical replicates. These definitions determine the ultimate standards required for making submissions to certain journals. In this article, we examine possible outcomes of different experimental study designs on PVAT control of arterial tone using isolated arteries. Based on experimental data, we determine the sample size and power of statistical analyses for such experiments. We discuss whether n -values should correspond to the number of arterial rings and analyze the resulting effects if those numbers are averaged to provide a single N -value per animal, or whether the hierarchical statistical method represents an alternative for analyzing such kind of data. Our analyses show that that the data (logEC 50 ) from (+) PVAT to (-) PVAT arteries are clustered. Intraclass correlation (ICC) was 31.4%. Moreover, it appeared that the hierarchical approach was better than regular statistical tests as the analyses revealed by a better goodness of fit (v 2 -2LL test). Based on our results, we propose to use at least three independent arterial rings from each from three animals or at least seven arterial rings from each from two animals for each group, i.e., (+) PVAT vs. (-) PVAT. Finally, we discuss a clinical situation where distinguishing between biological vs. technical replicates can lead to absurd situations in clinical decision makings. We conclude that discrimination between biological vs. technical replicates is helpful in experimental studies but is difficult to implement in everyday's clinical practice.

Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N 2 , 5% CO 2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O 2 , 5% CO 2 ). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels K Ca3.1 and K Ca2.1 , the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.

Background: Multiple studies have shown that an NO-induced activation of vascular smooth muscle BK channels contributes to the NO-evoked dilation in many blood vessels. In vivo, NO is released continuously. NO attenuates vessel constrictions and, therefore, exerts an anticontractile effect. It is unknown whether the anticontractile effect of continuously present NO is mediated by BK channels., Methods and Results: This study tested the hypothesis that BK channels mediate the vasodilatory effect of continuously present NO. Experiments were performed on rat and mouse tail and rat saphenous arteries using isometric myography and FURA-2 fluorimetry. Continuously present NO donors, as well as endogenous NO, attenuated methoxamine-induced vasoconstrictions. This effect was augmented in the presence of the BK channel blocker iberiotoxin. Moreover, the contractile effect of iberiotoxin was reduced in the presence of NO donors. The effect of the NO donor sodium nitroprusside was abolished by an NO scavenger and by a guanylyl cyclase inhibitor. In addition, the effect of sodium nitroprusside was reduced considerably by a protein kinase G inhibitor, but was not altered by inhibition of H 2 S generation. Sodium nitroprusside attenuated the intracellular calcium concentration response to methoxamine. Furthermore, sodium nitroprusside strongly reduced methoxamine-induced calcium influx, which depends entirely on L-type calcium channels. It did not affect methoxamine-induced calcium release., Conclusions: In summary, this study demonstrates the following: (1) continuously present NO evokes a strong anticontractile effect on rat and mouse arteries; (2) the iberiotoxin-induced augmentation of the effect of NO is associated with an NO-induced reduction of the effect of iberiotoxin; and (3) NO evoked a reduction of calcium influx via L-type calcium channels., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

The anti-contractile effect of perivascular adipose tissue (PVAT) is an important mechanism in the modulation of vascular tone in peripheral arteries. Recent evidence has implicated the XE991-sensitive voltage-gated KV (KCNQ) channels in the regulation of arterial tone by PVAT. However, until now the in vivo pharmacology of the involved vascular KV channels with regard to XE991 remains undetermined, since XE991 effects may involve Ca(2+) activated BKCa channels and/or voltage-dependent KV1.5 channels sensitive to diphenyl phosphine oxide-1 (DPO-1). In this study, we tested whether KV1.5 channels are involved in the control of mesenteric arterial tone and its regulation by PVAT. Our study was also aimed at extending our current knowledge on the in situ vascular pharmacology of DPO-1 and XE991 regarding KV1.5 and BKCa channels, in helping to identify the nature of K(+) channels that could contribute to PVAT-mediated relaxation. XE991 at 30 μM reduced the anti-contractile response of PVAT, but had no effects on vasocontraction induced by phenylephrine (PE) in the absence of PVAT. Similar effects were observed for XE991 at 0.3 μM, which is known to almost completely inhibit mesenteric artery VSMC KV currents. 30 μM XE991 did not affect BKCa currents in VSMCs. Kcna5 (-/-) arteries and wild-type arteries incubated with 1 μM DPO-1 showed normal vasocontractions in response to PE in the presence and absence of PVAT. KV current density and inhibition by 30 μM XE991 were normal in mesenteric artery VSMCs isolated from Kcna5 (-/-) mice. We conclude that KV channels are involved in the control of arterial vascular tone by PVAT. These channels are present in VSMCs and very potently inhibited by the KCNQ channel blocker XE991. BKCa channels and/or DPO-1 sensitive KV1.5 channels in VSMCs are not the downstream mediators of the XE991 effects on PVAT-dependent arterial vasorelaxation. Further studies will need to be undertaken to examine the role of other KV channels in the phenomenon.

Objectives: Sympathetic denervation enhances agonist-induced vasoconstriction. This effect may involve altered function of signaling mechanisms such as Rho kinase (Rock) and L-type Ca channels downstream from vasoconstrictor receptors. We tested if enhanced Rock and L-type calcium channel activation contribute to exaggerated norepinephrine-induced vasoconstrictions in renal and mesenteric resistance arteries after sympathectomy., Methods: Rats underwent neonatal sympathectomy or sham sympathectomy. Resistance arteries were investigated by small vessel myography. Vascular Rock and L-type Ca channel expression as well as Rock activation were investigated by quantitative real-time PCR and Western blot. Vascular smooth muscle cell (VSMC) membrane potential was recorded with microelectrodes., Results: Sympathetic denervation enhanced norepinephrine sensitivity in renal and mesenteric arteries. Both, Rock inhibition or L-type Ca inhibition shifted the norepinephrine concentration-response curve to the right. This effect was more pronounced in renal than in mesenteric arteries from sympathectomized vs. sham-sympathectomized animals. The L-type Ca channel activator S-(-)-BayK8644 elicited strong vasoconstrictions only in renal arteries from sympathectomized rats. Rock activity and L-type Ca channel α-subunit expression were similar in renal arteries from sympathectomized and sham-sympathectomized animals. VSMC membrane potential was -57.5 ± 2.0 and -64.3 ± 0.3 mV (P < 0.01), respectively, in renal arteries from sympathectomized and from sham-sympathectomized rats. Depolarization enhanced and KATP channel activation abolished S-(-)-BayK8644-induced contractions in renal arteries from sympathectomized rats., Conclusion: Sympathetic denervation enhances L-type Ca channel-dependent signaling in renal but not in mesenteric arteries. This effect may be partly explained by the decreased VSMC membrane potential in denervated renal arteries.

The aim of this study is to reveal the regulatory role of cystathionine gamma-lyase (CSE), the main source of hydrogen sulphide (H 2 S) in perivascular adipose tissue (PVAT), of diabetic rats. Diabetes was induced in male rats by a single intraperitoneal injection of streptozotocin. Animals with glucose levels above 20 mmol/L were determined as diabetic. The rat gracilis arteries (a. gracilis) were dissected with or without PVAT. In all in vitro experiments endothelium-denuded preparations were used for isometric contraction measurements. Increasing concentrations of 5-hydroxytryptamine (5-HT) from 10 -10 to 10 -5 mol/L were applied to induce gradual increase in force of contractions of circular artery segments. The relaxing effect of CSE was inhibited by DL-propargyl glycine (PGG). The presence of PVAT decreases the contractile response to 5-HT of a. gracilis from control rats. This response is reversed in contraction studies in the same rat artery from diabetic rats. DL-PPG (1 mmol/L) induced significant increase of the force of contraction in artery preparations with PVAT from control rats in the whole range of 5-HT. In contrast, PGG had a relaxing effect in high concentrations of 5-HT (10 -6 and 10 -5 mol/L) in diabetic rat arteries with PVAT. It is concluded that in skeletal muscle artery from diabetic rats, a mediator related to H 2 S is released from PVAT. This paracrine mediator increases the maximal force of contraction of endothelium-denuded preparations at higher concentrations of 5-HT.

The intrinsic ability of resistance arteries to respond to transmural pressure is the single most important determinant of their function. Despite an ever-growing catalogue of signalling pathways that underlie the myogenic response, it remains an enigmatic mechanism. The myogenic response's mechanistic diversity has largely been attributed to 'hard-wired' differences across species and vascular beds; however, emerging evidence suggests that the mechanistic basis for the myogenic mechanism is, in fact, 'plastic'. This means that the myogenic response can change quantitatively (i.e. change in magnitude) and qualitatively (i.e. change in mechanistic basis) in response to environmental challenges (e.g. disease conditions). Consequently, understanding the dynamics of how the myogenic response capitalizes on its mechanistic diversity is key to unlocking clinically viable interventions. Using myogenic sphingosine-1-phosphate (S1P) signalling as an example, this review illustrates the remarkable plasticity of the myogenic response. We propose that currently unidentified 'organizational programmes' dictate the contribution of individual signalling pathways to the myogenic response and introduce the concept that certain signalling elements act as 'divergence points' (i.e. as the potential higher level regulatory sites). In the context of pressure-induced S1P signalling, the S1P-generating enzyme sphingosine kinase 1 serves as a divergence point, by orchestrating the calcium-dependent and -independent signalling pathways underlying microvascular myogenic responsiveness. By acting on divergence points, the proposed 'organizational programmes' could form the basis for the flexible recruitment and fine-tuning of separate signalling streams that underlie adaptive changes to the myogenic response and its distinctiveness across species and vascular beds.

Endothelial cells (EC) control vascular smooth muscle cell (VSMC) tone by release of paracrine factors. VSMC may also influence the EC layer, and therefore, the present study hypothesized that the opening of large-conductance Ca(2+) activated K(+) (BK(Ca)) channels may indirectly modulate EC hyperpolarization and nitric oxide (NO) release via myoendothelial gap junctions (MEGJ). To address this hypothesis 'in situ' EC ion current recordings, isolated VSMC patch clamp recordings, and simultaneous measurements of NO concentration and relaxation were conducted using segments of the rat superior mesenteric artery. In arteries constricted by α(1)-adrenoceptor activation, ACh (1 μM) evoked EC outward currents, vasorelaxation, and NO release. In contrast to preincubation with iberiotoxin (IbTx, 100nM) application of IbTx after ACh decreased EC outward currents, NO release and vasorelaxation. Furthermore, in phenylephrine (Phe)-contracted arteries treated with a gap junction uncoupler, cabenoxolone (CBX), IbTx failed to decrease ACh-evoked EC outward currents. In addition, CBX decreased EC outward currents, time constant of the capacitative transients, input capacitance, and increased input resistance. In isolated VSMC CBX did not affect BK(Ca) currents. Immunohistochemistry revealed only BK(Ca) channel positive staining in the VSMC layer. Therefore, the present results suggest that BK(Ca) channels are expressed in the VSMC, and that Phe by activation of VSMC BK(Ca) channels modulates ACh-evoked EC outward currents, NO release and vasorelaxation via MEGJ in rat superior mesenteric artery., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Growing evidence suggests that mechanisms which regulate the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca2+ sensitivity is suited to partially uncouple intracellular Ca2+ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca2+-dependent processes. Following a brief review of 'classical' Ca2+-dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca2+ sensitivity. For the purpose of this review, Ca2+ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin light-chain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca2+ sensitivity will be discussed. Although Ca2+ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca2+-dependent 'initiation/trigger' phase followed by a Ca2+-independent 'maintenance' phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca2+-dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca2+ sensitivity, as an equal and complimentary partner of Ca2+-dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.

The aim of this study was to investigate the effect of CART peptide on cardiac performance and on the physiological signalling pathways involved using Rana ridibunda frog heart preparations in vivo. The CART peptide, when injected into the venous sinus, significantly and reproducibly increased the force of frog heart contractions by up to 33.0 +/- 6.4% during the first 15 min after its application but did not influence the chronotropic activity of the frog heart. The positive inotropic effect was entirely blocked by prazosin, pertussis toxin, R(p)-adenosine 3',5'-cyclic monophosphorothioate, autosauvagine 30 or metyrapone, as well as by extirpation of the pituitary gland, functional elimination of the inter-renal glands and long-lasting starvation, and was not observed on isolated heart preparations. Propranolol and double pithing were without significant effect on this phenomenon. It was concluded that: (i) CART peptide, administered to frogs in vivo, increases the force of heart contractions; (ii) this effect of the peptide is exerted via activation of the hypothalamic-pituitary-inter-renal gland axis through a corticoliberin-sensitive mechanism; (iii) CART augments the pumping function of the heart via a corticosteroid-dependent potentiation of myocardial alpha(1)-adrenoreceptors signalling; and (iv) prolonged food deprivation abolishes the positive inotropic effect of CART, suggesting the participation of endogenous CART in the physiological adaptation of the circulatory system to limitations of energy consumption.

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca(2+)-activated K(+) channels (K(Ca)), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K(Ca) (IK(Ca)/K(Ca)3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K(Ca)3.1. K(Ca)3.1-deficient mice (K(Ca)3.1(-/-)) were generated by conventional gene-targeting strategies. Endothelial K(Ca) currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K(Ca)3.1 gene abolished endothelial K(Ca)3.1 currents and significantly diminished overall current through K(Ca) channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K(Ca)3.1(-/-) mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K(Ca)3.1(-/-) mice. Moreover, the loss of K(Ca)3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K(Ca)3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 micromol/L noradrenaline was associated with a approximately 2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with alpha-toxin were relaxed with urocortin by 39+/-3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 micromol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 approximately 2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca(2+)-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.

The hypothesis that the amplitude of the myogenic response is modulated by factors released from nerve endings was tested in rat tail small arteries. A pressure myograph in conjunction with direct stimulation of nerve endings by electrical field stimulation (EFS) was used to determine rat small artery contractile reactions. Vessel pretreatment with 10(- 5) M phentolamine abolished EFS-induced reactions completely indicating that they are mediated mainly by an adrenoceptor agonist, probably noradrenaline. In the absence and presence of 10(- 5) M phentolamine, vessel diameter changes in the pressure range from 10 to 120 mmHg were not different. Vessel stimulation by (i) EFS, (ii) noradrenaline, (iii) selective stimulation of alpha1- and alpha2-receptors, (iv) serotonin, or (v) vasopressin significantly reduced the diameter change induced by stepping pressure from 10 to 40 mmHg compared to unstimulated, control vessels. Vessel diameter changes induced by stepping pressure from 40 to 80 and from 80 to 120 mmHg, however, were not different in vessels stimulated with EFS and noradrenaline compared to controls. In conclusion, these data show that factors released from unstimulated adrenergic nerve endings (i.e., not stimulated by EFS) are not involved in the myogenic response. In contrast, factors released upon stimulation of nerve endings can modulate the amplitude of the myogenic response, but only at low pressures. Thus, the pressure range for myogenic blood flow autoregulation is extended to lower pressures. Myogenic autoregulation of blood flow at physiological pressures is unaltered.

The hypothesis that Rho kinase is involved in myogenic reactivity was investigated in pressurized rat tail small arteries using videomicroscopic diameter determination and calcium fluorimetry. The potent Rho kinase inhibitor Y-27632 reversibly increased vessel diameter at 80 mmHg without changing the intracellular calcium concentration ([Ca](i)) shifting the relationship between diameter change and [Ca](i) to higher calcium levels. Neither endothelium removal nor inhibition of neural transmission affected the Y-27632-induced effect. Y-27632 at 3 x 10(-6) mol/l attenuated the myogenic response in the pressure range from 10 to 120 mmHg, shifting the relationship between vessel tone and [Ca](i) to higher calcium levels. In addition, the Y-27632-induced shift of the relationship between vessel tone and [Ca](i) was larger at 80 than at 10 mmHg. These results suggest that smooth muscle cell Rho kinase in rat tail small arteries 1) is in an active state partly determining the level of the myogenic tone, and 2) alters the strength of the myogenic response by changing calcium sensitivity, probably caused by the pressure-induced activation of the kinase.