- Email: [email protected]
Sodium-lithium countertransport activity in proximal tubule cells
AJH–April 2002–VOL. 15, NO. 4, PART 2
P-204 SODIUM-LITHIUM COUNTERTRANSPORT ACTIVITY IN PROXIMAL TUBULE CELLS Lisa A. Marshall, Mary J. MacLeod, James S. McLay. Medicine & Therapeutics, University of Aberdeen, Aberdeen, United Kingdom. This study was designed to determine whether renal proximal tubule cells display sodium - lithium countertransport (SLC) activity as activity has previously only been described in erythrocytes and more recently human skin fibroblasts. Proximal tubule (PT) cells were isolated from either Sprague-Dawley rats or New Zealand White rabbits by collagenase digestion followed by Percoll density gradient centrifugation. Rat PT cell SLC activity was determined in freshly isolated cell suspensions, rabbit PT cells were either cultured in 6 - well plates and used at Day 7 of culture or the cells were used in suspension. Cells were incubated in 150mM lithium, washed and re-suspended in either sodium-containing or sodium-free media. SLC activity was determined as the difference in lithium efflux between these 2 solutions. In cell suspensions, the Millipore Rapid Filtration Technique was used to determine lithium transport at short time intervals. In the rat PT cell suspensions, lithium uptake was shown to be time and concentration-dependent and the MTT assay confirmed that incubation with lithium was not toxic to the cells. Lithium efflux was significantly greater into 150mM sodium than into choline (0.404 vs 0.115 mmol lithium/mg protein/30s, respectively n⫽3) and a concentration dependency was observed. 0.1mM ouabain produced a slight inhibition of SLC activity (0.289 vs 0.205 mmol lithium/mg protein/30s n⫽3) but 0.1mM dimethylamiloride had no effect (0.325 vs 0.328 mmol lithium/mg protein/30s n⫽3). The transport of lithium was shown to be energy-independent as intracellular ATP depletion did not affect the SLC activity. A similar sodium-dependency was noted in freshly isolated rabbit PT cell suspensions. SLC activity was lower than that in the rat although longer efflux times were studied (0.112mmol lithium/mg protein/6 minutes n⫽3). The cultured rabbit PT cells demonstrated a time and concentrationdependent lithium uptake but lithium efflux was very similar into sodiumcontaining and sodium-free buffers (1.30mmol lithium/mg protein/30s vs 1.47mmol lithium/mg protein/30s, respectively n⫽3). No sodium-dependent lithium efflux was observed. Primary cultures of rabbit PT cells fail to demonstrate SLC activity but the results clearly demonstrate that SLC activity can be measured in freshly isolated renal proximal tubule cells. This is a functionally appropriate cell type in which to investigate SLC activity because of the relationship with essential hypertension. Key Words: Sodium-Lithium Countertransport, Proximal Tubule, Hypertension
P-205 THE ENDOTHELIUM-MODULATED BUT CYCLIC GMP-INDEPENDENT VASORELAXANT EFFECT OF H2S Rui Wang, Weimin Zhao, Xianfeng Sun. Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada. Hydrogen sulfide (H2S) is an important endogenous gas that possesses vasorelaxant property. The interaction of H2S with different cellular signaling pathways in vascular smooth muscle cells (SMCs), however, had not been systematically studied. In the present study, the mechanisms for the H2S-induced relaxation of phenylephrine-precontracted rat aortic tissues were further investigated. Removal of endothelium from vascular tissues reduced the H2S-induced vasorelaxation by shifting to the right of the concentration-response curve of H2S, but did not affect the maximal relaxation responses. Denervation of vascular tissues did not affect the © 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
POSTERS: Cell Membrane Transport/Ion Channels
103A
vasorelaxation induced by H2S. The sodium nitroprusside-induced relaxation of aortic tissues was completely abolished by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor. Instead of inhibition, ODQ potentiated the H2S-induced vasorelaxation. Inhibitors of cAMP, protein kinase C, and cyclo-oxygenase pathways did not affect the H2S-induced relaxation. In the absence of calcium from the bath solution, the relaxant effect of H2S was significantly attenuated. Prior exposure to H2S reduced the relaxant response of vascular tissues to nitric oxide. Our results suggest that the vasorelaxant effect of H2S was largely due to the interaction of H2S with smooth muscle cells and partially mediated by endothelium in an extracellular calcium entry-dependent manner. The mechanisms for the H2S-induced vasorelaxation were different from those of other gaseous vasoactive factors, such as nitric oxide or carbon monoxide that relax vascular smooth muscles by activating cGMP-dependent pathway. The interaction among these endogenous gases may provide fine tune of vascular tone. Supported by HSURC of Saskatchewan, Canada. Key Words: Hydrogen Sulfide, Vasorelaxation, cGMP
P-206 HYDROGEN SULFIDE DIFFERENTIALLY MODULATES SULFHYDRYL GROUPS ON RVSUR1 SUBUNIT IN HEK293 CELLS TRANSFECTED WITH RVKIR6.1/RVSUR1 CDNAS Kun Cao, Bo Jiang, Rui Wang. Physiology, University of Saskatchewan, Saskatoon, SK, Canada; Physiology, University of Saskatchewan, Saskatoon, SK, Canada; Physiology, University of Saskatchewan, Saskatoon, SK, Canada. Hydrogen sulfide (H2S) has been known as an endogenous opener of KATP channels in vascular smooth muscle cells. The purpose of present study is to further determine the underlying mechanisms for the interaction of H2S with KATP channel complex. Cloned rvKir6.1 and rvSUR1 cDNAs were transfected into HEK293 cells with standard procedures. Significant inward K⫹ currents were detected in HEK293 cells transfected with rvKir6.1 cDNA alone and these currents had no response to H2S stimulation (0.3 mM). Co-expression of rvKir6.1/rvSUR1 in HEK293 cells yielded a KATP current that was inhibited by increasing intracellular ATP concentration from 0 to 5 mM. At 50 M, H2S increased rvKir6.1/rvSUR1 KATP current from -389.9⫾90.0 to -495.2⫾108.6 pA (-150 mV; p⬍0.05; n⫽6). Thus, H2S stimulates KATP channels via a direct action on rvSUR1 subunit. The excitatory effect of H2S on KATP currents was reversible and could be inhibited by the KATP channel inhibitor glibenclamide (5 M). Intracellular application of the sulfhydryl-specific modifying agent N-ethylmaleimide (NEM) (1 mM) enhanced (p⬍0.01; n⫽5), whereas extracellular application of NEM (1 mM) inhibited, the stimulatory effect of H2S (50 M) on rvKir6.1/ rvSUR1 KATP currents. Our results suggested that, while H2S had no excitatory effect on rvKir6.1 subunit, it amplified the KATP currents at low concentrations by acting on rvSUR1 subunit in co-expressed rvKir6.1/rvSUR1 KATP channels. The stimulatory effect of H2S on extracellularly located sulfhydryl groups on rvSUR1 subunit is predominant under the physiological condition. On the other hand, intracellularly located sulfhydryl groups of rvSUR1 may be inhibited by H2S and the modification of intracellular sulfhydryl group abolished the inhibitory effect of H2S on KATP channels. Further investigations including mutagenesis study are needed to specify which cysteine residue or the disulfide bond between cysteine residues would be modulated by H2S on rvSUR1 subunit. Supported by NSERC and HSURC. Key Words: Hydrogen Sulfide, Sulfhydryl Group, ATP-Sensitive Potassium Channels 0895-7061/02/$22.00
P-204 SODIUM-LITHIUM COUNTERTRANSPORT ACTIVITY IN PROXIMAL TUBULE CELLS Lisa A. Marshall, Mary J. MacLeod, James S. McLay. Medicine & Therapeutics, University of Aberdeen, Aberdeen, United Kingdom. This study was designed to determine whether renal proximal tubule cells display sodium - lithium countertransport (SLC) activity as activity has previously only been described in erythrocytes and more recently human skin fibroblasts. Proximal tubule (PT) cells were isolated from either Sprague-Dawley rats or New Zealand White rabbits by collagenase digestion followed by Percoll density gradient centrifugation. Rat PT cell SLC activity was determined in freshly isolated cell suspensions, rabbit PT cells were either cultured in 6 - well plates and used at Day 7 of culture or the cells were used in suspension. Cells were incubated in 150mM lithium, washed and re-suspended in either sodium-containing or sodium-free media. SLC activity was determined as the difference in lithium efflux between these 2 solutions. In cell suspensions, the Millipore Rapid Filtration Technique was used to determine lithium transport at short time intervals. In the rat PT cell suspensions, lithium uptake was shown to be time and concentration-dependent and the MTT assay confirmed that incubation with lithium was not toxic to the cells. Lithium efflux was significantly greater into 150mM sodium than into choline (0.404 vs 0.115 mmol lithium/mg protein/30s, respectively n⫽3) and a concentration dependency was observed. 0.1mM ouabain produced a slight inhibition of SLC activity (0.289 vs 0.205 mmol lithium/mg protein/30s n⫽3) but 0.1mM dimethylamiloride had no effect (0.325 vs 0.328 mmol lithium/mg protein/30s n⫽3). The transport of lithium was shown to be energy-independent as intracellular ATP depletion did not affect the SLC activity. A similar sodium-dependency was noted in freshly isolated rabbit PT cell suspensions. SLC activity was lower than that in the rat although longer efflux times were studied (0.112mmol lithium/mg protein/6 minutes n⫽3). The cultured rabbit PT cells demonstrated a time and concentrationdependent lithium uptake but lithium efflux was very similar into sodiumcontaining and sodium-free buffers (1.30mmol lithium/mg protein/30s vs 1.47mmol lithium/mg protein/30s, respectively n⫽3). No sodium-dependent lithium efflux was observed. Primary cultures of rabbit PT cells fail to demonstrate SLC activity but the results clearly demonstrate that SLC activity can be measured in freshly isolated renal proximal tubule cells. This is a functionally appropriate cell type in which to investigate SLC activity because of the relationship with essential hypertension. Key Words: Sodium-Lithium Countertransport, Proximal Tubule, Hypertension
P-205 THE ENDOTHELIUM-MODULATED BUT CYCLIC GMP-INDEPENDENT VASORELAXANT EFFECT OF H2S Rui Wang, Weimin Zhao, Xianfeng Sun. Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada. Hydrogen sulfide (H2S) is an important endogenous gas that possesses vasorelaxant property. The interaction of H2S with different cellular signaling pathways in vascular smooth muscle cells (SMCs), however, had not been systematically studied. In the present study, the mechanisms for the H2S-induced relaxation of phenylephrine-precontracted rat aortic tissues were further investigated. Removal of endothelium from vascular tissues reduced the H2S-induced vasorelaxation by shifting to the right of the concentration-response curve of H2S, but did not affect the maximal relaxation responses. Denervation of vascular tissues did not affect the © 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
POSTERS: Cell Membrane Transport/Ion Channels
103A
vasorelaxation induced by H2S. The sodium nitroprusside-induced relaxation of aortic tissues was completely abolished by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor. Instead of inhibition, ODQ potentiated the H2S-induced vasorelaxation. Inhibitors of cAMP, protein kinase C, and cyclo-oxygenase pathways did not affect the H2S-induced relaxation. In the absence of calcium from the bath solution, the relaxant effect of H2S was significantly attenuated. Prior exposure to H2S reduced the relaxant response of vascular tissues to nitric oxide. Our results suggest that the vasorelaxant effect of H2S was largely due to the interaction of H2S with smooth muscle cells and partially mediated by endothelium in an extracellular calcium entry-dependent manner. The mechanisms for the H2S-induced vasorelaxation were different from those of other gaseous vasoactive factors, such as nitric oxide or carbon monoxide that relax vascular smooth muscles by activating cGMP-dependent pathway. The interaction among these endogenous gases may provide fine tune of vascular tone. Supported by HSURC of Saskatchewan, Canada. Key Words: Hydrogen Sulfide, Vasorelaxation, cGMP
P-206 HYDROGEN SULFIDE DIFFERENTIALLY MODULATES SULFHYDRYL GROUPS ON RVSUR1 SUBUNIT IN HEK293 CELLS TRANSFECTED WITH RVKIR6.1/RVSUR1 CDNAS Kun Cao, Bo Jiang, Rui Wang. Physiology, University of Saskatchewan, Saskatoon, SK, Canada; Physiology, University of Saskatchewan, Saskatoon, SK, Canada; Physiology, University of Saskatchewan, Saskatoon, SK, Canada. Hydrogen sulfide (H2S) has been known as an endogenous opener of KATP channels in vascular smooth muscle cells. The purpose of present study is to further determine the underlying mechanisms for the interaction of H2S with KATP channel complex. Cloned rvKir6.1 and rvSUR1 cDNAs were transfected into HEK293 cells with standard procedures. Significant inward K⫹ currents were detected in HEK293 cells transfected with rvKir6.1 cDNA alone and these currents had no response to H2S stimulation (0.3 mM). Co-expression of rvKir6.1/rvSUR1 in HEK293 cells yielded a KATP current that was inhibited by increasing intracellular ATP concentration from 0 to 5 mM. At 50 M, H2S increased rvKir6.1/rvSUR1 KATP current from -389.9⫾90.0 to -495.2⫾108.6 pA (-150 mV; p⬍0.05; n⫽6). Thus, H2S stimulates KATP channels via a direct action on rvSUR1 subunit. The excitatory effect of H2S on KATP currents was reversible and could be inhibited by the KATP channel inhibitor glibenclamide (5 M). Intracellular application of the sulfhydryl-specific modifying agent N-ethylmaleimide (NEM) (1 mM) enhanced (p⬍0.01; n⫽5), whereas extracellular application of NEM (1 mM) inhibited, the stimulatory effect of H2S (50 M) on rvKir6.1/ rvSUR1 KATP currents. Our results suggested that, while H2S had no excitatory effect on rvKir6.1 subunit, it amplified the KATP currents at low concentrations by acting on rvSUR1 subunit in co-expressed rvKir6.1/rvSUR1 KATP channels. The stimulatory effect of H2S on extracellularly located sulfhydryl groups on rvSUR1 subunit is predominant under the physiological condition. On the other hand, intracellularly located sulfhydryl groups of rvSUR1 may be inhibited by H2S and the modification of intracellular sulfhydryl group abolished the inhibitory effect of H2S on KATP channels. Further investigations including mutagenesis study are needed to specify which cysteine residue or the disulfide bond between cysteine residues would be modulated by H2S on rvSUR1 subunit. Supported by NSERC and HSURC. Key Words: Hydrogen Sulfide, Sulfhydryl Group, ATP-Sensitive Potassium Channels 0895-7061/02/$22.00