Vladimir Matchkov - Academia.edu (original) (raw)

Papers by Vladimir Matchkov

AJP: Heart and Circulatory Physiology, 2014

L-type Ca(2+) channels (LTCCs) are important for vascular smooth muscle cell (VSMC) contraction, ... more L-type Ca(2+) channels (LTCCs) are important for vascular smooth muscle cell (VSMC) contraction, as well as VSMC differentiation, as indicated by loss of LTCCs during VSMC dedifferentiation. However, it is not clear whether loss of LTCCs is a primary event underlying phenotypic modulation or whether loss of LTCCs has significance for vascular structure. We used small interference RNA (siRNA) transfection in vivo to investigate the role of LTCCs in VSMC phenotypic expression and structure of rat mesenteric arteries. siRNA reduced LTCC mRNA and protein expression in rat mesenteric arteries 3 days after siRNA transfection to 12.7 ± 0.7% and 47.3 ± 13%, respectively: this was associated with an increased resting intracellular Ca(2+) concentration ([Ca(2+)]i). Despite the high [Ca(2+)]i, the contractility was reduced (tension development to norepinephrine was 3.5 ± 0.2 N/m and 0.8 ± 0.2 N/m for sham-transfected and downregulated arteries respectively; P < 0.05). Expression of contractile phenotype marker genes was reduced in arteries downregulated for LTCCs. Phenotypic changes were associated with a 45% increase in number of VSMCs and a consequent increase of media thickness and media area. Ten days after siRNA transfection arterial structure was again normalized. The contractile responses of LTCC-siRNA transfected arteries were elevated in comparison with matched controls 10 days after transfection. The study provides strong evidence for causal relationships between LTCC expression and VSMC contractile phenotype, as well as novel data addressing the complex relationship between VSMC contractility, phenotype, and vascular structure. These findings are relevant for understanding diseases, associated with phenotype changes of VSMC and vascular remodeling, such as atherosclerosis and hypertension.

BioMed research international, 2015

The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood... more The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood but activation of small- and intermittent-conductance Ca(2+)-activated K(+) channels (SKCa and IKCa) is important. Previous studies have suggested that the significance of IKCa depends on [Ca(2+)]out. Also it has been suggested that K(+) is important through localized [K(+)]out signaling causing activation of the Na(+),K(+)-ATPase and inward-rectifying K(+) channels (Kir). Here we tested the hypothesis that the modulating effect of [Ca(2+)]out on the EDH-like response depends on [K(+)]out. We addressed this possibility using isometric myography of rat mesenteric small arteries. When [K(+)]out was 4.2 mM, relaxation to acetylcholine (ACh) was stronger at 2.5 mM [Ca(2+)]out than at 1 mM [Ca(2+)]out. Inhibition of IKCa with TRAM34 suppressed the relaxations but did not change the relation between the relaxations at the low and high [Ca(2+)]out. This [Ca(2+)]out-dependence disappeared at 5....

British Journal of Pharmacology, 2015

Vasodilatation may contribute to the neuroprotective and vascular antiremodelling effect of the t... more Vasodilatation may contribute to the neuroprotective and vascular antiremodelling effect of the tissue transglutaminase (TG2) inhibitor cystamine. The present study hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlie cystamine vasodilatation. In rat mesenteric small arteries: RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca(2+) ([Ca(2+) ]i ) , K(+) currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain (MLC2). RT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited curves for phenylephrine, 5-hydroxytryptamine, U46619, and for extracellular K(+) . Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of phospholipase C suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca(2+) ]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca(2+) ]i , and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of voltage-gated potassium KV 7 channels, XE991 and linopirdine inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr(855) phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr(855) and MLC2 phosphorylation. Our findings suggest that cystamine induces vasodilatation by inhibition of receptor-coupled TG2 leading to opening of KV channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine.

American Journal of Physiology - Cell Physiology, 2015

The extracellular calcium-sensing receptor, CaSR, is expressed in blood vessels where its role is... more The extracellular calcium-sensing receptor, CaSR, is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative (wild-type, WT) and Cre-positive (SM22α)CaSR(∆flox/∆flox) (knock-out, KO) mice showed and endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared to WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT, but only relaxation in KO aortae. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared to WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either NO-synthase inhibition with L-NAME or by a high salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated i... more 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 patchclamp 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 M BAY K8644, 0.4 M ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calciumactivated chloride current with an absolute requirement for cyclic GMP (EC 50 6.4 M). 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 Ϫ Ͼ Br Ϫ Ͼ I Ϫ Ͼ Cl Ϫ Ͼ acetate Ͼ F Ϫ ϾϾ aspartate, but the conductance sequence was I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ Ͼ acetate Ͼ F Ϫ Ͼ aspartate ϭ SCN Ϫ . 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.

American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 2015

Major depression is known to be associated with cardiovascular abnormalities and oxidative stress... more Major depression is known to be associated with cardiovascular abnormalities and oxidative stress has been suggested to play a role. We tested the hypothesis that antidepressant treatment reduces oxidative stress, and endothelial dysfunctions in the chronic mild stress (CMS) model of depression in rats. Rats with >30% reduction in sucrose intake after 4 weeks of CMS were defined in the study as CMS-susceptible and compared to unstressed controls. Sixteen CMS-susceptible and 8 unstressed rats were treated during weeks 5 to 8 of the CMS protocol with escitalopram. Escitalopram-treated rats with >20% recovery in the sucrose consumption during the last 2 weeks of treatment were defined as escitalopram responders. Rats which did not reach these criteria were defined as escitalopram non-responders. In the open field test escitalopram responders demonstrated anxiolytic effect of treatment. In mesenteric small arteries escitalopram affected neither NO nor COX-1 mediated vasodilation. Escitalopram potentiated endothelium-dependent hyperpolarization-like response which was suppressed in the vehicle-treated CMS-susceptible rats, and reduced COX-2 dependent relaxation which was elevated in the vehicle-treated CMS-susceptible rats. Escitalopram did not affect blood pressure and heart rate which were elevated in the vehicle-treated CMS-susceptible rats. Oxidative stress markers were changed in association with CMS in liver, heart, and brain. Escitalopram normalized oxidative stress markers in the majority of tissues. This study demonstrates that the antidepressant effect of escitalopram is associated with partial improvement of endothelial function in small arteries affecting COX-2 and endothelium-dependent hyperpolarization-like pathways.

Environmental Medicine

Adrenoreactivity of rat hindlimb vessels was studied in experiments with constant-pressure saline... more Adrenoreactivity of rat hindlimb vessels was studied in experiments with constant-pressure saline perfusion. An original mathematical model was applied to evaluate the mechanism of changes in vascular tone regulation. A 3-week suspension resulted in decreased responses to sympathetic nerve stimulation, as well as to exogenous noradrenaline, the latter effect being registered when the pressure level was close to normal. Mathematical simulation indicated that long-term suspension induces both structural and functional changes in the vascular bed of the hind limbs, one of which is a disorder in the myogenic mechanisms of vascular tone regulation. In suspended rats, suppression of the myogenic response can be one of the reasons for decreased vessel reactivity to constrictor stimuli and, consequently, for disturbances in blood flow regulation in skeletal muscles of the hind limbs.

British journal of pharmacology, Jan 25, 2015

T16Ainh -A01, CaCCinh -A01, and MONNA are identified as selective inhibitors of the TMEM16A calci... more T16Ainh -A01, CaCCinh -A01, and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. Isolated resistance arteries were maintained in a myograph and tension recording in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as charge carrier. Rodent arteries pre-constricted with noradrenaline or U46619 were concentration-dependently relaxed by T16Ainh -A01 (0.1-10μM): IC50 and maximum relaxation were equivalent ±chloride (30 minute aspartate substitution) and the T16Ainh -A01-induced vasorelaxation ±chloride was accompanied by membrane hyperpolarization and lower...

Current opinion in pharmacology, 2015

Ca(2+)-activated Cl(-) channels (CaCCs) have numerous functions in the body and are potential pla... more Ca(2+)-activated Cl(-) channels (CaCCs) have numerous functions in the body and are potential players in the control of blood pressure. The CaCCs represent a heterologous group including at least two protein families; TMEM16 and bestrophins. CaCCs expression has been shown in the kidney, the heart and blood vessels. Agonist-stimulated Ca(2+)-activated Cl(-) current is important for secretion in kidney epithelia, generation of a repolarizing current in the heart and amplification of excitation-contraction coupling in vascular smooth muscle cells. Changes in CaCC expression are shown in association with hypertension, kidney cysts, sudden cardiac death and arrhythmias. This review discusses recent advances in studies concerning the role of CaCC for blood pressure control with focus on the kidney, the heart and blood vessels.

Pflügers Archiv : European journal of physiology, 2014

The presence of Ca(2+)-activated Cl(-) channels (CaCCs) in vascular smooth muscle cells (SMCs) is... more The presence of Ca(2+)-activated Cl(-) channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca(2+)-activated Cl(-) currents (I Cl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent I Cl(Ca) depends on bestrophin expression, while the "classical" I Cl(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, [Ca(2+)]i, and SMC membrane potential were measured in isolated arterial segments. I Cl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent I Cl(Ca) and the "classical" I Cl(Ca) in SMCs. TMEM16A downregulation also r...

Channels, 2014

The presence of Ca(2+)-activated Cl(–) currents (I(Cl(Ca))) in vascular smooth muscle cells (VSMC... more The presence of Ca(2+)-activated Cl(–) currents (I(Cl(Ca))) in vascular smooth muscle cells (VSMCs) is well established. ICl(Ca) are supposedly important for arterial contraction by linking changes in [Ca(2+)]i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I(Cl(Ca)). Two distinct I(Cl(Ca)) are characterized in VSMCs; the cGMP-dependent I(Cl(Ca)) dependent upon bestrophin expression and the ‘classical’ Ca(2+)-activated Cl(–) current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I(Cl(Ca)). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl(–) channels. It is suggested that TMEM16A expression modulates voltage-gated Ca(2+) influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.

BioMed Research International, 2014

Endothelium is the endocrine organ essential for maintenance of homeostasis in the entire body. E... more Endothelium is the endocrine organ essential for maintenance of homeostasis in the entire body. Endothelium operates by a broad spectrum of signaling molecules controlling the contractile state of vascular smooth muscles and cardiomyocytes; long distance intercellular synchronization within the vascular wall; adhesive, coagulant, and rheological properties of blood; and permeability of the vascular wall. Dysfunctions in these signaling pathways result in loss of the important homeostatic functions as well as in engagement of the endothelium into activities leading to pathologies. This represents the two sides of the coin, where endothelium can be either a protective or "health-threatening" organ depending on the signaling involved. Endothelial dysfunction (ED) was observed in aging as well as in major lifestyle-related diseases suggesting that the endothelium can serve as a target for prevention and treatment of various diseases. Significant progress achieved in the identification of new endotheliumdependent signaling pathways and characterization of their role in various pathological states has been made since the identification of nitric oxide (NO) as an endothelium-derived relaxing factor. This special issue aims to highlight this progress and stimulate further this development. Variable functions of the endothelium are presented in six reviews, seven research articles, and two clinical studies.

ABSTRACT This work presents a polydimethylsiloxane (PDMS) system (named microbridle) monolithical... more ABSTRACT This work presents a polydimethylsiloxane (PDMS) system (named microbridle) monolithically integrating optical (microlenses, air mirrors etc.) and mechanical elements (cantilever) for the real time monitoring of dilatation and contraction events in small resistance arteries. Structural (response to intraluminal pressure changes) and functional properties (response to vasoactive substances) of mesenteric arteries were determined with the microbridle structure and compared to those obtained with conventional myograph systems. Both systems provide comparable data although microbridles were found advantageous in terms of precision, resolution (below the μm) and reliability for the poor contrast between sample and surrounding medium that impede reliable myography recordings.

Journal of cardiovascular pharmacology, Jan 2, 2014

Major depression and cardiovascular diseases are two of the most prevalent health problems in wes... more Major depression and cardiovascular diseases are two of the most prevalent health problems in western society and an association between them is generally accepted. Although the specific mechanism behind this comorbidity remains to be elucidated, it is clear that it has a complex multifactorial character including a number of neuronal, humoral, immune and circulatory pathways. Depression-associated cardiovascular abnormalities associate with cardiac dysfunctions as well as with changes in peripheral resistance. While cardiac dysfunction in association with depression has been studied in detail, little attention was given to structural and functional changes in resistance arteries responsible for blood pressure control and tissue perfusion. This review discusses recent achievements in studies of depression-associated abnormalities in resistance arteries in humans and animal experimental models. The changes in arterial structure, contractile and relaxing functions associated with depr...

Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2008

Vascular smooth muscle cells (SMCs) exhibit different types of calcium dynamics. Static vascular ... more Vascular smooth muscle cells (SMCs) exhibit different types of calcium dynamics. Static vascular tone is associated with unsynchronized calcium waves and the developed force depends on the number of recruited cells. Global calcium transients synchronized among a large number of cells cause rhythmic development of force known as vasomotion. We present experimental data showing a considerable heterogeneity in cellular calcium dynamics in the vascular wall. In stimulated vessels, some SMCs remain quiescent, whereas others display waves of variable frequency. At the onset of vasomotion, all SMCs are enrolled into synchronized oscillation. Simulations of coupled SMCs show that the experimentally observed cellular recruitment, the presence of quiescent cells and the variation in oscillation frequency may arise if the cell population is phenotypically heterogeneous. In this case, quiescent cells can be entrained at the onset of vasomotion by the collective driving force from the synchronized oscillations in the membrane potential of the surrounding cells. Partial synchronization arises with an increase in the concentration of cyclic guanosine monophosphate, but in a heterogeneous cell population complete synchronization also requires a high-conductance pathway that provides strong coupling between the cells.

Pflügers Archiv - European Journal of Physiology, 2005

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a ... more In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca(2+)-activated Cl(-) conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl(-) conductance has properties distinct from those of the 'classical' Ca(2+)-activated Cl(-) conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca(2+)-activated Cl(-) current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The 'classical' Ca(2+)-activated Cl(-) conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.

Pflügers Archiv - European Journal of Physiology, 2008

The possibility that Ca2+-activated Cl− conductances (CaCCs) contribute to oscillations in vascul... more The possibility that Ca2+-activated Cl− conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I Cl(Ca)) and cGMP-dependent (I Cl(Ca,cGMP)) chloride conductances are important. The effect of anion substitution and Cl− channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl− o replacement with aspartate blocked vasomotion while 36mM SCN− o (substituted for Cl−) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca2+]i disappeared with 36mM SCN−. DIDS and Zn2+ blocked I Cl(Ca,cGMP) but not I Cl(Ca). Vasomotion was not sensitive to DIDS and Zn2+, and DIDS and Zn2+ induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn2+ was sensitive to 36mM SCN− o. The anion substitution data indicate that Cl− is crucial for the V m and [Ca2+]i oscillations underlying vasomotion. The Cl− channel blocker data are consistent with both CaCCs being important.

AJP: Heart and Circulatory Physiology, 2014

L-type Ca(2+) channels (LTCCs) are important for vascular smooth muscle cell (VSMC) contraction, ... more L-type Ca(2+) channels (LTCCs) are important for vascular smooth muscle cell (VSMC) contraction, as well as VSMC differentiation, as indicated by loss of LTCCs during VSMC dedifferentiation. However, it is not clear whether loss of LTCCs is a primary event underlying phenotypic modulation or whether loss of LTCCs has significance for vascular structure. We used small interference RNA (siRNA) transfection in vivo to investigate the role of LTCCs in VSMC phenotypic expression and structure of rat mesenteric arteries. siRNA reduced LTCC mRNA and protein expression in rat mesenteric arteries 3 days after siRNA transfection to 12.7 ± 0.7% and 47.3 ± 13%, respectively: this was associated with an increased resting intracellular Ca(2+) concentration ([Ca(2+)]i). Despite the high [Ca(2+)]i, the contractility was reduced (tension development to norepinephrine was 3.5 ± 0.2 N/m and 0.8 ± 0.2 N/m for sham-transfected and downregulated arteries respectively; P < 0.05). Expression of contractile phenotype marker genes was reduced in arteries downregulated for LTCCs. Phenotypic changes were associated with a 45% increase in number of VSMCs and a consequent increase of media thickness and media area. Ten days after siRNA transfection arterial structure was again normalized. The contractile responses of LTCC-siRNA transfected arteries were elevated in comparison with matched controls 10 days after transfection. The study provides strong evidence for causal relationships between LTCC expression and VSMC contractile phenotype, as well as novel data addressing the complex relationship between VSMC contractility, phenotype, and vascular structure. These findings are relevant for understanding diseases, associated with phenotype changes of VSMC and vascular remodeling, such as atherosclerosis and hypertension.

BioMed research international, 2015

The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood... more The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood but activation of small- and intermittent-conductance Ca(2+)-activated K(+) channels (SKCa and IKCa) is important. Previous studies have suggested that the significance of IKCa depends on [Ca(2+)]out. Also it has been suggested that K(+) is important through localized [K(+)]out signaling causing activation of the Na(+),K(+)-ATPase and inward-rectifying K(+) channels (Kir). Here we tested the hypothesis that the modulating effect of [Ca(2+)]out on the EDH-like response depends on [K(+)]out. We addressed this possibility using isometric myography of rat mesenteric small arteries. When [K(+)]out was 4.2 mM, relaxation to acetylcholine (ACh) was stronger at 2.5 mM [Ca(2+)]out than at 1 mM [Ca(2+)]out. Inhibition of IKCa with TRAM34 suppressed the relaxations but did not change the relation between the relaxations at the low and high [Ca(2+)]out. This [Ca(2+)]out-dependence disappeared at 5....

British Journal of Pharmacology, 2015

Vasodilatation may contribute to the neuroprotective and vascular antiremodelling effect of the t... more Vasodilatation may contribute to the neuroprotective and vascular antiremodelling effect of the tissue transglutaminase (TG2) inhibitor cystamine. The present study hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlie cystamine vasodilatation. In rat mesenteric small arteries: RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca(2+) ([Ca(2+) ]i ) , K(+) currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain (MLC2). RT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited curves for phenylephrine, 5-hydroxytryptamine, U46619, and for extracellular K(+) . Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of phospholipase C suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca(2+) ]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca(2+) ]i , and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of voltage-gated potassium KV 7 channels, XE991 and linopirdine inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr(855) phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr(855) and MLC2 phosphorylation. Our findings suggest that cystamine induces vasodilatation by inhibition of receptor-coupled TG2 leading to opening of KV channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine.

American Journal of Physiology - Cell Physiology, 2015

The extracellular calcium-sensing receptor, CaSR, is expressed in blood vessels where its role is... more The extracellular calcium-sensing receptor, CaSR, is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative (wild-type, WT) and Cre-positive (SM22α)CaSR(∆flox/∆flox) (knock-out, KO) mice showed and endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared to WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT, but only relaxation in KO aortae. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared to WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either NO-synthase inhibition with L-NAME or by a high salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated i... more 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 patchclamp 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 M BAY K8644, 0.4 M ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calciumactivated chloride current with an absolute requirement for cyclic GMP (EC 50 6.4 M). 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 Ϫ Ͼ Br Ϫ Ͼ I Ϫ Ͼ Cl Ϫ Ͼ acetate Ͼ F Ϫ ϾϾ aspartate, but the conductance sequence was I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ Ͼ acetate Ͼ F Ϫ Ͼ aspartate ϭ SCN Ϫ . 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.

American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 2015

Major depression is known to be associated with cardiovascular abnormalities and oxidative stress... more Major depression is known to be associated with cardiovascular abnormalities and oxidative stress has been suggested to play a role. We tested the hypothesis that antidepressant treatment reduces oxidative stress, and endothelial dysfunctions in the chronic mild stress (CMS) model of depression in rats. Rats with >30% reduction in sucrose intake after 4 weeks of CMS were defined in the study as CMS-susceptible and compared to unstressed controls. Sixteen CMS-susceptible and 8 unstressed rats were treated during weeks 5 to 8 of the CMS protocol with escitalopram. Escitalopram-treated rats with >20% recovery in the sucrose consumption during the last 2 weeks of treatment were defined as escitalopram responders. Rats which did not reach these criteria were defined as escitalopram non-responders. In the open field test escitalopram responders demonstrated anxiolytic effect of treatment. In mesenteric small arteries escitalopram affected neither NO nor COX-1 mediated vasodilation. Escitalopram potentiated endothelium-dependent hyperpolarization-like response which was suppressed in the vehicle-treated CMS-susceptible rats, and reduced COX-2 dependent relaxation which was elevated in the vehicle-treated CMS-susceptible rats. Escitalopram did not affect blood pressure and heart rate which were elevated in the vehicle-treated CMS-susceptible rats. Oxidative stress markers were changed in association with CMS in liver, heart, and brain. Escitalopram normalized oxidative stress markers in the majority of tissues. This study demonstrates that the antidepressant effect of escitalopram is associated with partial improvement of endothelial function in small arteries affecting COX-2 and endothelium-dependent hyperpolarization-like pathways.

Environmental Medicine

Adrenoreactivity of rat hindlimb vessels was studied in experiments with constant-pressure saline... more Adrenoreactivity of rat hindlimb vessels was studied in experiments with constant-pressure saline perfusion. An original mathematical model was applied to evaluate the mechanism of changes in vascular tone regulation. A 3-week suspension resulted in decreased responses to sympathetic nerve stimulation, as well as to exogenous noradrenaline, the latter effect being registered when the pressure level was close to normal. Mathematical simulation indicated that long-term suspension induces both structural and functional changes in the vascular bed of the hind limbs, one of which is a disorder in the myogenic mechanisms of vascular tone regulation. In suspended rats, suppression of the myogenic response can be one of the reasons for decreased vessel reactivity to constrictor stimuli and, consequently, for disturbances in blood flow regulation in skeletal muscles of the hind limbs.

British journal of pharmacology, Jan 25, 2015

T16Ainh -A01, CaCCinh -A01, and MONNA are identified as selective inhibitors of the TMEM16A calci... more T16Ainh -A01, CaCCinh -A01, and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. Isolated resistance arteries were maintained in a myograph and tension recording in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as charge carrier. Rodent arteries pre-constricted with noradrenaline or U46619 were concentration-dependently relaxed by T16Ainh -A01 (0.1-10μM): IC50 and maximum relaxation were equivalent ±chloride (30 minute aspartate substitution) and the T16Ainh -A01-induced vasorelaxation ±chloride was accompanied by membrane hyperpolarization and lower...

Current opinion in pharmacology, 2015

Ca(2+)-activated Cl(-) channels (CaCCs) have numerous functions in the body and are potential pla... more Ca(2+)-activated Cl(-) channels (CaCCs) have numerous functions in the body and are potential players in the control of blood pressure. The CaCCs represent a heterologous group including at least two protein families; TMEM16 and bestrophins. CaCCs expression has been shown in the kidney, the heart and blood vessels. Agonist-stimulated Ca(2+)-activated Cl(-) current is important for secretion in kidney epithelia, generation of a repolarizing current in the heart and amplification of excitation-contraction coupling in vascular smooth muscle cells. Changes in CaCC expression are shown in association with hypertension, kidney cysts, sudden cardiac death and arrhythmias. This review discusses recent advances in studies concerning the role of CaCC for blood pressure control with focus on the kidney, the heart and blood vessels.

Pflügers Archiv : European journal of physiology, 2014

The presence of Ca(2+)-activated Cl(-) channels (CaCCs) in vascular smooth muscle cells (SMCs) is... more The presence of Ca(2+)-activated Cl(-) channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca(2+)-activated Cl(-) currents (I Cl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent I Cl(Ca) depends on bestrophin expression, while the "classical" I Cl(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, [Ca(2+)]i, and SMC membrane potential were measured in isolated arterial segments. I Cl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent I Cl(Ca) and the "classical" I Cl(Ca) in SMCs. TMEM16A downregulation also r...

Channels, 2014

The presence of Ca(2+)-activated Cl(–) currents (I(Cl(Ca))) in vascular smooth muscle cells (VSMC... more The presence of Ca(2+)-activated Cl(–) currents (I(Cl(Ca))) in vascular smooth muscle cells (VSMCs) is well established. ICl(Ca) are supposedly important for arterial contraction by linking changes in [Ca(2+)]i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I(Cl(Ca)). Two distinct I(Cl(Ca)) are characterized in VSMCs; the cGMP-dependent I(Cl(Ca)) dependent upon bestrophin expression and the ‘classical’ Ca(2+)-activated Cl(–) current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I(Cl(Ca)). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl(–) channels. It is suggested that TMEM16A expression modulates voltage-gated Ca(2+) influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.

BioMed Research International, 2014

Endothelium is the endocrine organ essential for maintenance of homeostasis in the entire body. E... more Endothelium is the endocrine organ essential for maintenance of homeostasis in the entire body. Endothelium operates by a broad spectrum of signaling molecules controlling the contractile state of vascular smooth muscles and cardiomyocytes; long distance intercellular synchronization within the vascular wall; adhesive, coagulant, and rheological properties of blood; and permeability of the vascular wall. Dysfunctions in these signaling pathways result in loss of the important homeostatic functions as well as in engagement of the endothelium into activities leading to pathologies. This represents the two sides of the coin, where endothelium can be either a protective or "health-threatening" organ depending on the signaling involved. Endothelial dysfunction (ED) was observed in aging as well as in major lifestyle-related diseases suggesting that the endothelium can serve as a target for prevention and treatment of various diseases. Significant progress achieved in the identification of new endotheliumdependent signaling pathways and characterization of their role in various pathological states has been made since the identification of nitric oxide (NO) as an endothelium-derived relaxing factor. This special issue aims to highlight this progress and stimulate further this development. Variable functions of the endothelium are presented in six reviews, seven research articles, and two clinical studies.

ABSTRACT This work presents a polydimethylsiloxane (PDMS) system (named microbridle) monolithical... more ABSTRACT This work presents a polydimethylsiloxane (PDMS) system (named microbridle) monolithically integrating optical (microlenses, air mirrors etc.) and mechanical elements (cantilever) for the real time monitoring of dilatation and contraction events in small resistance arteries. Structural (response to intraluminal pressure changes) and functional properties (response to vasoactive substances) of mesenteric arteries were determined with the microbridle structure and compared to those obtained with conventional myograph systems. Both systems provide comparable data although microbridles were found advantageous in terms of precision, resolution (below the μm) and reliability for the poor contrast between sample and surrounding medium that impede reliable myography recordings.

Journal of cardiovascular pharmacology, Jan 2, 2014

Major depression and cardiovascular diseases are two of the most prevalent health problems in wes... more Major depression and cardiovascular diseases are two of the most prevalent health problems in western society and an association between them is generally accepted. Although the specific mechanism behind this comorbidity remains to be elucidated, it is clear that it has a complex multifactorial character including a number of neuronal, humoral, immune and circulatory pathways. Depression-associated cardiovascular abnormalities associate with cardiac dysfunctions as well as with changes in peripheral resistance. While cardiac dysfunction in association with depression has been studied in detail, little attention was given to structural and functional changes in resistance arteries responsible for blood pressure control and tissue perfusion. This review discusses recent achievements in studies of depression-associated abnormalities in resistance arteries in humans and animal experimental models. The changes in arterial structure, contractile and relaxing functions associated with depr...

Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2008

Vascular smooth muscle cells (SMCs) exhibit different types of calcium dynamics. Static vascular ... more Vascular smooth muscle cells (SMCs) exhibit different types of calcium dynamics. Static vascular tone is associated with unsynchronized calcium waves and the developed force depends on the number of recruited cells. Global calcium transients synchronized among a large number of cells cause rhythmic development of force known as vasomotion. We present experimental data showing a considerable heterogeneity in cellular calcium dynamics in the vascular wall. In stimulated vessels, some SMCs remain quiescent, whereas others display waves of variable frequency. At the onset of vasomotion, all SMCs are enrolled into synchronized oscillation. Simulations of coupled SMCs show that the experimentally observed cellular recruitment, the presence of quiescent cells and the variation in oscillation frequency may arise if the cell population is phenotypically heterogeneous. In this case, quiescent cells can be entrained at the onset of vasomotion by the collective driving force from the synchronized oscillations in the membrane potential of the surrounding cells. Partial synchronization arises with an increase in the concentration of cyclic guanosine monophosphate, but in a heterogeneous cell population complete synchronization also requires a high-conductance pathway that provides strong coupling between the cells.

Pflügers Archiv - European Journal of Physiology, 2005

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a ... more In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca(2+)-activated Cl(-) conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl(-) conductance has properties distinct from those of the 'classical' Ca(2+)-activated Cl(-) conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca(2+)-activated Cl(-) current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The 'classical' Ca(2+)-activated Cl(-) conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.

Pflügers Archiv - European Journal of Physiology, 2008

The possibility that Ca2+-activated Cl− conductances (CaCCs) contribute to oscillations in vascul... more The possibility that Ca2+-activated Cl− conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I Cl(Ca)) and cGMP-dependent (I Cl(Ca,cGMP)) chloride conductances are important. The effect of anion substitution and Cl− channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl− o replacement with aspartate blocked vasomotion while 36mM SCN− o (substituted for Cl−) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca2+]i disappeared with 36mM SCN−. DIDS and Zn2+ blocked I Cl(Ca,cGMP) but not I Cl(Ca). Vasomotion was not sensitive to DIDS and Zn2+, and DIDS and Zn2+ induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn2+ was sensitive to 36mM SCN− o. The anion substitution data indicate that Cl− is crucial for the V m and [Ca2+]i oscillations underlying vasomotion. The Cl− channel blocker data are consistent with both CaCCs being important.