Dataset Information

Smooth muscle Ca(2+) -activated and voltage-gated K+ channels modulate conducted dilation in rat isolated small mesenteric arteries.

ABSTRACT: OBJECTIVE:? To assess the influence of blocking smooth muscle large conductance Ca(2+) -activated K+ channels and voltage-gated K+ channels on the conducted dilation to ACh and isoproterenol. MATERIALS AND METHODS:? Rat mesenteric arteries were isolated with a bifurcation, triple-cannulated, pressurized and imaged using confocal microscopy. Phenylephrine was added to the superfusate to generate tone, and agonists perfused into a sidebranch to evoke local dilation and subsequent conducted dilation into the feed artery. RESULTS:? Both ACh- and isoproterenol-stimulated local and conducted dilation with similar magnitudes of decay with distance along the feed artery (2000?m: ?15% maximum dilation). The gap junction uncoupler carbenoxolone prevented both conducted dilation and intercellular spread of dye through gap junctions. IbTx, TEA or 4-AP, blockers of large conductance Ca(2+) -activated K+ channels and voltage-gated K+ channels, did not affect conducted dilation to either agonist. A combination of either IbTx or TEA with 4-AP markedly improved the extent of conducted dilation to both agonists (2000?m: >50% maximum dilation). The enhanced conducted dilation was reflected in the hyperpolarization to ACh (2000?m: Control, 4±1?mV, n?=?3; TEA with 4-AP, 14±3mV, n=4), and was dependent on the endothelium. CONCLUSIONS:? These data show that activated BK(Ca) and K(V) -channels serve to reduce the effectiveness of conducted dilation.

SUBMITTER: Beleznai TZ

PROVIDER: S-EPMC4826745 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Smooth muscle Ca(2+) -activated and voltage-gated K+ channels modulate conducted dilation in rat isolated small mesenteric arteries.

Beleznai Timea Z TZ Yarova Polina L PL Yuill Kathryn H KH Dora Kim A KA

Microcirculation (New York, N.Y. : 1994) 20110801 6

<h4>Objective</h4> To assess the influence of blocking smooth muscle large conductance Ca(2+) -activated K+ channels and voltage-gated K+ channels on the conducted dilation to ACh and isoproterenol.<h4>Materials and methods</h4> Rat mesenteric arteries were isolated with a bifurcation, triple-cannulated, pressurized and imaged using confocal microscopy. Phenylephrine was added to the superfusate to generate tone, and agonists perfused into a sidebranch to evoke local dilation and subsequent co ...[more]

PMID: 21535295

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Project description:Background and purposeVasodilatation may contribute to the neuroprotective and vascular anti-remodelling effect of the tissue transglutaminase 2 (TG2) inhibitor cystamine. Here, we hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlies cystamine vasodilatation.Experimental approachWe used rat mesenteric small arteries and RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca(2+) ([Ca(2+)]i ), K(+) currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain, in our experiments.Key resultsRT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited responses to phenylephrine, 5-HT and U46619 and for extracellular potassium. Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of PLC suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca(2+)]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca(2+)]i , and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of Kv 7 channels, XE991 and linopirdine, inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr(855) phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr(855) and myosin regulatory light chain phosphorylation.Conclusions and implicationsCystamine induced vasodilatation by inhibition of receptor-coupled TG2, leading to opening of Kv channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine.

Project description:BackgroundStudies of the structure-function relationship in proteins for which no 3D structure is available are often based on inspection of multiple sequence alignments. Many functionally important residues of proteins can be identified because they are conserved during evolution. However, residues that vary can also be critically important if their variation is responsible for diversity of protein function and improved phenotypes. If too few sequences are studied, the support for hypotheses on the role of a given residue will be weak, but analysis of large multiple alignments is too complex for simple inspection. When a large body of sequence and functional data are available for a protein family, mature data mining tools, such as machine learning, can be applied to extract information more easily, sensitively and reliably. We have undertaken such an analysis of voltage-gated potassium channels, a transmembrane protein family whose members play indispensable roles in electrically excitable cells.ResultsWe applied different learning algorithms, combined in various implementations, to obtain a model that predicts the half activation voltage of a voltage-gated potassium channel based on its amino acid sequence. The best result was obtained with a k-nearest neighbor classifier combined with a wrapper algorithm for feature selection, producing a mean absolute error of prediction of 7.0 mV. The predictor was validated by permutation test and evaluation of independent experimental data. Feature selection identified a number of residues that are predicted to be involved in the voltage sensitive conformation changes; these residues are good target candidates for mutagenesis analysis.ConclusionMachine learning analysis can identify new testable hypotheses about the structure/function relationship in the voltage-gated potassium channel family. This approach should be applicable to any protein family if the number of training examples and the sequence diversity of the training set that are necessary for robust prediction are empirically validated. The predictor and datasets can be found at the VKCDB web site.

Dataset Information

Smooth muscle Ca(2+) -activated and voltage-gated K+ channels modulate conducted dilation in rat isolated small mesenteric arteries.

Publications

Smooth muscle Ca(2+) -activated and voltage-gated K+ channels modulate conducted dilation in rat isolated small mesenteric arteries.

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