Claire M. Peppiatt-Wildman

Diverse properties of store-operated TRPC channels activated by protein kinase C in vascular myocytes

In vascular smooth muscle, store‐operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)‐induced whole‐cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin‐1, CPA and the cell‐permeant Ca2+ chelator BAPTA‐AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mm[Ca2+]o the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mm[Ca2+]o the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13‐dibutyrate (PDBu), the diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti‐TRPC1 and ‐TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti‐TRPC6 blocked SOCs only in coronary artery and anti‐TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

Multiple activation mechanisms of store‐operated TRPC channels in smooth muscle cells

Store‐operated channels (SOCs) are plasma membrane Ca2+‐permeable cation channels which are activated by agents that deplete intracellular Ca2+ stores. In smooth muscle SOCs are involved in contraction, gene expression, cell growth and proliferation. Single channel recording has demonstrated that SOCs with different biophysical properties are expressed in smooth muscle indicating diverse molecular identities. Moreover it is apparent that several gating mechanisms including calmodulin, protein kinase C and lysophospholipids are involved in SOC activation. Evidence is accumulating that TRPC proteins are important components of SOCs in smooth muscle. More recently Orai and STIM proteins have been proposed to underlie the well‐described calcium‐release‐activated current (ICRAC) in non‐excitable cells but at present there is little information on the role of Orai and STIM proteins in smooth muscle. In addition it is likely that different TRPC subunits coassemble as heterotetrameric structures to form smooth muscle SOCs. In this brief review we summarize the diverse properties and gating mechanisms of SOCs in smooth muscle. We propose that the heterogeneity of the properties of these conductances in smooth muscle results from the formation of heterotetrameric TRPC structures in different smooth muscle preparations.

Endothelin-1 activates a Ca2+-permeable cation channel with TRPC3 and TRPC7 properties in rabbit coronary artery myocytes

In the present work we used patch pipette techniques to study the properties of a novel Ca2+‐permeable cation channel activated by the potent coronary vasoconstrictor endothelin‐1 (ET‐1) in freshly dispersed rabbit coronary artery myocytes. With cell‐attached recording bath application of 10 nm ET‐1 evoked cation channel currents (Icat) with subconductance states of about 18, 34 and 51 and 68 pS, and a reversal potential of 0 mV. ET‐1 evoked channel activity when extracellular Ca2+ was the charge carrier, illustrating significant Ca2+ permeability. ET‐1‐induced responses were inhibited by the ETA receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) also stimulated Icat, but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG‐ or ET‐1‐induced Icat. Inositol 1,4,5‐trisphosphate (IP3) did not activate Icat, but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti‐TRPC3 and anti‐TRPC7 antibodies to inside‐out patches markedly inhibited ET‐1‐evoked Icat, but antibodies to TRPC1, C4, C5 and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma, whereas TRPC3 was distributed throughout the myocyte, and moreover co‐localization of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd3+, La3+ and extracellular Ca2+ inhibited Icat with IC50 values of 2.45 μm, 3.8 μm, 7.36 μm and 22 μm, respectively. These results suggest that in rabbit coronary artery myocytes ET‐1 evokes a Ca2+‐permeable non‐selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance.

Sodium-Dependent Regulation of Renal Amiloride-Sensitive Currents by Apical P2 Receptors

The epithelial sodium channel (ENaC) plays a major role in the regulation of sodium balance and BP by controlling Na(+) reabsorption along the renal distal tubule and collecting duct (CD). ENaC activity is affected by extracellular nucleotides acting on P2 receptors (P2R); however, there remain uncertainties over the P2R subtype(s) involved, the molecular mechanism(s) responsible, and their physiologic role. This study investigated the relationship between apical P2R and ENaC activity by assessing the effects of P2R agonists on amiloride-sensitive current in the rat CD. Using whole-cell patch clamp of principal cells of split-open CD from Na(+)-restricted rats, in combination with immunohistochemistry and real-time PCR, we found that activation of metabotropic P2R (most likely the P2Y(2) and/or (4) subtype), via phospholipase C, inhibited ENaC activity. In addition, activation of ionotropic P2R (most likely the P2X(4) and/or (4/6) subtype), via phosphatidylinositol-3 kinase, either inhibited or potentiated ENaC activity, depending on the extracellular Na(+) concentration; therefore, it is proposed that P2X(4) and/or (4/6) receptors might function as apical Na(+) sensors responsible for local regulation of ENaC activity in the CD and could thereby help to regulate Na(+) balance and systemic BP.

The energy use associated with neural computation in the cerebellum

The brains energy supply determines its information processing power, and generates functional imaging signals, which are often assumed to reflect principal neuron spiking. Using measured cellular properties, we analysed how energy expenditure relates to neural computation in the cerebellar cortex. Most energy is used on information processing by non-principal neurons: Purkinje cells use only 18% of the signalling energy. Excitatory neurons use 73% and inhibitory neurons 27% of the energy. Despite markedly different computational architectures, the granular and molecular layers consume approximately the same energy. The blood vessel area supplying glucose and O2 is spatially matched to energy consumption. The energy cost of storing motor information in the cerebellum was also estimated.

Renal pericytes: regulators of medullary blood flow

Regulation of medullary blood flow (MBF) is essential in maintaining normal kidney function. Blood flow to the medulla is supplied by the descending vasa recta (DVR), which arise from the efferent arterioles of juxtamedullary glomeruli. DVR are composed of a continuous endothelium, intercalated with smooth muscle‐like cells called pericytes. Pericytes have been shown to alter the diameter of isolated and in situ DVR in response to vasoactive stimuli that are transmitted via a network of autocrine and paracrine signalling pathways. Vasoactive stimuli can be released by neighbouring tubular epithelial, endothelial, red blood cells and neuronal cells in response to changes in NaCl transport and oxygen tension. The experimentally described sensitivity of pericytes to these stimuli strongly suggests their leading role in the phenomenon of MBF autoregulation. Because the debate on autoregulation of MBF fervently continues, we discuss the evidence favouring a physiological role for pericytes in the regulation of MBF and describe their potential role in tubulo‐vascular cross‐talk in this region of the kidney. Our review also considers current methods used to explore pericyte activity and function in the renal medulla.

Extracellular nucleotides affect pericyte-mediated regulation of rat in situ vasa recta diameter.

Aim:  We hypothesized that extracellular nucleotides, established as being released from renal tubular epithelial cells, act at pericytes to regulate vasa recta capillary diameter.

Nucleotides Downregulate Aquaporin 2 via Activation of Apical P2 Receptors

Vasopressin regulates water reabsorption in the collecting duct, but extracellular nucleotides modulate this regulation through incompletely understood mechanisms. We investigated these mechanisms using immortalized mouse collecting duct (mpkCCD) cells. Basolateral exposure to dDAVP induced AQP2 localization to the apical membrane, but co-treatment with ATP internalized AQP2. Because plasma membrane-bound P2 receptors (P2R) mediate the effects of extracellular nucleotides, we examined the abundance and localization of P2R in mpkCCD cells. In the absence of dDAVP, P2Y(1) and P2Y(4) receptors localized to the apical membrane, whereas P2X(2), P2X(4), P2X(5), P2X(7), P2Y(2), P2Y(11), and P2Y(12) receptors localized to the cytoplasm. dDAVP induced gene expression of P2X(1), which localized to the apical domain, and led to translocation of P2X(2) and P2Y(2) to the apical and basolateral membranes, respectively. In co-expression experiments, P2R activation decreased membrane AQP2 and AQP2-mediated water permeability in Xenopus oocytes expressing P2X(2), P2Y(2,) or P2Y(4) receptors, but not in oocytes expressing other P2R subtypes. In summary, these data suggest that AQP2-mediated water transport is downregulated not only by basolateral nucleotides, mediated by P2Y(2) receptors, but also by luminal nucleotides, mediated by P2X(2) and/or P2Y(4) receptors.

An Intact Kidney Slice Model to Investigate Vasa Recta Properties and Function in situ

Background: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. Results: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E2) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.

Multiphoton Imaging of the Functioning Kidney

Translating discoveries made in isolated renal cells and tubules to the in vivo situation requires the assessment of cellular function in intact live organs. Multiphoton imaging is a form of fluorescence microscopy that is ideally suited to working with whole tissues and organs, but adequately loading cells with fluorescence dyes in vivo remains a challenge. We found that recirculation of fluorescence dyes in the rat isolated perfused kidney (IPK) resulted in levels of intracellular loading that would be difficult to achieve in vivo. This technique allowed the imaging of tubular cell structure and function with multiphoton microscopy in an intact, functioning organ. We used this approach to follow processes in real time, including (1) relative rates of reactive oxygen species (ROS) production in different tubule types, (2) filtration and tubular uptake of low-molecular-weight dextrans and proteins, and (3) the effects of ischemia-reperfusion injury on mitochondrial function and cell structure. This study demonstrates that multiphoton microscopy of the isolated perfused kidney is a powerful technique for detailed imaging of cell structure and function in an intact organ.