Vladimir V. Matchkov

Vasomotion – what is currently thought?

This minireview discusses vasomotion, which is the oscillation in tone of blood vessels leading to flowmotion. We will briefly discuss the prevalence of vasomotion and its potential physiological and pathophysiological relevance. We will also discuss the models that have been suggested to explain how a coordinated oscillatory activity of the smooth muscle tone can occur and emphasize the role of the endothelium, the handling of intracellular Ca2+ and the role of smooth muscle cell ion conductances. It is concluded that vasomotion is likely to enhance tissue dialysis, although this concept still requires more experimental verification, and that an understanding at the molecular level for the pathways leading to vasomotion is beginning to emerge.

Junctional and nonjunctional effects of heptanol and glycyrrhetinic acid derivates in rat mesenteric small arteries

Heptanol, 18α‐glycyrrhetinic acid (18αGA) and 18β‐glycyrrhetinic acid (18βGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca2+‐sensitive dyes), membrane potential and – as a measure of intercellular coupling – input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch‐clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. Heptanol (150 μM) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 μM did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 18βGA (30 μM) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18αGA (100 μM) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. We conclude that in mesenteric small arteries, heptanol and 18βGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18βGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18αGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.

A Cyclic GMP–dependent Calcium-activated Chloride Current in Smooth-muscle Cells from Rat Mesenteric Resistance Arteries

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 μM BAY K8644, 0.4 μM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 μM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN− > Br− > I− > Cl− > acetate > F− >> aspartate, but the conductance sequence was I− > Br− > Cl− > acetate > F− > aspartate = SCN−. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.

Disruption of Na+,HCO3− Cotransporter NBCn1 (slc4a7) Inhibits NO-Mediated Vasorelaxation, Smooth Muscle Ca2+ Sensitivity, and Hypertension Development in Mice

Background— Disturbances in pH affect artery function, but the mechanistic background remains controversial. We investigated whether Na+,HCO3− cotransporter NBCn1, by regulating intracellular pH (pHi), influences artery function and blood pressure regulation. Methods and Results— Knockout of NBCn1 in mice eliminated Na+,HCO3− cotransport and caused a lower steady-state pHi in mesenteric artery smooth muscle and endothelial cells in situ evaluated by fluorescence microscopy. Using myography, arteries from NBCn1 knockout mice showed reduced acetylcholine-induced NO-mediated relaxations and lower rho-kinase-dependent norepinephrine-stimulated smooth muscle Ca2+ sensitivity. Acetylcholine-stimulated NO levels (electrode measurements) and N-nitro-l-arginine methyl ester–sensitive l-arginine conversion (radioisotope measurements) were reduced in arteries from NBCn1 knockout mice, whereas relaxation to NO-donor S-nitroso-N-acetylpenicillamine, acetylcholine-induced endothelial Ca2+ responses (fluorescence microscopy), and total and Ser-1177 phosphorylated endothelial NO-synthase expression (Western blot analyses) were unaffected. Reduced NO-mediated relaxations in arteries from NBCn1 knockout mice were not rescued by superoxide scavenging. Phosphorylation of myosin phosphatase targeting subunit at Thr-850 was reduced in arteries from NBCn1 knockout mice. Evaluated by an in vitro assay, rho-kinase activity was reduced at low pH. Without CO2/HCO3−, no differences in pHi, contraction or relaxation were observed between arteries from NBCn1 knockout and wild-type mice. Based on radiotelemetry and tail-cuff measurements, NBCn1 knockout mice were mildly hypertensive at rest, displayed attenuated blood pressure responses to NO-synthase and rho-kinase inhibition and were resistant to developing hypertension during angiotensin-II infusion. Conclusions— Intracellular acidification of smooth muscle and endothelial cells after knockout of NBCn1 inhibits NO-mediated and rho-kinase–dependent signaling in isolated arteries and perturbs blood pressure regulation.

Bestrophin-3 (Vitelliform Macular Dystrophy 2–Like 3 Protein) Is Essential for the cGMP-Dependent Calcium-Activated Chloride Conductance in Vascular Smooth Muscle Cells

Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca2+-activated Cl− currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca2+-activated Cl− current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a “classic” (cGMP-independent) Ca2+-activated Cl− current and to have characteristics distinct from those previously known for Ca2+-activated Cl− currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca2+-activated Cl− current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca2+-activated Cl− current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca2+-activated Cl− current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca2+-activated Cl− current, whereas the magnitude of the classic Ca2+-activated Cl− current was not affected. The majority of previous suggestions that bestrophins are a new Cl− channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca2+-activated Cl− current in smooth muscles in the intact vascular wall.

Vascular smooth muscle cell phenotype is defined by Ca2+-dependent transcription factors

Ca2+ is an important second messenger in vascular smooth muscle cells (VSMCs). Therefore, VSMCs exercise tight control of the intracellular Ca2+ concentration ([Ca2+]i) by expressing a wide repertoire of Ca2+ channels and transporters. The presence of several pathways for Ca2+ influx and efflux provides many possibilities for controlling [Ca2+]i in a spatial and temporal manner. Intracellular Ca2+ has a dual role in VSMCs; first, it is necessary for VSMC contraction; and, second, it can activate multiple transcription factors. These factors are cAMP response element‐binding protein, nuclear factor of activated T lymphocytes, and serum response factor. Furthermore, it was recently reported that the C‐terminus of voltage‐dependent L‐type Ca2+ calcium channels can regulate transcription in VSMCs. Transcription regulation in VSMCs modulates the expression patterns of genes, including genes coding for contractile and cytoskeleton proteins, and those promoting proliferation and cell growth. Depending on their gene expression, VSMCs can exist in different functional states or phenotypes. The majority of healthy VSMCs show a contractile phenotype, characterized by high contractile ability and a low proliferative rate. However, VSMCs can undergo phenotypic modulation with different physiological and pathological stimuli, whereby they start to proliferate, migrate, and synthesize excessive extracellular matrix. These events are associated with injury repair and angiogenesis, but also with the development of cardiovascular pathologies, such as atherosclerosis and hypertension. This review discusses the currently known Ca2+‐dependent transcription factors in VSMCs, their regulation by Ca2+ signalling, and their role in the VSMC phenotype.

Intracellular Ca2+ Signalling and Phenotype of Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) express considerable plasticity in their phenotype and even can change their phenotype in vivo depending on the functional demand. In addition to contractile phenotype, VSMCs can be proliferative, migrating and/or synthetic. Importantly, contractile and non-contractile phenotypes differ significantly in their intracellular Ca²⁺ signalling, which is a consequence of difference in expression of Ca²⁺ transport proteins. Contractile VSMCs express Ca²⁺ transporters, including voltage-gated L-type Ca²⁺ channels and SERCA2a pump, which maintain low resting cytosolic Ca²⁺ and allow dynamic changes of Ca²⁺ in the spatial and temporal domain, while non-contractile VSMCs have significantly reduced voltage dependence of Ca²⁺ entry. These changes associated with phenotypic switch are consequences of changes in gene expression programmes, where the expression of phenotype-specific proteins and other proteins is suppressed. Importantly, Ca²⁺ -sensitive transcription factors, including serum response factor, cAMP response element-binding protein and nuclear factor of activated T lymphocytes, which are important for this phenotype switch, can be activated by different types of Ca²⁺ signalling. Thus, different Ca²⁺ transport proteins not only control averaged intracellular Ca²⁺ but also through their differences in the character of the Ca²⁺ signal modulate the activity of transcription factors and thus initiate phenotype switch. The essential stimuli for phenotype switch are unknown, but intracellular Ca²⁺ is an important second messenger in the cell transcription programme. This article reviews the relationship between intracellular Ca²⁺ signalling and VSMC phenotype.

KV7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries

Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca2+ concentration ([Ca2+]i) by opening of K channels and release of H2S.

TMEM16A knockdown abrogates two different Ca2+-activated Cl− currents and contractility of smooth muscle in rat mesenteric small arteries

The presence of Ca2+-activated Cl− channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca2+-activated Cl− currents (ICl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent ICl(Ca) depends on bestrophin expression, while the “classical” ICl(Ca) is not. Downregulation of bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca2+]i, and SMC membrane potential were measured in isolated arterial segments. ICl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent ICl(Ca) and the “classical” ICl(Ca) in SMCs. TMEM16A downregulation also reduced expression of bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K+-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca2+]i. Surprisingly, K+-induced depolarization was unchanged but Ca2+ entry was reduced. We suggested that this is due to reduced expression of the L-type Ca2+ channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs ICl(Ca) and vascular function and suggests an interaction between TMEM16A and L-type Ca2+ channels.

Interaction Between Na+/K+-Pump and Na+/Ca2+-Exchanger Modulates Intercellular Communication

Ouabain, a specific inhibitor of the Na+/K+-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na+/K+-pump and the Na+/Ca2+-exchanger leading to an increase in the intracellular calcium concentration ([Ca2+]i) in discrete areas near the plasma membrane. [Ca2+]i in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na+/K+-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca2+]i near the membrane, or by ATP depletion. Reduction of Na+/K+-pump activity by removal of extracellular potassium ([K+]o) also uncoupled cells, but only after inhibition of KATP channels. Inhibition of the Na+/Ca2+-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na+ and clamping of [Ca2+]i at low concentrations prevented the uncoupling. The experiments suggest that the Na+/K+-pump may affect gap junction conductivity via localized changes in [Ca2+]i through modulation of Na+/Ca2+-exchanger activity.