Maksym I. Harhun

Participation of KCNQ (Kv7) potassium channels in myogenic control of cerebral arterial diameter

KCNQ gene expression was previously shown in various rodent blood vessels, where the products of KCNQ4 and KCNQ5, Kv7.4 and Kv7.5 potassium channel subunits, respectively, have an influence on vascular reactivity. The aim of this study was to determine if small cerebral resistance arteries of the rat express KCNQ genes and whether Kv7 channels participate in the regulation of myogenic control of diameter. Quantitative reverse transcription polymerase chain reaction (QPCR) was undertaken using RNA isolated from rat middle cerebral arteries (RMCAs) and immunocytochemistry was performed using Kv7 subunit‐specific antibodies and freshly isolated RMCA myocytes. KCNQ4 message was more abundant than KCNQ5=KCNQ1, but KCNQ2 and KCNQ3 message levels were negligible. Kv7.1, Kv7.4 and Kv7.5 immunoreactivity was present at the sarcolemma of freshly isolated RMCA myocytes. Linopirdine (1 μm) partially depressed, whereas the Kv7 activator S‐1 (3 and/or 20 μm) enhanced whole‐cell Kv7.4 (in HEK 293 cells), as well as native RMCA myocyte Kv current amplitude. The effects of S‐1 were voltage‐dependent, with progressive loss of stimulation at potentials of >−15 mV. At the concentrations employed linopirdine and S‐1 did not alter currents due to recombinant Kv1.2/Kv1.5 or Kv2.1/Kv9.3 channels (in HEK 293 cells) that are also expressed by RMCA myocytes. In contrast, another widely used Kv7 blocker, XE991 (10 μm), significantly attenuated native Kv current and also reduced Kv1.2/Kv1.5 and Kv2.1/Kv9.3 currents. Pressurized arterial myography was performed using RMCAs exposed to intravascular pressures of 10–100 mmHg. Linopirdine (1 μm) enhanced the myogenic response at ≥20 mmHg, whereas the activation of Kv7 channels with S‐1 (20 μm) inhibited myogenic constriction at >20 mmHg and reversed the increased myogenic response produced by suppression of Kv2‐containing channels with 30 nm stromatoxin (ScTx1). These data reveal a novel contribution of KCNQ gene products to the regulation of myogenic control of cerebral arterial diameter and suggest that Kv7 channel activating drugs may be appropriate candidates for the development of an effective therapy to ameliorate cerebral vasospasm.

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE KCNQ‐encoded voltage‐gated potassium channels (Kv7) have recently been identified as important anti‐constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

Downregulation of Kv7.4 Channel Activity in Primary and Secondary Hypertension

BACKGROUND Voltage-gated potassium (K(+)) channels encoded by KCNQ genes (Kv7 channels) have been identified in various rodent and human blood vessels as key regulators of vascular tone; however, nothing is known about the functional impact of these channels in vascular disease. We ascertained the effect of 3 structurally different activators of Kv7.2 through Kv7.5 channels (BMS-204352, S-1, and retigabine) on blood vessels from normotensive and hypertensive animals. METHODS AND RESULTS Precontracted thoracic aorta and mesenteric artery segments from normotensive rats were relaxed by all 3 Kv7 activators, with potencies of BMS-204352=S-1>retigabine. We also tested these agents in the coronary circulation using the Langendorff heart preparation. BMS-204352 and S-1 dose dependently increased coronary perfusion at concentrations between 0.1 and 10 μmol/L, whereas retigabine was effective at 1 to 10 μmol/L. In addition, S-1 increased K(+) currents in isolated mesenteric artery myocytes. The ability of these agents to relax precontracted vessels, increase coronary flow, or augment K(+) currents was impaired considerably in tissues isolated from spontaneously hypertensive rats (SHRs). Of the 5 KCNQ genes, only the expression of KCNQ4 was reduced (≈3.7 fold) in SHRs aorta. Kv7.4 protein levels were ≈50% lower in aortas and mesenteric arteries from spontaneously hypertensive rats compared with normotensive vessels. A similar attenuated response to S-1 and decreased Kv7.4 were observed in mesenteric arteries from mice made hypertensive by angiotensin II infusion compared with normotensive controls. CONCLUSIONS In 2 different rat and mouse models of hypertension, the functional impact of Kv7 channels was dramatically downregulated.

Interstitial cells in the vasculature

Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non‐contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c‐kit. These vascular cells have been termed “interstitial cells” by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca2+‐release events from endo/sarcoplasmic reticulum including fast localised Ca2+ transients (Ca2+ sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca2+ oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight‐seal patch‐clamp recording, and fluorescence confocal imaging techniques.

Function of Interstitial Cells of Cajal in the Rabbit Portal Vein

Interstitial cells of Cajal (ICCs) were identified in the intact fixed media of the rabbit portal vein (RPV) using c-kit staining. The following experiments were performed using single cell preparations of the enzyme-dispersed vessel. Surviving contacts between the processes of single ICCs and the bodies of smooth muscle cells (SMCs) were observed in electron micrographs and by confocal microscopy. Spontaneous rhythmical [Ca2+]i oscillations were observed in ICCs after loading with the calcium indicator fluo-3 and were associated with depolarizations of the ICCs recorded by tight-seal patch pipette. To investigate signal transmission from ICCs to SMCs in dispersed cell pairs, or within small surviving fragments of the ICC network, an ICC was stimulated under voltage-clamp, while changes in [Ca2+]i in the stimulated cell as well as in a closely adjacent SMC or ICCs were monitored using fast x- y confocal imaging of fluo-3 fluorescence. After stimulation of single voltage-clamped ICC by a depolarizing step similar in duration to depolarizations associated with spontaneous [Ca2+]i oscillations, a depolarization and transient elevation of [Ca2+]i was observed in a closely adjacent SMCs after a delay of up to 4 seconds. In contrast, signal transmission from ICC to ICC was much faster, the delay being less than 200 ms. These results suggest that the an ICC may, in addition to generating an electrical signal (such as a slow wave) and thereby acting as a pacemaker for vascular SMCs of RPV, also release some unknown diffusible substance, which depolarizes the SMCs.

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca2+ concentration ([Ca2+]i). Although it is well‐appreciated that the myocyte Ca2+ signalling system is composed of microdomains, little is known about the structure of the [Ca2+]i responses induced by P2X receptor stimulation in vascular myocytes.

Purinoreceptor‐mediated current in myocytes from renal resistance arteries

Background and purpose:  Ionotropic purinoreceptors (P2X) in renal vascular smooth muscle cells (RVSMCs) are involved in mediating the sympathetic control and paracrine regulation of renal blood flow (RBF). Activation of P2X receptors elevates [Ca2+]i in RVSMCs triggering their contraction, leading to renal vasoconstriction and decrease of RBF. The goal of the present work was to characterize the P2X receptor‐mediated ionic current (IP2X) and to identify the types of P2X receptors expressed in myocytes isolated from interlobar and arcuate arteries of rat kidney.

Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell

This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC‐like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea‐pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle α‐actin and smooth muscle myosin heavy chain (SM‐MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmis‐sion electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.

Interstitial cells from rat middle cerebral artery belong to smooth muscle cell type

It is now established that non‐contractile cells with thin filopodia, also called vascular interstitial cells (VICs), are constitutively present in the media of many, if not all, blood vessels. The aim of this study was to determine the type of cell lineage to which arterial VICs belong using immunocytochemical, and real‐time and reverse transcription PCR (RT‐PCR). Using RT‐PCR, we compared gene expression profiles of single VICs and smooth muscle cells (SMCs) freshly dispersed from rat middle cerebral artery. Both VICs and SMCs expressed the SMC marker, smooth muscle myosin heavy chain (SM‐MHC), but did not express fibroblast, pericyte, neuronal, mast cell, endothelial or stem cell markers. Freshly isolated VICs also did not express c‐kit, which is the marker for interstitial cells of Cajal in the gastrointestinal tract. Immunocytochemical labelling of contractile proteins showed that VICs and SMCs expressed SM‐MHC similarly to the same degree, but VICs in contrast to SMCs had decreased expression of α‐SM‐actin and very low or no expression of calponin. Real‐time RT‐PCR was consistent with immunocytochemical experiments and showed that VICs had four times lower gene expression of calponin comparing to SMCs, which may explain VICs’ inability to contract. VICs had greater expression than SMCs of structural proteins such as non‐muscular β‐actin and desmin. The results obtained suggest that VICs represent a subtype of SMCs and may originate from the same precursor as SMCs, but later develop filopodia and a non‐contractile cell phenotype.

Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Vascular interstitial cells (VICs) are non‐contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro‐omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT‐PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM‐MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM‐MHC and αSM‐actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h‐calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC‐specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.