Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway.
ABSTRACT: This study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC).Isometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na+/K+-pump and Na+,K+,2Cl-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86Rb influx, respectively.NaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K+]o=30 mM). At 10-4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10-3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10-4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na+,K+,2Cl-cotransport, whereas the inhibition seen at 10-3 M NaHS was suppressed in the presence of K+ channel blocker TEA. In cultured SMC, 5×10-5 M NaHS increased Na+,K+,2Cl- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45Ca influx was enhanced in the presence of 10-4 M NaHS and suppressed under elevation of [NaHS] up to 10-3 M. 45Ca influx triggered by 10-4 M NaHS was abolished by bumetanide and L-type Ca2+ channel blocker nicardipine.Our results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels.
Project description:This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog alpha,beta-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X(1) receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y(6) receptors. Inhibition of Na(+), K(+), 2Cl(-) cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y(6)-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y(6)-mediated signaling should be examined further.
Project description:Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K(+) concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K(+) concentrations and was inhibited when K(+) and/or Cl(-) was absent from the medium. The results suggested that the well-documented Na(+)-K(+)-2Cl(-) cotransport system was responsible for the K(+) influx activated during the RVI. However, inhibitors of Na(+)-K(+)-2Cl(-) cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K(+) influx in T. foetus.
Project description:Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na(+)-K(+)-2Cl(-) cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (> or =100 microM). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC(50) approximately 20 microM, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC(50) approximately 8 microM), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 microM had no effect, and 20 microM caused 20% block of human Na(+)-K(+)-2Cl(-) cotransporter activity, in contrast to >90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl(-) currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.
Project description:Electroneutral Na+:Cl- cotransport systems are involved in a number of important physiological processes including salt absorption and secretion by epithelia and cell volume regulation. One group of Na+:Cl- cotransporters is specifically inhibited by the benzothiadiazine (thiazide) class of diuretic agents and can be distinguished from Na+:K+:2Cl- cotransporters based on a lack of K+ requirement and insensitivity to sulfamoylbenzoic acid diruetics like bumetanide. We report here the isolation of a cDNA encoding a thiazide-sensitive, electroneutral sodium-chloride cotransporter from the winter flounder urinary bladder using an expression cloning strategy. The pharmacological and kinetic characteristics of the cloned cotransporter are consistent with the properties of native thiazide-sensitive sodium-chloride cotransporters in teleost urinary bladder and mammalian renal distal tubule epithelia. The nucleotide sequence predicts a protein of 1023 amino acids (112 kDa) with 12 putative membrane-spanning regions, which is not related to other previously cloned sodium or chloride transporters. Northern hybridization shows two different gene products: a 3.7-kb mRNA localized only to the urinary bladder and a 3.0-kb mRNA present in several non-bladder/kidney tissues.
Project description:GABA, the main inhibitory neurotransmitter in the adult nervous system, evokes depolarizing membrane responses in immature neurons, which are crucial for the generation of early network activity. Although it is well accepted that depolarizing GABA actions are caused by an elevated intracellular Cl- concentration ([Cl-]i), the mechanisms of Cl- accumulation in immature neurons are still a matter of debate. Using patch-clamp, microfluorimetric, immunohistochemical, and molecular biological approaches, we studied the mechanism of Cl- uptake in Cajal-Retzius (CR) cells of immature [postnatal day 0 (P0) to P3] rat neocortex. Gramicidin-perforated patch-clamp and 6-methoxy-N-ethylquinolinium-microfluorimetric measurements revealed a steady-state [Cl-]i of approximately 30 mM that was reduced to values close to passive distribution by bumetanide or Na+-free solutions, suggesting a participation of Na+-K+-2Cl- cotransport isoform 1 (NKCC1) in maintaining elevated [Cl-]i. Expression of NKCC1 was found in CR cells on the mRNA and protein levels. To determine the contribution of NKCC1 to [Cl-]i homeostasis in detail, Cl- uptake rates were analyzed after artificial [Cl-]i depletion. Active Cl- uptake was relatively slow (47.2 +/- 5.0 microM/s) and was abolished by bumetanide or Na+-free solution. Accordingly, whole-cell patch-clamp recordings revealed a low Cl- conductance in CR cells. The low capacity of NKCC1-mediated Cl- uptake was sufficient to maintain excitatory GABAergic membrane responses, however, only at low stimulation frequencies. In summary, our results demonstrate that NKCC1 is abundant in CR cells of immature rat neocortex and that the slow Cl- uptake mediated by this transporter is sufficient to maintain high [Cl-]i required to render GABA responses excitatory.
Project description:Introduction: Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) is important in regulating intracellular K+ and Cl- homeostasis and cell volume. In this study, we investigated a role of NKCC1 in regulating glioma K+ influx and proliferation in response to apoptosis inducing chemotherapeutic drug temozolomide (TMZ). The efficacy of a new bumetanide (BMT)-derivative NKCC1 inhibitor STS66 [3-(butylamino)-2-phenoxy-5-[(2, 2, 2-trifluoroethylamino) methyl] benzenesulfonamide] in blocking NKCC1 activity was compared with well-established NKCC1 inhibitor BMT. Methods: NKCC1 activity in cultured mouse GL26 and SB28-GFP glioma cells was measured by Rb+ (K+) influx. The WNK1-SPAK/OSR1-NKCC1 signaling and AKT/ERK-mTOR signaling protein expression and activation were assessed by immunoblotting. Cell growth was determined by bromodeoxyuridine (BrdU) incorporation assay, MTT proliferation assay, and cell cycle analysis. Impact of STS66 and BMT on cell Rb+ influx and growth was measured in glioma cells treated with or without TMZ. Results: Rb+ influx assay showed that 10 ?M BMT markedly decreased the total Rb+ influx and no additional inhibition detected at >10 ?M BMT. In contrast, the maximum effects of STS66 on Rb+ influx inhibition were at 40-60 ?M. Both BMT and STS66 reduced TMZ-mediated NKCC1 activation and protein upregulation. Glioma cell growth can be reduced by STS66. The most robust inhibition of glioma growth, cell cycle, and AKT/ERK signaling was achieved by the TMZ + STS66 treatment. Conclusion: The new BMT-derivative NKCC1 inhibitor STS66 is more effective than BMT in reducing glioma cell growth in part by inhibiting NKCC1-mediated K+ influx. TMZ + STS66 combination treatment reduces glioma cell growth via inhibiting cell cycle and AKT-ERK signaling.
Project description:By mediating the coupled movement of Na, K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2-kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.
Project description:Na+-alanine cotransport across the cell membrane in isolated rat hepatocytes was studied. Changes in the cell membrane potential associated with the transport of alanine interfere with determination of the Na+-alanine coupling ratio of the cotransport. With valinomycin present to 'clamp' the cell membrane potential, a coupling ratio of 1:1 for the Na+-alanine influx was obtained.
Project description:Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40-70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na(+)-Cl(-) cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by (86)Rb(+) influx in Xenopus laevis oocytes was markedly reduced by methyl-beta-cyclodextrin (MbetaCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MbetaCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.
Project description:Stress is a major risk factor for psychiatric disorders, such as depression, posttraumatic stress disorder, and schizophrenia. Early life stress, such as maternal separation, can have long-term effects on the development of the central nervous system and pathogenesis of psychiatric disorders. In the present study, we found that maternal separation increased the susceptibility to stress in adolescent rats, increased the expression of Na+/K+/2Cl- cotransporter 1 (NKCC1) on postnatal day 14, and increased the expression of K+/2Cl- cotransporter 2 (KCC2) and ?-aminobutyric acid A (GABAA) receptor subunits on postnatal day 40 in the hippocampus. NKCC1 inhibition by the U.S. Food and Drug Administration-approved drug bumetanide during the first two postnatal weeks rescued the depressive- and anxiety-like behavior that was induced by maternal separation and decreased the expression of NKCC1, KCC2 and GABAA receptor ?1 and ?2,3 subunits in the hippocampus. Bumetanide treatment during early development did not adversely affect body weight or normal behaviors in naive rats, or affect serum osmolality in adult rats. These results suggest that bumetanide treatment during early development may prevent the maternal separation-induced susceptibility to stress and impairments in GABAergic transmission in the hippocampus.