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

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Non-Contractile Cells with Thin Processes Resembling Interstitial Cells of Cajal Found in the Wall of Guinea-Pig Mesenteric Arteries

Arterial interstitial cells of Cajal (ICC)-like cells (AIL cells) with a multipolar, irregular, elongated shape and with numerous thin (often less than 1 μm), sometimes branching, processes with lengths up to ≈60 μm were isolated enzymatically from 1st to 7th order branches of guinea-pig mesenteric artery. Some of the processes of AIL cells were growing (average speed ≈0.15 μm min−1) and their growth was blocked by 10 μm latrunculin B, an inhibitor of actin polymerisation. Staining with BODIPY phalloidin, a fluorescent dye selective for F-actin, showed the presence of F-actin in the processes of AIL cells. Voltage clamp of single AIL cells revealed an inward current that was four times more dense than in myocytes and was abolished by 10 μm nicardipine, and an outward current carried exclusively by potassium ions that was reduced by 1 mm 4-aminopyridine and/or 100 nm iberiotoxin but unaffected by 10 nm dendrotoxin-K. Imaging of intracellular ionised calcium with fluo-4 using a laser scanning confocal microscope showed local or global calcium transients lasting several seconds in ≈28 % of AIL cells. When membrane current was recorded simultaneously, the calcium transients were found to correspond to long-lasting transient outward currents, which occurred at potentials positive to −40 mV. Unlike myocytes, AIL cells did not contract in response to 1 mm caffeine or 5 μm noradrenaline, although they responded with a [Ca2+]i increase. The segments of intact arteries did not stain for c-kit, a marker of ICCs. Single AIL cells stained positive for vimentin, desmin and smooth muscle myosin. The presence of ICC-like cells is demonstrated for the first time in the media of resistance arteries

Bimodal effects of the Kv7 channel activator retigabine on vascular K+ currents

Yeung SYM, Schwake M, Pucovský V, Greenwood IA. Bimodal effects of the K 7 channel activator retigabine on vascular K+ currents. British Journal of Pharmacology. 2008;155(1):62-72.Background and purpose: This study investigated the functional and electrophysiological effects of the Kv7 channel activator, retigabine, on murine portal vein smooth muscle. Experimental approach: KCNQ gene expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical experiments. Whole cell voltage clamp and current clamp were performed on isolated myocytes from murine portal vein. Isometric tension recordings were performed on whole portal veins. K+ currents generated by KCNQ4 and KCNQ5 expression were recorded by two-electrode voltage clamp in Xenopus oocytes. Key results: KCNQ1, 4 and 5 were expressed in mRNA derived from murine portal vein, either as whole tissue or isolated myocytes. Kv7.1 and Kv7.4 proteins were identified in the cell membranes of myocytes by immunocytochemistry. Retigabine (2–20 μM) suppressed spontaneous contractions in whole portal veins, hyperpolarized the membrane potential and augmented potassium currents at −20 mV. At more depolarized potentials, retigabine and flupirtine, decreased potassium currents. Both effects of retigabine were prevented by prior application of the Kv7 blocker XE991 (10 μM). Recombinant KCNQ 4 or 5 channels were only activated by retigabine or flupirtine. Conclusions and implications: The Kv7 channel activators retigabine and flupirtine have bimodal effects on vascular potassium currents, which are not seen with recombinant KCNQ channels. These results provide support for KCNQ4- or KCNQ5-encoded channels having an important functional impact in the vasculature

TRPC3 properties of a native constitutively active Ca2+-permeable cation channel in rabbit ear artery myocytes.

Previously we have described a constitutively active, Ca(2+)-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd(3+), La(3+) and Ca(2+) inhibited spontaneous channel activity in outside-out patches with IC(50) values of 6.8 μm, 25 nm, 1.5 μm and 0.124 mm, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance

Molecular expression and pharmacological identification of a role for Kv7 channels in murine vascular reactivity

BACKGROUND AND PURPOSE: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta. KEY RESULTS: All blood vessels expressed KCNQ1, 4 and 5 with hitherto ‘neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 –20 μM), flupirtine (20 μM) and meclofenamic acid (20 μM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 μM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 μM). CONCLUSIONS AND IMPLICATIONS: Murine blood vessels exhibit a distinctive KCNQ expression profile with ‘neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca2+ channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca2+ entry through VGCCs triggers RyR-mediated Ca2+ release via a Ca2+-induced Ca2+ 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 Ca2+ release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP3Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca2+]i. The initial abrupt sub-PM [Ca2+]i upstroke was all but abolished by block of VGCCs (by 5 μM nicardipine), depletion of intracellular Ca2+ stores (with 10 μM cyclopiazonic acid) or inhibition of IP3Rs (by 2 μM xestospongin C or 30 μM 2-APB), but was not affected by block of RyRs (by 50–100 μM tetracaine or 100 μM ryanodine). Inhibition of either IP3Rs 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 Ca2+ entry through VGCCs which evokes an initial IP3R-mediated Ca2+ release activated via a CICR mechanism