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BACKGROUND AND PURPOSE Pulmonary transepithelial Na+ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na+ channels and basolateral Na+K+ ATPase activity. EXPERIMENTAL APPROACH H441 human airway epithelial cells were used to examine the effects of hypoxia on Na+ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS. KEY RESULTS AMPK was activated by exposure to 3% or 0.2% O2 for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm−2) was added to the apical surface of cells grown at the air-liquid interface. Only 0.2% O2 activated AMPK in cells grown at the air-liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive Isc ( Iouabain) and apical amiloride-sensitive Na+ conductance ( GNa+). Modification of AMPK activity prevented the effect of hypoxia on Iouabain (Na+K+ ATPase) but not apical GNa+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical GNa+ (epithelial Na+ channels). CONCLUSIONS AND IMPLICATIONS Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na+ channels and basolateral Na+K+ ATPase activity to decrease transepithelial Na+ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions. [ABSTRACT FROM AUTHOR]

Background: Purinergic P2X receptors in vascular smooth muscle cells (VSMCs) play an important role in physiological stimulatory responses to the extracellularly released ATP. The aim of this work was to identify molecular P2X receptor subunits in VSMCs isolated from rat anterior, posterior and basilar arteries using a number of contemporary laboratory techniques., Methods: P2X mediated ionic currents were recorded using amphotericin B perforated patch clamp method. Gene expression analysis was performed using RT-PCR in manually collected VSMCs. The expression of proteins was confirmed by fluorescent immunocytochemistry., Results: Under voltage clamp conditions VSMCs stimulated by application of 10 μmol/l selective P2X receptor agonist αβ-meATP, the biphasic currents consisting of rapidly rising rapidly desensitizing and slowly desensitizing components were observed in freshly isolated myocytes from all three arteries. Using RT-PCR, the expression of genes encoding only P2X1 and P2X4 receptor subunits was detected in preparations from all three arteries. The expression of corresponding P2X1 and P2X4 receptor subunit proteins was confirmed in isolated VSMCs., Conclusions: Our work therefore identified that in major arteries of rat cerebral circulation VSMCs express only P2X1 and P2X4 receptors subunits. We can propose that these P2X receptor subunits participate in functional P2X receptor structures mediating ATP-evoked stimulatory responses in cerebral vascular myocytes in vivo., (Crown Copyright © 2015. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Vasomotion is the rhythmical changes in vascular tone of various blood vessels. It was proposed that in rabbit portal vein (RPV) the spontaneous contractile activity is driven by vascular interstitial cells (VICs), since RPV VICs generate rhythmical changes in intracellular Ca(2+) concentration ([Ca(2+)]i) associated with membrane depolarisation in these cells. In this work, using confocal imaging in Fluo-3 loaded RPV VICs we studied if generation of rhythmical [Ca(2+)]i changes is affected when Ca(2+) handling by mitochondria is compromised. We also visualised mitochondria in VICs using Mito Tracker Green fluorescent dye. Our results showed that freshly dispersed RPV VICs generated rhythmical [Ca(2+)]i oscillations with a frequency of 0.2-0.01 Hz. Imaging of VICs stained with Mito Tracker Green revealed abundant mitochondria in these cells with a higher density of the organelles in sub-plasmalemmar region compared to the central region of the cell. Oligomycin, an ATP synthase inhibitor, did not affect the amplitude and frequency of rhythmical [Ca(2+)]i oscillations. In contrast, two uncoupling agents, carbonylcyanide-3-chlorophenylhydrazone (CCCP) and carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP) effectively abolished rhythmical [Ca(2+)]i changes with simultaneous increase in basal [Ca(2+)]i in RPV VICs. These data suggest that in RPV VICs mitochondrial Ca(2+) handling is important for the generation of rhythmical [Ca(2+)]i changes which underlie the spontaneous rhythmical contractile activity in this vessel., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca(2+)]i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca(2+)]i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Background and Purpose: Current knowledge states that vasoconstrictor responses to ATP are mediated by rapidly desensitizing ligand-gated P2X1 receptors in vascular smooth muscle cells (VSMCs). However, ATP is implicated in contributing to pathological conditions involving sustained vasoconstrictor response such as cerebral vasospasm. The purpose of this study is to test the hypothesis that the stimulation of VSMC P2XR receptors (P2XRs) contributes to ATP-evoked sustained vasoconstrictions in rat middle cerebral arteries (RMCAs)., Methods: Reverse transcription- polymerase chain reaction, Western blot, and immunocytochemistry were used to analyze expression of mRNA and proteins in RMCAs VSMCs. Ionic currents and calcium responses were investigated using patch-clamp and confocal imaging techniques, respectively. Functional responses were confirmed using wire myography., Results: Expression of mRNA and protein for P2X1R and P2X4R subunits was identified in RMCA VSMCs. Confocal imaging in fluo-3-loaded VSMCs showed that ATP and a selective P2XR agonist, αβmeATP, evoked similar dose-dependent increases in [Ca(2+)]i. Patch-clamp experiments identified 2 components of P2XR-mediated currents: consisting of a fast desensitizing phase mediated by homomeric P2X1Rs and a slowly desensitizing phase involving heteromeric P2X1/4Rs. Isometric tension measurements showed that ≈80%:20% of initial ATP-evoked vasoconstriction in RMCA is mediated by homomeric P2X1Rs and heteromeric P2X1/4Rs, respectively. The sustained slowly desensitizing and rapidly recovering from desensitization responses are mediated by heteromeric P2X1/4Rs., Conclusions: This study reveals for the first time that apart from rapidly desensitizing homomeric P2X1Rs, heteromeric P2X1/4Rs contribute to the sustained component of the purinergic-mediated vasoconstriction in RMCA. Our study, therefore, identifies possible novel targets for therapeutical intervention in cerebral circulation., (© 2014 American Heart Association, Inc.)

Background: ATP is one of the principal sympathetic neurotransmitters which contracts vascular smooth muscle cells (SMCs) via activation of ionotropic P2X receptors (P2XRs). We have recently demonstrated that contraction of the guinea pig small mesenteric arteries evoked by stimulation of P2XRs is sensitive to inhibitors of IP3 receptors (IP3Rs). Here we analyzed contribution of IP3Rs and ryanodine receptors (RyRs) to [Ca(2+)]i transients induced by P2XR agonist αβ-meATP (10 μM) in single SMCs from these vessels., Methods: The effects of inhibition of L-type Ca(2+) channels (VGCCs), RyRs and IP3Rs (5 μM nicardipine, 100 μM tetracaine and 30 μM 2-APB, respectively) on αβ-meATP-induced [Ca(2+)]i transients were analyzed using fast x-y confocal Ca(2+) imaging., Results: The effect of IP3R inhibition on the [Ca(2+)]i transient was significantly stronger (67 ± 7%) than that of RyR inhibition (40 ± 5%) and was attenuated by block of VGCCs. The latter indicates that activation of VGCCs is linked to IP3R-mediated Ca(2+) release. Immunostaining of RyRs and IP3Rs revealed that RyRs are located mainly in deeper sarcoplasmic reticulum (SR) while sub-plasma membrane (PM) SR elements are enriched with type 1 IP3Rs. This structural peculiarity makes IP3Rs more accessible to Ca(2+) entering the cell via VGCCs. Thus, IP3Rs may serve as an "intermediate amplifier" between voltage-gated Ca(2+) entry and RyR-mediated Ca(2+) release., Conclusions: P2X receptor activation in mesenteric artery SMCs recruits IP3Rs-mediated Ca(2+) release from sub-PM SR, which is facilitated by activation of VGCCs. Sensitivity of IP3R-mediated release to VGCC antagonists in vascular SMCs makes this mechanism of special therapeutic significance., (Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Various in vitro models are used for studying phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of vascular smooth muscle cells (cVSMCs) is most often used for this purpose. On the other hand, vascular interstitial cells (VICs) are native phenotypically modulated VSMCs present in blood vessels under normal physiological conditions. The aim of this work has been to compare the difference in expression of a number of VSMC-specific markers, which are commonly used for the characterisation of phenotypic modulation of VSMCs, between freshly dispersed VSMCs, VICs and cVSMCs from rat abdominal aorta. Our experiments show that VICs are present in the rat aorta and express markers of VSMCs. Both VICs and cVSMCs display the presence of sparse individual stress fibres enriched in alpha smooth muscle actin (αSM-actin), whereas in VSMCs, this protein is more densely packed. Compared with contractile VSMCs, both VICs and cVSMCs display decreased expression of VSMC-specific markers such as smoothelin, myosin light chain kinase and SM22α; however, the expression of two major cytoskeletal and contractile proteins (smooth muscle myosin heavy chain and αSM-actin) was downregulated in cVSMCs but not in VICs compared with contractile VSMCs. These results suggest different mechanisms for the phenotypic modulation of cVSMCs and VICs. VICs might therefore represent a novel convenient model for studying molecular mechanisms that govern the phenotypic modulation of VSMCs.

Background: There is growing evidence suggesting involvement of L-type voltage-gated Ca2+ channels (VGCCs) in purinergic signaling mechanisms. However, detailed interplay between VGCCs and P2X receptors in intracellular Ca2+ mobilization is not well understood. This study examined relative contribution of the Ca2+ entry mechanisms and induced by this entry Ca2+ release from the intracellular stores engaged by activation of P2X receptors in smooth muscle cells (SMCs) from the guinea-pig small mesenteric arteries., Methods: P2X receptors were stimulated by the brief local application of αβ-meATP and changes in [Ca2+]i were monitored in fluo-3 loaded SMCs using fast x-y confocal Ca2+ imaging. The effects of the block of L-type VGCCs and/or depletion of the intracellular Ca2+ stores on αβ-meATP-induced [Ca2+]i transients were analyzed., Results: Our analysis revealed that Ca2+ entry via L-type VGCCs is augmented by the Ca2+-induced Ca2+ release significantly more than Ca2+ entry via P2X receptors, even though net Ca2+ influxes provided by the two mechanisms are not significantly different., Conclusions: Thus, arterial SMCs upon P2X receptor activation employ an effective mechanism of the Ca2+ signal amplification, the major component of which is the Ca2+ release from the SR activated by Ca2+ influx via L-type VGCCs. This signaling pathway is engaged by depolarization of the myocyte membrane resulting from activation of P2X receptors, which, being Ca2+ permeable, per se form less effective Ca2+ signaling pathway. This study, therefore, rescales potential targets for therapeutic intervention in purinergic control of vascular tone.

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., (© 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

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 Ca(2+) concentration ([Ca(2+) ](i) ). Although it is well-appreciated that the myocyte Ca(2+) signalling system is composed of microdomains, little is known about the structure of the [Ca(2+) ](i) responses induced by P2X receptor stimulation in vascular myocytes., Experimental Approaches: Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca(2+) signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP)., Key Results: RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP(3) R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca(2+) ](i) transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L(-1) αβ-meATP triggered an abrupt Ca(2+) release from sub-plasmalemmal ('junctional') sarcoplasmic reticulum enriched with IP(3) Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca(2+) channels (VGCCs) or IP(3) Rs suppressed the sub-plasmalemmal [Ca(2+) ](i) upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP(3) R inhibition on the sub-plasmalemmal [Ca(2+) ](i) upstroke was attenuated following block of VGCCs., Conclusions and Implications: Depolarization of RVSMCs following P2X receptor activation induces IP(3) R-mediated Ca(2+) release from sub-plasmalemmal ('junctional') sarcoplasmic reticulum, which is activated mainly by Ca(2+) influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: KCNQ-encoded voltage-gated potassium channels (K(v) 7) 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., Experimental Approach: Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K(v) 7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels., Key Results: KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K(v) 7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K(v) 7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K(+) currents. In contrast, the K(v) 7.1-specific blocker chromanol 293B did not affect vascular tone. Two K(v) 7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K(v) 7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991., Conclusions and Implications: This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo., Linked Article: This article is commented on by Mani and Byron, pp. 38-41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Geometric measures of localized calcium release (LCR) events have been used to understand their biophysical basis. We found power law scaling between three such metrics-maximum amplitude (MA), mass above half-maximum amplitude (MHM), and area at half-maximum amplitude (AHM). In an effort to understand this scaling a minimal analytic model was employed to simulate LCR events recorded by confocal line scan. The distribution of logMHM as a function of logAHM, pMHM(pAHM), was dependent on model parameters such as channel open time, current size, line scan offset, and apparent diffusion coefficient. The distribution of log[MHM/AHM] as a function of logMA, p[MHM/AHM](pMA), was invariant, reflecting the gross geometry of the LCR event. The findings of the model were applied to real LCR line scan data from rabbit portal vein myocytes, rat cerebral artery myocytes, and guinea pig fundus knurled cells. pMHM(pAHM) could be used to distinguish two populations of LCR events in portal vein, even at the scale of "calcium noise," and to calculate the relative current of the two. The relative current was 2. pMHM(pAHM) could also be used to study pharmacological effects. The pMHM(pAHM) distribution of knurled cell LCR events was markedly contracted by ryanodine, suggesting a reduction in channel open time. The p[MHM/AHM](pMA) distributions were invariant across all cell types and were consistent with the model, underlying the common physical basis of their geometry. The geometric scaling of LCR events demonstrated here may help with their mechanistic characterization.

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 microm) partially depressed, whereas the Kv7 activator S-1 (3 and/or 20 microm) 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 microm), 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 microm) enhanced the myogenic response at 20 mmHg, whereas the activation of Kv7 channels with S-1 (20 microm) 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.

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 [Ca(2+)](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 (I(P2X)) and to identify the types of P2X receptors expressed in myocytes isolated from interlobar and arcuate arteries of rat kidney., Experimental Approach: The expression of P2X receptors in isolated RVSMCs was analysed by reverse transcription (RT)-PCR. I(P2X) and membrane potential were recorded using the amphotericin B-perforated patch method., Key Results: RT-PCR analysis on single RVSMCs showed the presence of genes encoding P2X1 and P2X4 receptors. Under voltage clamp conditions, the selective P2X receptor agonist alphabeta-methylene ATP (alphabeta-meATP) evoked I(P2X) similar to that induced by ATP. Under current clamp conditions, both ATP and alphabeta-meATP evoked a spike-like membrane depolarization followed by a sustained depolarization, linking P2X receptors in RVSMCs to sympathetic control of renal vascular tone. A selective antagonist of P2X1 receptors, NF279, reduced I(P2X) amplitude by approximately 65% concentration-dependently manner within the nanomolar to sub-micromolar range. The residual current was resistant to micromolar concentrations of NF279, but was inhibited by sub-millimolar to millimolar concentrations of NF279., Conclusions and Implications: Two types of functional P2X receptors, monomeric P2X1 and heteromeric P2X1/4 receptors, are expressed in RVSMCs. Our study has identified important targets for possible pharmacological intervention in the sympathetic control of renal circulation.

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 alpha-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 beta-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.

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

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 alpha-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 transmission electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.

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.

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.

A rise in intracellular ionised calcium concentration ([Ca(2+)](i)) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca(2+) sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca(2+)](i) upon myocyte excitation; (2) spontaneous Ca(2+) sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca(2+)-dependent K(+) and Cl(-) channels sense the local changes in [Ca(2+)](i) during a calcium spark and thereby couple changes in [Ca(2+)](i) within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP(3)Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.

Voltage-gated components of the outward current in single smooth muscle cells isolated from the epididymal part of the rat vas deferens were studied using amphotericin B perforated patch-clamp techniques. The complex kinetics of the net outward current elicited by positive voltage steps from -80 mV to +40 mV suggested the presence of several components. Bath application of 200 nM charybdotoxin, a potent blocker of large-conductance, Ca(2+)-dependent K(+) channels (BK(Ca)), reduced the current amplitude significantly. When BK(Ca) channels were suppressed, fast-inactivating (I(K,f)) and delayed rectifying (I(K,dr)) components of the outward current were identified. I(K,f) was characterized by fast kinetics of current decay, negative steady-state activation and inactivation dependencies and sensitivity to 4-aminopyridine with an apparent K(d) of 0.32 mM, properties similar to those of the A-type K(+) current. In contrast, I(K,dr) activated and inactivated at more positive potentials. The time constant of activation of I(K,dr) was voltage dependent with an e-fold decrease per 21 mV depolarization. I(K,dr) was inhibited by clofilium, a blocker of voltage-gated K(+) channels, with an IC(50) of 12 micro M and was not blocked by 5 mM 4-aminopyridine. The possible significance of the voltage-gated currents is discussed.

Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40-70 microm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 microm, and spindle-shaped cells from 40 to 300 microm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90-150 microm). The cell membrane capacitance was 46.5+/-2.2 pF in SMCs, 39.7+/-2.4 pF in spindle-shaped ICs and 27.8+/-0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca(2+) sparks and Ca(2+) waves. Ca(2+) waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca(2+)](i) transients (lasting about 2s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1-10 microM), caffeine (1-10 mM) or high-K(+) solution (60mM) led to a rise of [Ca(2+)](i) in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current-clamp conditions was -46.5+/-2.0 mV in spindle-shaped ICs and -45.6+/-2.7 mV in SMCs. Under voltage-clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca(2+) current, a set of K(+) currents, including spontaneous transient outward currents (STOCs) but no Na(+) current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.

K+ current which take part in the controlling of membrane potential in myocytes isolated from rat resistance mesenteric arteries have been investigated using conventional patch clamp method. The mean resting potential of myocytes was--37 mV. Charybdotoxin (200 nM)--selective blocker of large conductance Ca(2+)-activated K+ (KCa) channels--inhibited transmembrane outward K+ current by 60%. 1 mM of tetraethylammonium inhibited outward K+ current same as 200 nM of charybdotoxin, also it inhibited spontaneous spike-like hyperpolarizations and did not affect the membrane potential. Transmembrane current had a 4 aminopyridine (4-AP) sensitive component of delayed rectifier current (KV). Addition of 5 mM of 4-AP evoked membrane depolarization with mean significance of 12.0 +/- 1.5 mV in 5 from 7 single myocytes which had resting potential in the range of -50 ... -35 mV. The obtained results suggest that large conductance KCa channels do not determine the resting potential, but may serve as a negative feedback mechanism at the considerable membrane depolarization. In contrast, 4-AP sensitive KV current take part in the controlling of the resting membrane potential of single myocytes from rat resistance mesenteric arteries.