Veronika Golubinskaya

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.

Reduced anti-contractile effect of perivascular adipose tissue on mesenteric small arteries from spontaneously hypertensive rats: role of Kv7 channels.

Perivascular adipose tissue (PVAT) has been shown to produce vasoactive substances and regulate vascular tone. This function of PVAT has been reported to be altered in hypertension. However, the underlying mechanisms are not fully understood. In this study we used age-matched normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) as well as Sprague-Dawley rats and tested effects of PVAT on mesenteric small arteries. Vessels were mounted in a Mulvany-Halpern myograph and cumulative concentration-response relations to noradrenaline were determined in the presence or absence of PVAT. We found that PVAT has an anti-contractile effect on mesenteric small vessels, irrespective of strains. A reduced effect of PVAT was observed in SHR compared to WKY rats; the difference between strains was eliminated by 10 μM XE991, a blocker of Kv7 (KCNQ) voltage-dependent potassium channels. The anti-contractile effect of PVAT was not affected by depolarizing smooth muscle cells with high K(+) solution. Sensitivities to exogenous vasodilators acetylcholine or sodium nitroprusside were not potentiated but reduced in vessels with PVAT. Our results suggest that the reduced anti-contractile effect of PVAT in SHR correlates with a deficiency in Kv7 channels. Diffusion hindrance of PVAT is also a factor that should be considered in investigations on rat mesenteric small arteries.

Endothelial function in a mouse model of myeloperoxidase deficiency.

Myeloperoxidase (MPO) activity is suggested to reduce the function of vascular nitric oxide, thereby contributing to endothelial dysfunction, although data in rodents are inconclusive. We examined vascular contractile and relaxant responses in MPO-deficient (MPO−/−) and wild-type mice to investigate the role for myeloperoxidase in the development of endothelial dysfunction. Carotid and saphenous arteries were taken from 8-month-old mice and studied in a myograph. Responses of carotid arteries to phenylephrine, high potassium, or acetylcholine (Ach) were statistically not different from controls. Treatment with lipopolysaccharide (LPS; to enhance endothelial dysfunction) reduced responses to Ach in MPO−/− but did not affect responses in wild-type. In response to high concentrations of Ach, carotid arteries responded with transient contractions, which were not different between the groups and not affected by LPS treatment. Saphenous arteries from MPO−/− had smaller normalized diameters and developed less contractile force. Vessels from MPO−/− were less sensitive to Ach than controls. These data suggest that mature MPO-deficient mice do not show enhanced endothelial function compared to wild-type mice, even when provoked with LPS treatment. The EDHF response appears to be reduced in MPO deficiency.

Phase resetting of arterial vasomotion by burst stimulation of perivascular nerves

Arteries display cyclic diameter variations, vasomotion. In vivo, these rhythmic contractions are modulated by the influence of sympathetic nerves. In this study, we investigated the effect of burst stimulation of intramural nerves in vitro on the vasomotion of rat mesenteric small arteries. Vessels were mounted for isometric force measurement. After initiation of vasomotion with noradrenaline (0.5–2 µM), periarterial sympathetic nerves were stimulated electrically (10 impulses at 20 Hz) at approximately half-minute intervals. With a delay of 2–3 s, a neurogenic burst caused a brief contraction of the vascular smooth muscle and altered the period of the current vasomotion cycle. The effect on amplitude decayed rapidly and was practically not apparent in the next vasomotion cycle after the burst. With respect to period, stimulation at increasing intervals from the trough in force of vasomotion caused gradual prolongation of the cycle until a critical interval was reached, after which cycle duration was reduced instead. Since subsequent cycles were not affected, a change in phase remained. When two segments of oscillating arteries were mounted in a two-vessel myograph, simultaneously applied bursts of impulses synchronized their oscillation. The data suggest that changes in neural activity are able to make different vessels oscillate in phase, thereby coordinating vasomotion in different parts of the vascular tree, possibly explaining the synchronicity of vasomotion in different vascular beds that can be observed in vivo.

The role of purinergic and adrenergic transmitters of the sympathetic system in the control of arterial blood pressure variability

Variability of mean arterial pressure (MAP) was examined in chronically instrumented, conscious, freely moving rats with pharmacologically altered efferent sympathetic influences on the cardiovascular system. MAP was recorded for 30 min beat-to-beat, using a computer under both control and experimental conditions: after administration of adrenoceptor antagonists (prazosin or phentolamine) or under P2X receptor inactivation produced either by desensitization with alpha, beta-methylene ATP or by PPADS blockade. Inhibition of adrenergic sympathetic effects on the cardiovascular system produced long-lasting and stable decrease in MAP. Prazosin did not modify MAP variability whereas phentolamine enhanced it. Under P2X receptor desensitization MAP decreased, the hypotensive effect being accompanied by a significant increase in MAP variability. A similar increase in MAP variability was observed after PPADS administration, while MAP level was not changed. Administration of PPADS in combination with phentolamine increased MAP variability more significantly than each of the drugs given separately. Changes in MAP variability under the various experimental conditions were not consistently correlated with changes in heart rate variability. We propose that ATP, being a mediator of sympathetic vasoconstriction, participates in baroreceptor-induced stabilization of MAP level.

Low-Frequency Blood Pressure Oscillations in Mesenteric Vessels in Conscious Rats

Accessible online at: www.karger.com/journals/jvr This work was supported by grants 99–04–49634 and 98–04–49222 from the Russian Foundation for Basic Research. Systemic arterial pressure is determined by a complex interaction between the general control of the circulation and locally acting mechanisms which serve the local demands of the vascular region. The general control can be studied by measuring fluctuations in the systemic mean arterial blood pressure (sysMAP) and cardiac output. Specific mechanisms, characterizing a given vascular region, can be studied by measuring local blood pressure simultaneously with sysMAP. By comparing these two signals the activity of local mechanisms can be elucidated. The present work was aimed at studying the characteristics of tone regulation in the mesenteric vascular bed. Recently, a new technique was developed for recording the mean intravascular pressure at the base of the mesenteric arcade (mesMAP; at the point where the arcade runs closest to the intestinal wall) in conscious, unrestrained rats [1, 2]. This technique showed that, in the rat mesenteric vascular bed, arteries proximal to the mesenteric arcade (with diameter of 1200 Ìm) contribute substantially to the control of peripheral resistance. In the present study we applied this approach for the investigation of short-term blood pressure variability. This allowed us to observe regular oscillations of mesenteric vascular tone, probably resulting from vasomotion-like vessel activity.

Bestrophin-3 is differently expressed in normal and injured mouse glomerular podocytes

Bestrophins are putative calcium‐activated chloride channels. Recently, cell‐protective functions for Bestrophin‐3 (Best3) were proposed. Best3 exists in different splice variants. We have here examined expression, alternative splicing and localization of Best3 in mouse podocytes under normal conditions and during endoplasmic reticulum (ER) stress.

Internet Discussion Forum

The study by Steeds et al. [1] was selected for this forum since it investigates not only the association between polymorphisms and diseases but also the possible functional consequences. Specifically, the authors investigate two gene polymorphisms of components of the renin angiotensin system (RAS) which have been previously associated with cardiovascular disorders. The D allele of the angiotensin converting enzyme (ACE) I/D polymorphism as well as the C allele of the A1166-C-polymorphism of the angiotensin II (AT1) receptor gene have been previously associated with myocardial infarction and changes in vascular structure. The authors ask the important question whether these associations between genotype and disease have a functional correlate at the level of the vascular wall. For the study, resistance arteries were obtained after colon surgery and studied in a small vessel wire myograph. Vascular function was characterized using a number of stimuli and the genotypes of the individual samples were determined. Results revealed no significant effect of the ACE I/D polymorphism on vascular function. The AT1 polymorphism was only associated with an increase to prostaglandin F2alpha but not with any other changes. The authors conclude that neither polymorphisms are associated with any significant functional vascular changes in mesenteric resistance arteries in subjects without cardiovascular disease.

Potassium-Channel-Independent Relaxing Influence of Adipose Tissue on Mouse Carotid Artery

Since the cardiovascular consequences of obesity reportedly vary in different types of obesity, we investigated the influence of adipose tissue from different locales on the phenylephrine-induced tone of the mouse carotid artery. Vessels were mounted in a Mulvany-Halpern-type wire myograph, and adipose tissue, from the back (brown) or mesenteric or inguinal subcutaneous (white), was placed around the artery. Contractile responses to phenylephrine were not affected by brown adipose tissue but were reduced (p < 0.001) by either type of white adipose tissue, with no difference between the 2 locales. The relaxing effect persisted in the presence of the Kv7 channel inhibitor XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), the KATP channel inhibitor glibenclamide (1 µM), or the KV channel inhibitor 4-amino pyridine (1 mM), as well as after elevation of the extracellular potassium concentration to 30 mM. Contractions of rat carotid artery were equally reduced by mouse and rat subcutaneous adipose tissue. Thus, white, but not brown, adipose tissue reduces the adrenergic contractions of the carotid artery with no differences between the locales of origin, and the effect appears largely independent of potassium channels.