Genetic control of renal thiazide receptor response to dietary NaCl and hypertension. (1/196)

Excess NaCl increases blood pressure in some strains of animals but not others. An 8% NaCl diet did not change renal thiazide receptor (TZR) density in two salt-resistant normotensive rat strains (Wistar-Kyoto and Sprague-Dawley) [Fanestil, D. D., D. A. Vaughn, and P. Blakely. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R1241-R1245, 1997]. However, the renal response to salt differs in normal and hypertensive kidneys [Rettig, R., N. Bandelow, O. Patschan, B. Kuttler, B. Frey, and A. Uber. J. Hum. Hypertens. 10: 641-644, 1996]. Therefore, we examined two strains with salt-aggravated hypertension. Renal TZR did not change when Dahl-S (salt sensitive) animals became hypertensive with 8% dietary NaCl. In contrast, renal TZR decreased 34%, whereas blood pressure increased further, in SHR with 8% dietary NaCl. Blood pressure increased after NG-nitro-L-arginine in SHR, but renal TZR did not change, indicating the salt-induced decrease in TZR in SHR cannot be attributed nonspecifically to elevated arterial pressure. We conclude that the renal response to NaCl-induced increases in blood pressure can be genetically modulated independently of the genes that mediate either the primary hypertension or the salt sensitivity of the hypertension. This finding may be of use in future studies directed at identifying genotypes associated with salt-dependent hypertension.  (+info)

Dietary magnesium, not calcium, regulates renal thiazide receptor. (2/196)

This study reports for the first time a relationship between dietary Mg and the renal thiazide-sensitive Na-Cl cotransporter (TZR, measured by saturation binding with 3H-metolazone). Ion-selective electrodes measured plasma ionized magnesium (PMg++), calcium (PCa++), and potassium (PK+). Restricting dietary Mg for 1 wk decreased PMg++ 18%, TZR 25%, and renal excretion of magnesium (UMg) and calcium (UCa) more than 50% without changing PCa++, PK+, or plasma aldosterone. A low Mg diet for 1 d significantly decreased PMg++, TZR, UMg and UCa. Return of dietary Mg after 5 d of Mg restriction restored PMg++ and TZR toward normal. In the control, Mg-deficient, and Mg-repleting animals, TZR correlated with PMg++ (r = 0.86) and with UMg (r = 0.87) but not UCa (r = 0.09). Increasing oral intake of Mg for 1 wk increased PMg++ 14%, TZR 32%, UMg 74%, and UCa more than fourfold without changing PCa++ or PK+. In contrast, increasing dietary Ca content from 0.02% to 1.91% did not change TZR, but increased UCa fivefold without changing PCa++. Hormonal mediators (if any) involved in the relationship between dietary Mg and TZR remain to be elucidated, as does the relationship between TZR and tubular reabsorption of Mg.  (+info)

A kinetic model of the thiazide-sensitive Na-Cl cotransporter. (3/196)

The aim of this study was to construct a numerical model of the thiazide-sensitive Na-Cl cotransporter (TSC) that can predict kinetics of thiazide binding and substrate transport of TSC. We hypothesized that the mechanisms underlying these kinetic properties can be approximated by a state diagram in which the transporter has two binding sites, one for sodium and another for chloride and thiazide. On the basis of the state diagram, a system of linear equations that should be satisfied in the steady state was postulated. Numerical solution of these equations yielded model prediction of kinetics of thiazide binding and substrate transport. Rate constants, which determine transitional rates between states, were systematically adjusted to minimize a penalty function that was devised to quantitatively estimate the difference between model predictions and experimental results. With the resultant rate constants, the model could simulate the following experimental results: 1) dissociation constant of thiazide in the absence of sodium and chloride; 2) inhibitory effect of chloride on thiazide binding; 3) stimulatory effect of sodium on thiazide binding; 4) combined effects of sodium and chloride on thiazide binding; 5) dependence of sodium influx on extracellular sodium and chloride; and 6) inhibition of sodium influx by extracellular thiazide. We conclude that known kinetic properties of TSC can be predicted by a model which is based on a state diagram.  (+info)

Altered expression of Na transporters NHE-3, NaPi-II, Na-K-ATPase, BSC-1, and TSC in CRF rat kidneys. (4/196)

In chronic renal failure (CRF), reduction in renal mass leads to an increase in the filtration rates of the remaining nephrons and increased excretion of sodium per nephron. To address the mechanisms involved in the increased sodium excretion, we determined the total kidney levels and the densities per nephron of the major renal NaCl transporters in rats with experimental CRF. Two weeks after 5/6 nephrectomy (reducing the total number of nephrons to approximately 24 +/- 8%), the rats were azotemic and displayed increased Na excretion. Semiquantitative immunoblotting revealed significant reduction in the total kidney levels of the proximal tubule Na transporters NHE-3 (48% of control), NaPi-II (13%), and Na-K-ATPase (30%). However, the densities per nephron of NHE-3, NaPi-II, and Na-K-ATPase were not significantly altered in remnant kidneys, despite the extensive hypertrophy of remaining nephrons. Immunocytochemistry confirmed the reduction in NHE-3 and Na-K-ATPase labeling densities in the proximal tubule. In contrast, there was no significant reduction in the total kidney levels of the thick ascending limb and distal convoluted tubule NaCl transporters BSC-1 and TSC, respectively. This corresponded to a 3.6 and 2.5-fold increase in densities per nephron, respectively (confirmed by immunocytochemistry). In conclusion, in this rat CRF model: 1) increased fractional sodium excretion is associated with altered expression of proximal tubule Na transporter expression (NHE-3, NaPi-II, and Na-K-ATPase), consistent with glomerulotubular imbalance in the face of increased single-nephron glomerular filtration rate; and 2) compensatory increases in BSC-1 and TSC expression per nephron occur in distal segments.  (+info)

GAT1 (GABA:Na+:Cl-) cotransport function. Steady state studies in giant Xenopus oocyte membrane patches. (5/196)

Neurotransmitter transporters are reported to mediate transmembrane ion movements that are poorly coupled to neurotransmitter transport and to exhibit complex "channel-like" behaviors that challenge the classical "alternating access" transport model. To test alternative models, and to develop an improved model for the Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporter, GAT1, we expressed GAT1 in Xenopus oocytes and analyzed its function in detail in giant membrane patches. We detected no Na+- or Cl--dependent currents in the absence of GABA, nor did we detect activating effects of substrates added to the trans side. Outward GAT1 current ("reverse" transport mode) requires the presence of all three substrates on the cytoplasmic side. Inward GAT1 current ("forward" transport mode) can be partially activated by GABA and Na+ on the extracellular (pipette) side in the nominal absence of Cl-. With all three substrates on both membrane sides, reversal potentials defined with specific GAT1 inhibitors are consistent with the proposed stoichiometry of 1GABA:2Na+:1Cl-. As predicted for the "alternating access" model, addition of a substrate to the trans side (120 mM extracellular Na+) decreases the half-maximal concentration for activation of current by a substrate on the cis side (cytoplasmic GABA). In the presence of extracellular Na+, the half-maximal cytoplasmic GABA concentration is increased by decreasing cytoplasmic Cl-. In the absence of extracellular Na+, half-maximal cytoplasmic substrate concentrations (8 mM Cl-, 2 mM GABA, 60 mM Na+) do not change when cosubstrate concentrations are reduced, with the exception that reducing cytoplasmic Cl- increases the half-maximal cytoplasmic Na+ concentration. The forward GAT1 current (i.e., inward current with all extracellular substrates present) is inhibited monotonically by cytoplasmic Cl- (Ki, 8 mM); cytoplasmic Na+ and cytoplasmic GABA are without effect in the absence of cytoplasmic Cl-. In the absence of extracellular Na+, current-voltage relations for reverse transport current (i.e., outward current with all cytoplasmic substrates present) can be approximated by shallow exponential functions whose slopes are consistent with rate-limiting steps moving 0.15-0.3 equivalent charges. The slopes of current-voltage relations change only little when current is reduced four- to eightfold by lowering each cosubstrate concentration; they increase twofold upon addition of 100 mM Na+ to the extracellular (pipette) side.  (+info)

GAT1 (GABA:Na+:Cl-) cotransport function. Kinetic studies in giant Xenopus oocyte membrane patches. (6/196)

To explain cotransport function, the "alternating access" model requires that conformational changes of the empty transporter allow substrates to bind alternatively on opposite membrane sides. To test this principle for the GAT1 (GABA:Na+:Cl-) cotransporter, we have analyzed how its charge-moving partial reactions depend on substrates on both membrane sides in giant Xenopus oocyte membrane patches. (a) "Slow" charge movements, which require extracellular Na+ and probably reflect occlusion of Na+ by GAT1, were defined in three ways with similar results: by application of the high-affinity GAT1 blocker (NO-711), by application of a high concentration (120 mM) of cytoplasmic Cl-, and by removal of extracellular Na+ via pipette perfusion. (b) Three results indicate that cytoplasmic Cl- and extracellular Na+ bind to the transporter in a mutually exclusive fashion: first, cytoplasmic Cl- (5-140 mM) shifts the voltage dependence of the slow charge movement to more negative potentials, specifically by slowing its "forward" rate (i.e., extracellular Na+ occlusion); second, rapid application of cytoplasmic Cl- induces an outward current transient that requires extracellular Na+, consistent with extracellular Na+ being forced out of its binding site; third, fast charge-moving reactions, which can be monitored as a capacitance, are "immobilized" both by cytoplasmic Cl- binding and by extracellular Na+ occlusion (i.e., by the slow charge movement). (c) In the absence of extracellular Na+, three fast (submillisecond) charge movements have been identified, but no slow components. The addition of cytoplasmic Cl- suppresses two components (tau < 1 ms and 13 micros) and enables a faster component (tau < 1 micros). (d) We failed to identify charge movements of fully loaded GAT1 transporters (i.e., with all substrates on both sides). (e) Under zero-trans conditions, inward (forward) GAT1 current shows pronounced pre-steady state transients, while outward (reverse) GAT1 current does not. (f) Turnover rates for reverse GAT1 transport (33 degrees C), calculated from the ratio of steady state current magnitude to total charge movement magnitude, can exceed 60 s(-1) at positive potentials.  (+info)

GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model. (7/196)

We have developed an alternating access transport model that accounts well for GAT1 (GABA:Na+:Cl-) cotransport function in Xenopus oocyte membranes. To do so, many alternative models were fitted to a database on GAT1 function, and discrepancies were analyzed. The model assumes that GAT1 exists predominantly in two states, Ein and E(out). In the Ein state, one chloride and two sodium ions can bind sequentially from the cytoplasmic side. In the Eout state, one sodium ion is occluded within the transporter, and one chloride, one sodium, and one gamma-aminobutyric acid (GABA) molecule can bind from the extracellular side. When Ein sites are empty, a transition to the Eout state opens binding sites to the outside and occludes one extracellular sodium ion. This conformational change is the major electrogenic GAT1 reaction, and it rate-limits forward transport (i.e., GABA uptake) at 0 mV. From the Eout state, one GABA can be translocated with one sodium ion to the cytoplasmic side, thereby forming the *Ein state. Thereafter, an extracellular chloride ion can be translocated and the occluded sodium ion released to the cytoplasm, which returns the transporter to the Ein state. GABA-GABA exchange can occur in the absence of extracellular chloride, but a chloride ion must be transported to complete a forward transport cycle. In the reverse transport cycle, one cytoplasmic chloride ion binds first to the Ein state, followed by two sodium ions. One chloride ion and one sodium ion are occluded together, and thereafter the second sodium ion and GABA are occluded and translocated. The weak voltage dependence of these reactions determines the slopes of outward current-voltage relations. Experimental results that are simulated accurately include (a) all current-voltage relations, (b) all substrate dependencies described to date, (c) cis-cis and cis-trans substrate interactions, (d) charge movements in the absence of transport current, (e) dependencies of charge movement kinetics on substrate concentrations, (f) pre-steady state current transients in the presence of substrates, (g) substrate-induced capacitance changes, (h) GABA-GABA exchange, and (i) the existence of inward transport current and GABA-GABA exchange in the nominal absence of extracellular chloride.  (+info)

The pathophysiological and molecular basis of Bartter's and Gitelman's syndromes. (8/196)

Molecular defects affecting the transport of sodium, potassium and chloride in the nephron through the ROMK K+ channel, Na+/K+/2Cl- cotransporter, the Na+/Cl- cotransporter and chloride channel have been identified in patients with Bartter's and Gitelman's syndromes. Defects of the angiotensin II type I receptor and CFTR have also being described. These defects are simple (i.e., most are single amino acid substitutions) but affect key elements in tubular transport. The simplicity of the genetic defects may explain why the inheritance of these conditions remains unclear in most kindreds (i.e., not just recessive or dominant) and emphasises the crucial importance of the conformational structure of these channels. Application of this molecular information will allow the early genetic identification of patients with these syndromes and enable us to differentiate between the various disorders at a functional level. It may also identify a subgroup in which the heterozygous form may make patients potentially exquisitely sensitive to diuretics.  (+info)