Vascular Myogenic Tone Research Articles

Diminished Rbfox1 increases vascular constriction by dynamically regulating alternative splicing of CaV1.2 calcium channel in hypertension.

Calcium influx from depolarized CaV1.2 calcium channels triggers the contraction of vascular smooth muscle cells (VSMCs), which is important for maintaining vascular myogenic tone and blood pressure. The function of CaV1.2 channel can be subtly modulated by alternative splicing (AS), and its aberrant splicing involves in the pathogenesis of multiple cardiovascular diseases. The RNA-binding protein Rbfox1 is reported to regulate the AS events of CaV1.2 channel in the neuronal development, but its potential roles in vascular CaV1.2 channels and vasoconstriction remain undefined. Here, we detect Rbfox1 is expressed in rat vascular smooth muscles. Moreover, the protein level of Rbfox1 is dramatically decreased in the hypertensive small arteries from spontaneously hypertensive rats in comparison with normotensive ones from Wistar-Kyoto rats. In VSMCs, Rbfox1 could dynamically regulate the AS of CaV1.2 exons 9* and 33. By whole-cell patch clamp, we identify knockdown of Rbfox1 induces the hyperpolarization of CaV1.2 current-voltage relationship curve in VSMCs. Furthermore, siRNA-mediated knockdown of Rbfox1 increases the K+-induced constriction of rat mesenteric arteries. In summary, our results indicate Rbfox1 modulates vascular constriction by dynamically regulating CaV1.2 alternative exons 9* and 33. Therefore, our work elucidates the underlying mechanisms for CaV1.2 channels regulation and provides a potential therapeutic target for hypertension.

Pregnancy Increases Ca2+ Sparks/Spontaneous Transient Outward Currents and Reduces Uterine Arterial Myogenic Tone.

Spontaneous transient outward currents (STOCs) at physiological membrane potentials of vascular smooth muscle cells fundamentally regulate vascular myogenic tone and blood flow in an organ. We hypothesize that heightened STOCs play a key role in uterine vascular adaptation to pregnancy. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Ca2+ sparks were measured by confocal microscopy, and STOCs were determined by electrophysiological recording in smooth muscle cells. Percentage of Ca2+ spark firing myocytes increased dramatically at the resting condition in uterine arterial smooth muscle of pregnant animals, as compared with nonpregnant animals. Pregnancy upregulated the expression of RyRs (ryanodine receptors) and significantly boosted Ca2+ spark frequency. Ex vivo treatment of uterine arteries of nonpregnant sheep with estrogen and progesterone imitated pregnancy-induced RyR upregulation. STOCs occurred at much more negative membrane potentials in uterine arterial myocytes of pregnant animals. STOCs in uterine arterial myocytes were diminished by inhibiting large-conductance Ca2+-activated K+ (BKCa) channels and RyRs, thus functionally linking Ca2+ sparks and BKCa channel activity to STOCs. Pregnancy and steroid hormone treatment significantly increased STOCs frequency and amplitude in uterine arteries. Of importance, inhibition of STOCs with RyR inhibitor ryanodine eliminated pregnancy- and steroid hormone-induced attenuation of uterine arterial myogenic tone. Thus, the present study demonstrates a novel role of Ca2+ sparks and STOCs in the regulation of uterine vascular tone and provides new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

Distribution and Function of Neuropeptides W/B Signaling System.

Neuropeptide W (NPW) and neuropeptide B (NPB) are two structurally and functionally related regulatory peptides, which are highly expressed in several brain regions and, additionally, in some peripheral tissues. Nevertheless, their distributions in the tissues are not similar. They act on target tissues via two subtypes of G protein-coupled receptors which are designated as NPBWR1 (GPR7) and NPBWR2 (GPR8), respectively, and possess different binding affinities. NPB activates NPBWR1, whereas NPW stimulates both the receptors with similar potency. Both of these peptides takes a part in the central regulation of neuroendocrine axes, feeding behavior, energy homeostasis, cardiovascular functions, circadian rhythm, pain sensation, modulation of inflammatory pain, and emotions. Over the past few years, studies have shown that NPB is also involved in sleep regulation. On the contrary, NPW participates in regulation of vascular myogenic tone, inhibits gastric tension sensitive vagal afferents and insulin secretion. Also, expression of NPW in the stomach is regulated by feeding. Abovementioned findings clearly demonstrate the functional diversity among NPW versus NPB signaling systems. In this review, signal transduction pathways of NPW/NPB are critically evaluated and observed together with mapping of expression of their signaling systems.

PIEZO1 Channel Is a Potential Regulator of Synovial Sarcoma Cell-Viability.

Detection of mechanical stress is essential for diverse biological functions including touch, audition, and maintenance of vascular myogenic tone. PIEZO1, a mechano-sensing cation channel, is widely expressed in neuronal and non-neuronal cells and is expected to be involved in important biological functions. Here, we examined the possibility that PIEZO1 is involved in the regulation of synovial sarcoma cell-viability. Application of a PIEZO1 agonist Yoda1 effectively induced Ca2+ response and cation channel currents in PIEZO1-expressing HEK (HEK-Piezo1) cells and synovial sarcoma SW982 (SW982) cells. Mechanical stress, as well as Yoda1, induced the activity of an identical channel of conductance with 21.6 pS in HEK-Piezo1 cells. In contrast, Yoda1 up to 10 μM had no effects on membrane currents in HEK cells without transfecting PIEZO1. A knockdown of PIEZO1 with siRNA in SW982 cells abolished Yoda1-induced Ca2+ response and significantly reduced cell cell-viability. Because PIEZO1 is highly expressed in SW982 cells and its knockdown affects cell-viability, this gene is a potential target against synovial sarcoma.

Loss of Vascular Myogenic Tone in miR-143/145 Knockout Mice Is Associated With Hypertension-Induced Vascular Lesions in Small Mesenteric Arteries.

Pressure-induced myogenic tone is involved in autoregulation of local blood flow and confers protection against excessive pressure levels in small arteries and capillaries. Myogenic tone is dependent on smooth muscle microRNAs (miRNAs), but the identity of these miRNAs is unclear. Furthermore, the consequences of altered myogenic tone for hypertension-induced damage to small arteries are not well understood. The importance of smooth muscle-enriched microRNAs, miR-143/145, for myogenic tone was evaluated in miR-143/145 knockout mice. Furthermore, hypertension-induced vascular injury was evaluated in mesenteric arteries in vivo after angiotensin II infusion. Myogenic tone was abolished in miR-143/145 knockout mesenteric arteries, whereas contraction in response to calyculin A and potassium chloride was reduced by ≈30%. Furthermore, myogenic responsiveness was potentiated by angiotensin II in wild-type but not in knockout mice. Angiotensin II administration in vivo elevated systemic blood pressure in both genotypes. Hypertensive knockout mice developed severe vascular lesions characterized by vascular inflammation, adventitial fibrosis, and neointimal hyperplasia in small mesenteric arteries. This was associated with depolymerization of actin filaments and fragmentation of the elastic laminae at the sites of vascular lesions. This study demonstrates that miR-143/145 expression is essential for myogenic responsiveness. During hypertension, loss of myogenic tone results in potentially damaging levels of mechanical stress and detrimental effects on small arteries. The results presented herein provide novel insights into the pathogenesis of vascular disease and emphasize the importance of controlling mechanical factors to maintain structural integrity of the vascular wall.

Aberrant Splicing Induced by Dysregulated Rbfox2 Produces Enhanced Function of CaV1.2 Calcium Channel and Vascular Myogenic Tone in Hypertension.

Calcium influx from activated voltage-gated calcium channel CaV1.2 in vascular smooth muscle cells is indispensable for maintaining myogenic tone and blood pressure. The function of CaV1.2 channel can be optimized by alternative splicing, one of post-transcriptional modification mechanisms. The splicing factor Rbfox2 is known to regulate the CaV1.2 pre-mRNA alternative splicing events during neuronal development. However, Rbfox2's roles in modulating the key function of vascular CaV1.2 channel and in the pathogenesis of hypertension remain elusive. Here, we report that the proportion of CaV1.2 channels with alternative exon 9* is increased by 10.3%, whereas that with alternative exon 33 is decreased by 10.5% in hypertensive arteries. Surprisingly, the expression level of Rbfox2 is increased ≈3-folds, presumably because of the upregulation of a dominant-negative isoform of Rbfox2. In vascular smooth muscle cells, we find that knockdown of Rbfox2 dynamically increases alternative exon 9*, whereas decreases exon 33 inclusion of CaV1.2 channels. By patch-clamp studies, we show that diminished Rbfox2-induced alternative splicing shifts the steady-state activation and inactivation curves of vascular CaV1.2 calcium channel to hyperpolarization, which makes the window current potential to more negative. Moreover, siRNA-mediated knockdown of Rbfox2 increases the pressure-induced vascular myogenic tone of rat mesenteric artery. Taken together, our data indicate that Rbfox2 modulates the functions of vascular CaV1.2 calcium channel by dynamically regulating the expressions of alternative exons 9* and 33, which in turn affects the vascular myogenic tone. Therefore, our work suggests a key role for Rbfox2 in hypertension, which provides a rational basis for designing antihypertensive therapies.

The Role of NADPH Oxidase in Regulating Cerebral Vascular Myogenic Tone

The myogenic response refers to the intrinsic ability of arteries to constrict to elevated pressure. The underlying mechanism has yet to be elucidated but recent evidence suggests that the angiotensin II type 1 receptor (AT1R) is a key mechanosensor, linking intravascular pressure to tone development. One intriguing aspect of this receptor is its ability to activate NADPH oxidase (Nox), an enzyme responsible for the production of reactive oxygen species. The goal of this study was to ascertain the role of Nox in myogenic tone development. Briefly, rat cerebral arteries were mounted in a myograph, pressurized to 60mmHg and diameter monitored prior to and following the application of apocynin (a general Nox inhibitor) or ML171 (a Nox1 specific inhibitor). Myogenic constriction progressively rose as vessels were pressurized from 20 to 60 mmHg. These pressure‐induced responses were attenuated by apocynin in a concentration dependent manner (37% vs 24% at 60 mmHg) irrespective of whether the endothelium was present or absent. ML171 (30μM) was equally effective at moderating the myogenic response (36% vs 24%); control experiments showed no discernible role for Nox2. Similar attenuating effects were observed in human cerebral arteries. These findings indicate Nox1 is expressed in rat cerebral arterial smooth muscle and may be a regulator of pressure‐induced responses. While the precise downstream mechanism has yet to be resolved, the L‐type Ca2+ channel is a likely target of Nox regulation. This work sheds new light on mechanosensation and how unique signaling proteins impact the ability of resistance arteries to respond to intravascular pressure.Support or Funding InformationThis work is supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) for DG Welsh. DG Welsh is a Rorabeck chair in molecular neuroscience and vascular biology at the University of Western Ontario. AM Hashad is a Vanier scholar (CIHR) and was supported by a salary studentship from Alberta Innovates Health solutions (AIHS).

Studying the Effects of Bioactive Lipids on TRPV4 Activation

Blood pressure regulation relies on the ability of mechanoelectrical transduction proteins to transform variations in physical (e.g., pressure changes) and chemical stimuli (e.g., lipids) into electrical signals, which in turn regulate vascular tone. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel that has been implicated in the control of blood pressure, osmotic regulation, and vascular myogenic tone. TRPV4 is widely distributed in the vasculature, being found in smooth muscles, vascular endothelial cells, and perivascular sensory neurons. The precise molecular mechanism underlying TRPV4 activation by mechanical stimuli (e.g., shear stress) or bioactive lipids (e.g., epoxyeicosatrienoic acid [EET]) remains unknown. Moreover, whether TRPV4 is directly or indirectly activated by physical and chemical stimuli is a topic of debate. A more systematic analysis of the activation mechanism requires the examination of TRPV4 in a controlled lipid environment. Here, we examine the role of upstream lipid pathways on TRPV4 in the animal model C. elegans. Worms allow for easy and accurate manipulation of lipid content, the ability to incorporate lipids back via diet supplementation, and a collection of lipid-deficient mutants. To determine the mechanisms by which endogenous molecules modulate TRPV4, we use a transgenic worm that expresses rat (r) TRPV4 in a sensory neuron. Chemical activation of transgenic rTRPV4 worm (but not wild-type) by GSK1016790A (a selective agonist) elicits withdrawal responses. Behavior of rTRPV4 transgenic worms crossed with strains lacking fatty acids and lipids suggest that long fatty acids are required for rTRPV4-mediated withdrawal responses. These findings support the role of bioactive lipids as cofactors of TRPV4 activation.

Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

Increased Serca Pump Expression is Associated with Slow Termination of Calcium Sparks and Delayed Local Recovery in Vascular Smooth Muscle Cells of Hyperthyroid Rats

In vascular smooth muscle cells of cerebral arteries (VSMCs), spontaneous and repetitive local Ca2+ signals produced by the Ca2+ channel/Ryanodine Receptor (RyRs), known as Ca2+ sparks, are involved in the regulation of vascular myogenic tone. However, the molecular mechanisms behind the frequency and properties of Ca2+ sparks in VSMCs are still under study. Considering that in VSMCs, spontaneous Ca2+ sparks are generated at preferred subcellular locations, we have analyzed the spatio-temporal properties of repetitive same-location Ca2+ sparks, its time-dependent recovery (or restitution) and the regulation that luminal Ca2+ might exert in this phenomenon by using the experimental model of hyperthyroid rat (T3).Ca2+ sparks recorded in VSMCs from T3 rats showed similar frequency, amplitude, and Ca2+ flux compared with those recorded in control cells. However, Ca2+ spark properties such as duration, size, time-to-peak, and k decay were significantly increased in T3 cells; and as a consequence, spark-mediated Ca2+ leak was augmented by 3.3-fold. In both experimental groups, amplitude recovery of repetitive Ca2+ sparks was an all-or-none phenomenon and did not show any time-dependency. Histogram distribution of spark-to-spark delays from T3 cells showed a 4-fold reduction in shorter delays (0-200 ms), with the shortest spark-to-spark delay of 115.9 ms in T3 cells and of 67.2 ms in control cells. Although SERCA pump expression was increased in vascular tissue from T3 rats, both basal Ca2+ concentration and intracellular SR Ca2+ load, determined by caffeine, remained unchanged. Our data suggest that in VSMCs of T3 rats spontaneous Ca2+ sparks decrease SR Ca2+ content not only by increasing its frequency but also by delaying its termination to counteract SR Ca2+ overload resulting from the overexpression of SERCA pump.

Abnormal Ca2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice

Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca2+ spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from −50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca2+ spark/STOC coupling in CASMCs of diabetic animals.

Abstract 209: Hemeoxygenase-1 Induction Restores Beta ENaC Expression in Ischemic Placentas

Preeclampsia is a pregnancy-specific disease characterized by hypertension, proteinuria, and vascular abnormalities; the etiology of which is under active investigation. Utilizing the rat model of reduced uterine perfusion pressure (RUPP), our lab previously demonstrated that induction of hemeoxygenase-1 (HO-1) reduces blood pressure and oxidative stress in placental ischemic rats. While epithelial sodium channel proteins (specifically β-ENaC) are important in maintaining vascular myogenic tone and have been implicated in cytotropholast invasion, the effect of placental ischemia and induction of HO-1 on β-ENaC expression in placentas have not been established. Therefore, the purpose of this study is to test the hypothesis that placental β-ENaC expression is reduced in ischemic placentas and restored by HO-1 induction. Normal pregnant and RUPP rats were treated with the HO-1 inducer, cobalt protoporphyrin (CoPP) (n=4 per group/treatment). Blood pressure was measured and placentas were harvested on day 19 of gestation. Blood pressure increased from 101±1 to 141±4 mmHg (p<0.0001) in response to the RUPP procedure but was reduced by HO-1 induction (117±2 mmHg). RUPP rats also had fewer pups compared to normal pregnant rats (14±1 vs. 4±2 pups). HO-1 induction did not prevent the reduction in pup number. Placental β-ENaC protein expression was reduced by 62% in the RUPPs compared to the normal pregnant rats (p=0.036). HO-1 induction restored placental expression of β-ENaC to the levels of the normal pregnant rats. These findings suggest that HO-1 may exert its protective effects by altering the placental environment through changes in β-ENaC expression.

Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors

Hypertension is a cardiovascular risk factor present in over two-thirds of people over age 60 in North America; elevated blood pressure correlates with increased risk of heart attack, stroke and progression to heart and kidney failure. Current therapies are insufficient to control blood pressure in almost half of these patients. The mineralocorticoid receptor (MR), acting in the kidney, is known to regulate blood pressure through aldosterone binding and stimulation of sodium retention. However, recent studies support the concept that the MR also has extrarenal actions and that defects in sodium handling alone do not fully explain the development of hypertension and associated cardiovascular mortality. We and others have identified functional MR in human vascular smooth muscle cells (SMCs), suggesting that vascular MR might directly regulate blood pressure. Here we show that mice with SMC-specific deficiency of the MR have decreased blood pressure as they age without defects in renal sodium handling or vascular structure. Aged mice lacking MR in SMCs (SMC-MR) have reduced vascular myogenic tone, agonist-dependent contraction and expression and activity of L-type calcium channels. Moreover, SMC-MR contributes to angiotensin II–induced vascular oxidative stress, vascular contraction and hypertension. This study identifies a new role for vascular MR in blood pressure control and in vascular aging and supports the emerging hypothesis that vascular tone contributes directly to systemic blood pressure.

PI3K in hypertension: a novel therapeutic target controlling vascular myogenic tone and target organ damage

In the past decade, several studies have characterized a number of cellular and molecular mechanisms that contribute to the regulation of the vascular myogenic response, thus affecting blood pressure regulation. Recently, phosphoinositide 3-kinase γ (PI3Kγ) has been identified as a main regulator of vascular myogenic tone and blood pressure, a result further strengthened by a highly significant genome-wide association of a single nucleotide polymorphism flanking this gene with blood pressure regulation, in a large human population. The goal of this review is to summarize the available information regarding the mechanism whereby PI3Kγ exerts blood pressure control, regulating myogenic tone at the level of L-type calcium channel in smooth muscle cells. Moreover, an overview of the pharmacological approaches available for targeting this signalling pathway shows that PI3Kγ is a suitable candidate for antihypertensive therapy, capable of lowering blood pressure. Finally, a survey of the studies dissecting the role of PI3Kγ in pathological conditions that are typically induced by hypertension in its target organs provides a more complete picture of the high potential of this novel therapeutic approach for fighting hypertension and, at the same time, its target organ damage, independently of blood pressure-lowering effects.

Role of K ATP and L-type Ca 2+ channel activities in regulation of ovine uterine vascular contractility: effect of pregnancy and chronic hypoxia

Our objective was to determine whether the pregnancy and high altitude long-term hypoxia-mediated changes in uterine artery contractility were regulated by K(ATP) and L-type Ca(2+) channel activities. Uterine arteries were isolated from nonpregnant and near-term pregnant ewes that had been maintained at sea level (∼300 m) or exposed to high altitude (3801 m) for 110 days. Isometric tension was measured in a tissue bath. Pregnancy increased diazoxide, but not verapamil-induced relaxations. Long-term hypoxia attenuated diazoxide-induced relaxations in near-term pregnant uterine arteries, but enhanced verapamil-induced relaxations in nonpregnant uterine arteries. Diazoxide decreased the maximal response (E(max)) of phenylephrine-induced contractions in near-term pregnant uterin arteries but not nonpregnant uterine arteries in normoxic sheep. In contrast, diazoxide had no effect on phenylephrine-induced E(max) in near-term pregnant uterine arteries but decreased it in nonpregnant uterine arteries in long-term hypoxia animals. Verapamil decreased the E(max) and pD(2) (-logEC(50)) of phenylephrine-induced contractions in both nonpregnant uterine arteries and near-term pregnant uterine arteries in normoxic and long-term hypoxia animals, except nonpregnant uterine arteries of normoxic animals in which verapamil showed no effect on the pD(2). The results suggest that pregnancy selectively increases K(ATP), but not L-type Ca(2+) channel activity. Long-term hypoxia decreases the K(ATP) channel activity, which may contribute to the enhanced uterine vascular myogenic tone observed in pregnant sheep at high altitude hypoxia.

ABNORMAL CALCIUM HOMEOSTASIS IN ARTERIAL MYOCYTES FROM MILAN HYPERTENSIVE STRAIN RATS: PP.29.177

Polymorphisms in the genes encoding adducin, a membrane-skeletal protein, are associated with hypertension in humans and rats (Milan hypertensive strain, MHS). MHS rats, with their control normotensive strain (MNS), are a model for a subgroup of patients with essential hypertension. The relationship between adducin gene polymorphisms and renal dysfunction has been well-studied, but the mechanisms that underlie vascular functional abnormalities are unclear. Ca2+ homeostasis plays a crucial role in the genesis of vascular myogenic tone, and increases in cytosolic Ca2+ concentration ([Ca2+]cyt) appear to underlie at least part of the increased peripheral vascular resistance in hypertension. We hypothesize that Ca2+ signaling mediated by Na/Ca exchanger (NCX) and TRPC-encoded receptor-operated Ca2+ channels is up-regulated in MHS mesenteric artery myocytes compared with that in MNS. The [Ca2+]cyt was determined with digital imaging of Fura-2-loaded freshly dissociated mesenteric artery smooth muscle cells (ASMCs). On average, the MNS rats had a mean systolic BP (SBP) of 113 ± 1 mmHg; the SBP of the MHS rats was significantly higher (144 ± 2 mmHg, P < 0.05). Freshly dissociated MHS ASMCs had significantly higher resting [Ca2+]cyt than did MNS ASMCs (97 ± 2 nM vs. 78 ± 3 nM, P < 0.05). Receptor-operated entry was activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba2+, OAG (100 μM)-induced elevations of cytosolic Ba2+ (Fura-2 340/380 nm ratio) were significantly larger in MHS ASMCs. The ATP (5 μM)-induced Ca2+ responses were also augmented in MHS ASMCs (729 ± 70 nM vs. 533 ± 44 nM, P < 0.05). These changes in MHS ASMCs correlate with ∼3-fold up-regulation of TRPC6, while expression of other TRPCs was not changed. Recently we showed that expression of TRPC6 and NCX1 in ASMCs is inter-related and TRPC6 and NCX1 are both up-regulated in ouabain hypertensive rats (Pulina et al., Amer. J. Physiol. 298(1):H263-H274, 2010). Here, we show that NCX1 is also robustly (∼13-fold) up-regulated in MHS ASMCs. The findings suggest that up-regulation of NCX1 and TRPC6 can represent a general downstream phenomenon intrinsic to various forms of hypertension.

Role of potassium channels in regulation of rat coronary arteriole tone

Activity of potassium (K +) channels plays a key role in regulating arterial tone and therefore blood flow. But little is known about the roles of K + channels in the coronary arterioles. The aim of the present study was to investigate the influences of several potassium channel blockers on resting vascular tone in isolated rat coronary arteriole. The coronary arterioles were carefully dissected, cannulated and pressurized. The inner diameter of vessels was recorded by a computerized diameter tracking system and the effects of K + channels on the development of vascular myogenic tone was tested by application of various K + channel inhibitors. Inhibition of voltage-dependent K + (K V) channels and inward rectifier K + (K ir) channels significantly increased the tone constriction. But inhibition of Ca 2+-activated K + (K Ca) channels and ATP-sensitive K + (K ATP) channels had no effect on the resting tone. In addition, inhibition of nitric oxide synthase (NOS) further decreased the tone diameter. Combined inhibition of K V, K ir and NOS induced the strongest tone constriction. Our results suggested that activity of K V and K ir channels, and basal nitric oxide (NO) contribute to the myogenic tone in rat coronary arteriole under resting conditions, but basal NO may not contribute to the activation of K V and K ir channels. K Ca and K ATP channels are likely to have a very low open probability under normal conditions.

Direct Chronic Effect of Steroid Hormones in Attenuating Uterine Arterial Myogenic Tone

Pregnancy is associated with a significant decrease in uterine vascular tone and an increase in uterine blood flow. The present study tested the hypothesis that estrogen and progesterone differentially regulate the extracellular signal–regulated kinase (ERK)1/2 and protein kinase C (PKC) signaling pathways in vascular smooth muscle, resulting in a decrease in uterine vascular myogenic tone in pregnancy. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Chronic treatment (48 hours) of nonpregnant uterine arteries with 17β-estradiol and progesterone caused a significant decrease in PKC-mediated contractions and pressure-induced myogenic tone. In accordance, treatment of near-term pregnant uterine arteries for 48 hours with ICI 182780 and RU 486 significantly increased PKC-induced contractions and myogenic tone. In contrast, acute treatment for 30 minutes had no effect on uterine artery contractility. An ERK1/2 inhibitor, PD098059, restored the chronic effect of steroids on PKC-mediated contractions in nonpregnant sheep. ERK1/2 protein and mRNA levels were greater in near-term pregnant as compared with nonpregnant uterine arteries. 17β-Estradiol and progesterone increased ERK1/2 protein in nonpregnant sheep. In agreement, ICI 182780 and RU 486 caused significant decreases in ERK1/2 protein in near-term pregnant sheep. Western blot showed 6 PKC isozymes, α, β I , β II , δ, ε, and ζ, in the uterine arteries. 17β-Estradiol and progesterone decreased the particulate:cytosolic ratios of PKCα, ε, and ζ, respectively, in nonpregnant sheep. ICI 182780 and RU 486 increased the ratios in near-term pregnant sheep. The results indicate a direct chronic effect of the steroid hormones in the upregulation of ERK1/2 expression and downregulation of the PKC signaling pathway, resulting in attenuated myogenic tone of the uterine artery in pregnancy.

Ubiquitous inactivation of TRPM4 leads to elevated blood pressure in mice

Recently we showed that transient receptor potential TRPM4 proteins act as Ca2+‐activated nonselective cation channels in mast cells that critically limit the driving force for Ca2+ influx, release of inflammatory mediators and anaphylactic responses. In addition to mast cells we detected TRPM4 proteins in heart, aortic endothelial cells, kidney and adrenal gland from mice. Earley et al suggested that TRPM4 is expressed in vascular smooth muscle cells and it is supposed to act as a mechanosensor regulating vascular myogenic tone.Telemetric blood pressure measurements reveal that mean arterial blood pressure (MAP) is elevated in TRPM4‐/‐ mice by 11mmHg under basal conditions. Blood pressure elevation is observed during the complete circadian period and most notable under resting conditions. Plasma aldosterone levels, renin mRNA expression in the kidney and renin plasma concentrations are not different in TRPM4‐/‐ mice and there are no differences in MAP in response to application of phenylephrine (PE), norepinephrine and L‐NAME. PE induced contraction and acetylcholine induced relaxation of aortic rings is not altered. Currently we analyse the contractility of resistance vessels with respect to smooth muscle‐dependent contraction and endothelium‐dependent relaxation. Inhibition of ganglionic transmission with hexamethonium abolishes the difference in MAP between both genotypes similarly like application of prazosin. Analysis of plasma catecholamine level revealed a significant elevation of epinephrine in TRPM4‐/‐ mice. These results suggest that an increased neurogenic tone contributes to the development of the hypertension.

Highlights From The Literature

Highlights From The Literature