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.

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.

Previously we have described a constitutively active, Ca2+ -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.

A voltage-gated Na + current was characterised in freshly dissociated mouse portal vein (PV) smooth muscle myocytes. The current was found superimposed upon the relatively slow L-type Ca 2+ current and was resistant to conventional Ca 2+ channel blockers but was abolished by external Na + replacement and tetrodotoxin (TTX, 1 µM). The molecular identity of the channel responsible for this conductance was determined by RT-PCR where only the transcripts for Na + channel genes SCN7a, 8a and 9a were detected. The presence of the protein counterparts to the SCN8a and 9a genes (NaV 1.6 and NaV 1.7 , respectively) on the individual smooth muscle myocytes were confirmed in immunocytochemistry, which showed diffuse staining around a predominantly plasmalemmal location. TTX inhibited the action potential in individual myocytes generated in the current clamp mode but isometric tissue tension experiments revealed that TTX (1 and 5 µM) had no effect on the inherent mouse PV rhythmicity. However, the Na + channel opener veratridine (10 and 50 µM) significantly increased the length of contraction and the interval between contractions. This effect was not influenced by pre-incubation with atropine, prazosin and propranolol, but was reversed by TTX (1 µM) and completely abolished by nicardipine (1 µM). Furthermore, preincubation with the reverse-mode Na + -Ca 2+ exchange blocker KB-R7943 (10 µM) also inhibited the veratridine response. We have established for the first time the molecular identity of the voltage-gated Na + channel in freshly dispersed smooth muscle cells and have shown that these channels can modulate contractility through a novel mechanism of action possibly involving reverse mode Na + -Ca 2+ exchange.

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 Ca 2+ -release events from endo/sarcoplasmic reticulum including fast localised Ca 2+ transients (Ca 2+ sparks) and longer and slower Ca 2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca 2+ 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.

Background and purpose: This study investigated the functional and electrophysiological effects of the K v 7 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 þ 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. K v 7.1 and K v 7.4 proteins were identified in the cell membranes of myocytes by immunocytochemistry. Retigabine (2-20 mM) suppressed spontaneous contractions in whole portal veins, hyperpolarized the membrane potential and augmented potassium currents at À20 mV. At more depolarized potentials, retigabine and flupirtine, decreased potassium currents. Both effects of retigabine were prevented by prior application of the K v 7 blocker XE991 (10 mM). Recombinant KCNQ 4 or 5 channels were only activated by retigabine or flupirtine. Conclusions and implications: The K v 7 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.

Background and purpose: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloridesensitive (I amiloride ) and ouabain-sensitive basolateral (I ouabain ) short circuit current in H441 lung epithelial cells. Experimental approach: H441 cells were grown on permeable filters at air interface. I amiloride , I ouabain and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Key results: Phenformin, AICAR and metformin increased AMPK (a1) activity and decreased I amiloride . The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I ouabain across H441 monolayers and decreased monolayer resistance. The decrease in I amiloride was closely related to I ouabain with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Conclusions and implications: Activation of a1-AMPK is associated with inhibition of apical amiloride-sensitive Na þ channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I ouabain, with potential secondary effects on apical Na þ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na þ K þ ATPase is an important component.

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