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Biomedical subjects

B Sacktor

Publications and source records attributed to B Sacktor.

At least 109 records · Page 6Linked to original sources
PubMed1978

Membrane potential-sensitive fluorescence changes during Na+-dependent D-glucose transport in renal brush border membrane vesicles.

When D-glucose was added to a suspension of renal brush border membrane vesicles equilibrated in a Na+-containing medium, there was a rapid transient increase in the fluorescence of the probe, 3,3'-dipropyl thiodicarbocyanine iodide (DiS-C3-(5)). This sugar-induced response was stereospecific for the D isomer, dependent on Na+, inhibited by phlorizin, and blocked by ionophores, valinomycin plus nigericin, which dissipate ionic gradients. The enhancement in fluorescence suggests the entrance into the vesicle of Na+, cotransported with the sugar. This would lead to the interior of the membrane vesicle becoming more positive, resulting in depolarization of the membrane potential. That the sugar induced the transport of Na+ was confirmed by direct measurement of 22Na+ uptake. Thus, the Na+-sugar co-transport system provides a mechanism for D-glucose to stimulate the flux of Na+ as well as for the Na+ electrochemical gradient to enhance the transport of D-glucose.

Animals↗
PubMed1978

The sodium electrochemical potential-mediated uphill transport of D-glucose in renal brush border membrane vesicles.

The uphill transport of D-glucose in renal brush border membrane vesicles was correlated with the Na+ electrochemical gradient. Each component of the electrochemical potential, the membrane potential (outside positive) or the Na+ chemical gradient, when assayed independently, supported the concentrative uptake of the sugar. When the two components were combined, the rates of D-glucose uptake were additive. Accumulation of D-glucose as a function of various Na+ gradients, in the absence of a membrane potential, suggests a 1:1 stoichiometry between sugar and Na+ uptake. These findings are consistent with the role of ionic gradients in energizing uphill solute transport.

Animals↗
PubMed1978

The stimulation by catecholamines of guanylate cyclase activity in a cell-free system.

Cytosolic guanylate cylase activity in cell-free preparations of the rabbit renal cortex was increased 3- to 5-fold by catecholamines. The plasma membrane-bound enzyme was not activated, although hormone receptors were present. Stimulation was augmented by NaN3, which by itself had little effect on the soluble enzyme activity. With a partially purified enzyme, activity was enhanced by 0.1 muM 1-epinephrine and activated half-maximally by about 1 muM. In decreasing potency, epinephrine greater than isoproterenol greater than norepinephrine greater than dopamine greater than catechol. Phenylephrine and metanephrine did not stimulate. 1-Epinephrine-stimulation of the enzyme was reversed by dialysis and the deactivated enzyme was reactivatable by a second exposure to the catecholamine. Activation by catecholamines was not stereospecific. Epinephrine-stimulated guanylate cyclase activity in the crude cytosolic fraction was partially inhibited by alpha-adrenergic antagonists, but neither alpha- nor beta-blockers inhibited when the partially purified enzyme was used; thus, leaving open the question of a role for typical alpha- or beta-adrenergic mechanisms in this regulation of the soluble enzyme. Adrenochrome was the most potent activator of the partially purified guanylate cyclase, being approximately 10-times more effective than epinephrine. Epinephrine and adrenochrome activated in the presence of reducing agents, i.e., ascorbate, DTT and N2, although the enzyme in a more SH-reduced form and in an oxygen-deficient medium had a decreased sensitivity to both effectors. Epinephrine activated soluble guanylate cyclase in several tissues, including cerebrum, cerebellum, brain stem, lung, heart, liver, ductus deferens and colon. Although the precise mechanism by which low concentrations of catecholamines stimulated guanylate cyclase activity is unknown and the physiological significance of the activation remains to be established, these findings direct attention to an interesting interaction of catecholamines with the cytosolic enzyme system and stress the need for further studies.

Adrenochrome↗
PubMed1977

Preparation of renal cortex basal-lateral and bursh border membranes. Localization of adenylate cyclase and guanylate cyclase activities.

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and gamma-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na+ + K+1)-ATPase. However, the specific activity of (Na+ + K+)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na+ + K+)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na+ + K+)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5'-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN3 plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na+ + K+)-ATPase, be used as an enzyme "marker" for the renal basal-lateral membrane.

Adenosine Triphosphate↗
PubMed1977

Transport of amino acids in renal brush border membrane vesicles. Uptake of the neutral amino acid L-alanine.

The transport of L-alanine was studied using membrane vesicles derived from the brush borders of the rabbit renal proximal tubule. Preincubation of the renal membranes with L-alanine, but not D-alanine, accelerated exchange diffusion of L-alanine, i.e. stereospecific counter transport. The equilibrium uptake of L-alanine decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, i.e. zero intravesicular space, indicates no uptake. These findings demonstrate that the uptake of L-alanine represents transport into membrane vesicles and not surface binding to the membrane. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium stimulated L-alanine uptake. Accumulation of the amino acid in the vesicles was maximal at 5 min and then decreased, indicating efflux. The final level of uptake in the presence of the Na+ gradient was identical with that obtained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the "overshoot" the uptake of L-alanine was slmost twice the final equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of L-alanine into renal brush border membrane vesicles against its concentration gradient. Stimulation of L-alanine uptake was specific for Na+. When the intravesicular medium contained no Na+, the rate of uptake enhanced with increases in the concentration of Na+ in the external medium. Increasing the Na+ gradient lowered the apparent Km for L-alanine. In the absence of the Na+ gradient, the rates of uptake of L- and D-alanine were identical. In the presence of the Na+ gradient, the rate of D-alanine uptake was stimulated, but significantly less than that of L-alanine. The uptake of L-alanine, at a given concentration of amino acid reflected the sum of the contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at about 2 mM L-alanine. The independent system exhibited minimal saturability and may itself represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations of L-alanine, the rate of the Na+ gradient-dependent uptake was 5-fold that in the absence of the gradient. Valinomycin enhanced the Na+ gradient-dependent uptake of L-alanine, provided a K+ gradient (vesicle greater than medium) was present. This finding indicates that the Na+ gradient-dependent transport of L-alanine into renal brush border membrane vesicles is an electrogenic process and suggests that the membrane potential is a determinant of L-alanine transport. In the presence of a Na+ gradient, the uptake of L-alanine was strongly inhibited by other neutral L-amino-acids. Imino acids and glycine also inhibited, but acidic and basic amino acids were without effect. In the absence of a Na+ gradient, little selective competition was found...

Alanine↗
PubMed1977

Transport of amino acids in renal brush border membrane vesicles. Uptake of L-proline.

The findings (a) stereospecific counter transport; (b) equilibrium uptake of L-proline decreased with increasing medium osmolarity; and (c) L-proline and Na+ were taken up into identical intravesicular spaces, indicate that uptake of L-proline by rabbit renal brush border membranes represents transport into membrane vesicles, not surface binding to the membrane. An Na+ gradient between extravesicular and intravesicular media stimulated the initial rate of L-proline uptake about 10 times. Accumulation of the imino acid was maximal at 2 min, then decreased until the equilibrium level was attained. At the peak of this "overshoot" uptake of L-proline was 3-fold greater than the final equilibrium value. These results suggest that the electrochemical Na+ gradient drives the transient movement of L-proline into the membrane vesicles against its concentration gradient. Stimulation of L-proline uptake was specific for Na+. Increasing the Na+ gradient lowered the apparent Km for L-proline. Efflux of L-proline from the membrane vesicles, like uptake, showed stimulatory effects when the Na+ gradient and L-proline were on the same side, and inhibitory effects when the Na+ gradient and the imino acid were on opposite sides of the membrane. Uptake of L-proline, at a given concentration, reflected the sum of contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at 4 mM L-proline. The independent system did not saturate but may represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations the rate of the Na+ gradient-dependent uptake was 5 times that in the absence of the gradient. In K+-loaded vesicles, valinomycin, but not nigericin, enhanced the Na+ gradient-dependent uptake of L-proline. Gramicidin diminished uptake. These findings indicate that the Na+ gradient-dependent transport of L-proline is an electrogenic process and suggest that the membrane potential is a determinant of L-proline transport. The Na+ gradient-dependent rate of L-proline uptake was strongly inhibited by other imino acids, suggesting that L-imino acids have a common transport system. Glycine and neutral amino acids inhibited the rate of L-proline uptake moderately, acidic amino acid and D-glucose were very weak inhibitors, and basic amino acids were without effect. In the absence of Na+, the rate of L-proline uptake was independent of the presence of other amino acids. These findings indicate that the brush border membrane is a site of amino acid recognition during vectorial transepithelial transport. It is proposed that imino and neutral amino acids inhibit transport of L-proline by competitive interaction with the L-proline "carriers" in addition to competition for the electrochemical Na+ gradient or membrane potential. The findings are relevant to the understanding of genetic amino acid transport disorders, such as iminoglycinuria.

Amino Acids↗