Biomedical subjects

S G Schultz

Publications and source records attributed to S G Schultz.

At least 19 recordsLinked to original sources

PubMed1992

A peptide from the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membranes of Necturus enterocytes.

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.

Amino Acid Sequence↗

PubMed1992

Effect of trypsin on a Ca(2+)-activated K+ channel reconstituted into planar phospholipid bilayers.

Exposure of the cytoplasmic side of calcium-activated, high (maxi)-conductance potassium [BK(Ca)] channels in basolateral membrane vesicles from rabbit colonocytes incorporated into planar phospholipid bilayers to trypsin rapidly reduces, but does not abolish, the sensitivity of this channel to activation by calcium without affecting its conductance or high selectivity for K+ over Cl-. The results of these studies also indicate that this BK(Ca) channel does not have intrinsic voltage-gating properties but that its voltage sensitivity is related to its ability to interact with calcium. This conclusion is consistent with the model proposed by Moczydlowski and Lattore (J. Gen. Physiol. 82: 511-542, 1983) for the role of membrane voltage in modulating the interaction between calcium and the BK(Ca) channel in rat skeletal muscle.

Animals↗

PubMed1991

Reconstitution of isolated Ca(2+)-activated K+ channel proteins from basolateral membranes of rabbit colonocytes.

Using calmodulin-affinity chromatography, we have isolated a fraction of proteins from solubilized basolateral membranes of rabbit colonocytes which when reconstituted into planar phospholipid bilayers disclosed Ca(2+)-activated single K+ channel activities. The properties of the reconstituted channels are identical to those of native membrane vesicles incorporated into these bilayers with respect to their high selectivity for K+ over C-, high ("maxi") conductance, voltage gating, and inhibition by trifluoperazine. Two-dimensional sodium dodecyl sulfate gel electrophoresis of these proteins revealed three major protein species with molecular masses of 120, 60, and 35 kDa, which constituted 70, 10, and 20%, respectively, of the total protein. The results of other studies strongly suggest that the 35-kDa protein may be the Ca(2+)-activated K+ channel protein in these membranes.

Animals↗

PubMed1989

Reconstitution of a calcium-activated potassium channel in basolateral membranes of rabbit colonocytes into planar lipid bilayers.

A highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989) J. Membrane Biol. 110:147-162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance. Ca2+-activated K+ channel. This channel is highly selective for K+ over Na+ and Cl-, displays voltage-gating similar to "maxi" K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K+ diffusion through the channel involves either the binding of a single K+ ion to a site within the channel or "single-filing" ("multi-ion occupancy"). Channel activity is inhibited by the venom from the scorpion Leiurus quinquestriatus, Ba2+, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed.

Analog-Digital Conversion↗

PubMed1989

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Basolateral membrane vesicles from Necturus enterocytes, highly (greater than 20-fold) enriched in Na+,K+-ATPase, were reconstituted into planar lipid bilayers. The principal channel activity observed is selective for K+ over Na+ and Cl-. This K+ channel is blocked by Ba2+ and Leiurus quinquestriatus venom but is not affected by Ca2+ over the range of 10(-3) to less than 10(-7) M and is not inhibited by charybdotoxin. L. quinquestriatus venom also markedly reduces the conductance of the basolateral membrane of intact villus cells of Necturus small intestine. The open-time probability (Po) of the channel displays a voltage-dependence characteristic of an "inward rectifier"; i.e., the channel inactivates when the basolateral membrane is depolarized and Po increases with increasing hyperpolarization of that barrier. Assuming that similar properties prevail under physiological conditions, this characteristic could provide, in part, an explanation for the parallelism between Na+-pump and K+-leak activities of the basolateral membrane observed in this epithelium. Thus, an increase in rheogenic Na+-pump activity at the basolateral membrane would hyperpolarize that barrier and, in turn, increase the open time of this K+ channel.

Animals↗

PubMed1988

Sodium-coupled glycine uptake by Ehrlich ascites tumor cells results in an increase in cell volume and plasma membrane channel activities.

The addition of 10 mM glycine to a physiological saline bathing Ehrlich ascites tumor cells is followed by a slow increase in cell volume that plateaus between 15 and 30 min at a level approximately equal to 17% greater than the control volume; this increase is not observed when glycine is added to cells suspended in a Na+-free saline. The results of studies using the patch-clamp technique in the cell-attached mode indicate that, 0.5-3 min after the addition of glycine to the bathing solution, there is a marked increase in the activity of single channels, which is almost all instances were previously present and operant in the plasma membrane. Successfully excised patches of membrane that contained a channel stimulated by glycine fell into two categories. Some became inactive within 15 sec in spite of the fact that the G omega seal remained intact. Others persisted for the lifetime of the seal. All of the persistent channels had an 11-fold selectivity for Cl- over K+ and a conductance of 23 pS when bathed by symmetrical 150 mM KCl solutions. Although the ionic specificities of the other channels have not been identified, there is reason to suspect that they might be K+ channels whose activities are dependent on factors lost when the patch is excised. Swelling induced by exposing these cells to a 50% hypotonic perfusate stimulated the activities of Cl- channels whose properties closely resemble those stimulated by the addition of glycine to the perfusate, strongly suggesting that the glycine-induced stimulation of Cl- channel activity is part of a volume-regulatory response to cell swelling. If the increase in channel activity induced by the addition of glycine to the perfusate is indeed a response to cell swelling, then this perfusate is indeed a response to cell swelling, then this volume-regulatory response must be extremely sensitive inasmuch as it appears to be "triggered" by an average increase in cell volume that does not exceed 5%.

Animals↗

PubMed1988

Localization of calmodulin in the enterocyte of Necturus small intestine.

Calmodulin was localized in the small intestine of Necturus maculosa at both the light- and electron-microscopic levels utilizing an affinity-purified, monospecific antibody and immunoperoxidase cytochemistry. The microvilli and basolateral membranes of the epithelium were highly reactive, the cytoplasm was moderately reactive. Connective tissue, endothelia, and muscularis of the submucosa were also reactive. The mitochondrial matrix, nuclear envelope, and terminal web, and mucous granules of goblet cells were unreactive. Omission of the primary antibody, substitution of immunoglobulin not bound to the affinity column or inappropriate immunoglobulin (sheep anti-viral src product) for the primary antibody, caused no immunoreaction product to be formed.

Animals↗

PubMed1987

Cell Na+ activities and transcellular Na+ absorption by descending colon from normal and Na+-deprived rabbits.

The relation between intracellular Na+ activities, (Na)c, determined employing Na+-selective microelectrodes, and the rates of active Na+ absorption, INa, by rabbit descending colon was examined when INa was varied over a wide range by chronic dietary Na+ deprivation. (Na)c averaged 13 mM and was independent of INa over a sixfold range. Further, the ratios of the slope resistance of the apical membrane (rm) to that of the basolateral membrane (rs) (i.e. rm/rs) in low-transporters (control diet) and high-transporters (Na+-deprived) did not differ significantly inspite of the fact that the Na+ conductance of the apical membranes of high-transporters was, on the average, three times greater than that of the low-transporters. These findings, together with the results reported by other laboratories, strongly suggest that the aldosterone-induced increase in the conductance of the apical membrane to Na+ and, in turn, the rate of entry of Na+ into the absorptive cells are followed by parallel increases in the ability of cells to extrude Na+ across the basolateral membrane in the absence of a sustained increase in (Na)c as well as the conductance of that barrier.

Animals↗

PubMed1986

Effect of hypertonicity on the increase in basolateral conductance of Necturus small intestine in response to Na+-sugar cotransport.

Exposure of Necturus small intestine to a galactose-containing perfusate that is 20% hypertonic compared to the galactose-free (control) perfusate results in a rapid depolarization of the electrical potential difference across the apical membrane, psi mc, and a decrease in the ratio of the resistance of the apical membrane to that of the basolateral membrane, (rm/rs); however, the slow repolarization of psi mc and increase in (rm/rs), observed under isotonic conditions, is blocked. These findings are consistent with the notion that the increase in the conductance of the basolateral membrane in response to Na+-coupled sugar (or amino acid) transport across the apical membrane may be a 'volume regulatory response' to cell swelling.

Animals↗

PubMed1986

Current-voltage relations of sodium-coupled sugar transport across the apical membrane of Necturus small intestine.

The current-voltage (I-V) relations of the rheogenic Na-sugar cotransport mechanism at the apical membrane of Necturus small intestine were determined from the relations between the electrical potential difference across the apical membrane, psi mc, and that across the entire epithelium, psi ms, when the latter was varied over the range +/- 200 mV, under steady conditions in the presence of galactose and after the current across the apical membrane carried by the cotransporter, ImSNa, is blocked by the addition of phloridzin to the mucosal solution. ImSNa was found to be strongly dependent upon psi mc over the range -50 mV less than psi mc less than EmSNa where EmSNa is the "zero current" or "reversal" potential. Over the range of values of psi mc encountered under physiological conditions the cotransporter may be modeled as a conductance in series with an electromotive force so that ImSNa = gmSNa (EmSNa - psi mc) where gmSNa is the contribution of this mechanism to the conductance of the apical membrane and is "near constant." In several instances ImSNa "saturated" at large hyperpolarizing or depolarizing values of psi mc. The values of EmSNa determined in the presence of 1, 5, and 15 mM galactose strongly suggest that if the Na-galactose cotransporters are kinetically homogeneous, the stoichiometry of this coupled process is unity. Finally, the shapes of the observed I-V relations are consistent with the predictions of a simple kinetic model which conforms with current notions regarding the mechanico-kinetic properties of this cotransport process.

Animals↗

PubMed1986

Transcellular sodium transport and intracellular sodium activities in rabbit gallbladder.

This study was designed to explore the relation between the rate of transcellular active Na+ transport by rabbit gallbladder epithelium, JNa, and the intracellular Na+ activity, (Na)c; the latter was determined by use of highly selective Na+ microelectrodes. The underlying strategy was based on the well-established observation that JNa is stimulated by the presence of bicarbonate in the bathing solutions. Our results confirm previous observations that the addition of bicarbonate to the bathing solutions results in a twofold increase in JNa. In the absence of bicarbonate, (Na)c averaged 16 mM. Within 2-4 min after the addition of bicarbonate to both bathing solutions, (Na)c increased to an average value of 22 mM and then gradually declined and by 15 min did not differ significantly from the value observed in the absence of bicarbonate. Thus, a twofold increase in JNa is not associated with an increase in (Na)c. These results are in accord with earlier observations on Necturus urinary bladder and small intestine and contradict the notion that an increase in the rate of active Na+ extrusion from the cell across the basolateral membrane in response to an increase in the rate of Na+ entry across the apical membrane is necessarily the result of a sustained increase in (Na)c.

Animals↗

PubMed1986

The application of two-dimensional centrifugation to clinical chemistry testing.

We have developed a new clinical chemistry analyzer, the VISION System, which uses centrifugal force to separate whole blood, measure reagent and plasma volumes, and complete all steps required for a spectrophotometric analysis. The system is based on a multichambered test pack containing liquid reagents, which can be centrifuged in two planes oriented at right angles to each other. The analyzer regulates the temperature, timing and optical measurements for up to 10 different test packs in the same run. We have demonstrated good precision and accuracy on 6 clinical chemistry analytes, 2 enzymes, potassium and theophylline using this system.

Blood Chemical Analysis↗

PubMed1985

Kinetics of the effect of amiloride on the permeability of the apical membrane of rabbit descending colon to sodium.

The effects of the addition of graded concentrations of amiloride, (A)m, to the mucosal bathing solution on the permeability of the apical membrane of rabbit descending colon to Na (PmNa) were determined when the Na activity in the mucosal bathing solution, (Na)m, was 18, 32 or 100 mM. PmNa was obtained from current-voltage relations determined on tissues bathed with a high-K serosal solution before and after the addition of a maximally inhibitory concentration of amiloride to the mucosal solution as described by Turnheim et al. (Turnheim, K., Thompson, S.M., Schultz, S.G. 1983. J. Membrane Biol. 76:299-309). The results indicate that: (1) As demonstrated previously (Turnheim et al., 1983), PmNa decreases with increasing (Na)m. (2) PmNa also decreases hyperbolically with increasing (A)m. Kinetic analyses of the effect of amiloride on PmNa are consistent with the conclusions that: (i) the stoichiometry between the interaction of amiloride with apical membrane receptors that results in a decrease in PmNa is one-for-one; (ii) there is no evidence for cooperativity between amiloride and these binding sites; (iii) the value of (A)m needed to halve PmNa at a fixed (Na)m is 0.6-1.0 microM; and, (iv) this value is independent of (Na)m over the fivefold range studied. These findings are consistent with the notion that the sites with which amiloride interacts to bring about closure of the channels through which Na crosses the apical membrane are kinetically distinct from the sites with which (Na)m interacts to bring about closure (i.e., "self-inhibition"). In short, the effects of (Na)m and (A)m on PmNa in this tissue appear to be independent and additive.

Amiloride↗

PubMed1985

Electrophysiological studies of sodium cotransport in epithelia: toward a cellular model.

During the past two decades, microelectrophysiological studies of small intestine and renal proximal tubule employing conventional as well as ion-selective microelectrodes have contributed significantly to our understanding of the nature of Na-coupled entry processes at the apical membrane as well as the overall workings of the simple model illustrated in FIGURE 1. These studies have unequivocally established the rheogenic and conductive nature of the Na-coupled sugar and amino-acid entry processes across the apical membrane of small intestine (and renal proximal tubule) and have, in addition, disclosed that the properties of the basolateral membrane respond to an increase in Na-coupled solute entry with an increase in the ability of the Na-K pump to extrude Na with little or no change in (Na)c32 and a parallel increase in the conductance of that barrier to K. Although these responses may be "triggered" by cell swelling, it is unclear how a cell "recognizes" minimal swelling and how this recognition, in turn, culminates in the observed changes in basolateral membrane pump-leak properties. Clearly, these findings have brought us to the interfaces between cell physiology and cell and molecular biology and have raised a number of intriguing questions that focus on the more global question: How do epithelial cells work?

Amino Acids↗

PubMed1985

Two-dimensional centrifugation for desk-top clinical chemistry.

We have developed a new system for clinical chemistry analysis, the Vision System, in which centrifugal force is used to separate whole blood, measure reagent and plasma volumes, and complete all steps required for a spectrophotometric analysis. The system is based on use of a multichambered plastic test pack containing liquid reagents, which can be centrifuged at 500 X g in two planes, oriented at right angles to each other. Alternating centrifugal fields allows liquid reagents and plasma to flow into highly precise measuring and mixing chambers. A unique flash lamp and diode array spectrometer provide for optical measurements of 10 test packs at as many as eight wavelengths simultaneously. The temperature of each individual test pack is controlled by using a flash lamp coupled to a liquid crystal temperature sensor. Microprocessor control allows as many as 10 different chemistry reactions to be measured simultaneously on whole-blood, plasma, or serum samples. Comparison with results by an established batch-photometric analyzer demonstrated excellent precision and accuracy for various clinical chemistry tests.

Blood Chemical Analysis↗

PubMed1979

Intracellular chloride activities and active chloride absorption in the intestinal epithelium of the winter flounder.

Intracellular chloride activities, (Cl)c, and the electrical potential difference across the mucosal membrane, psimc, were determined in the isolated small intestine of the winter flounder, using Cl-selective and conventional (KCl-filled) microelectrodes. In the presence of a Na-containing buffer psimc averages -69 mV and (Cl)c averages 24 mM, a value that is 3.4 times that predicted for an equilibrium distribution across the mucosal membrane. On the other hand, when the tissue is then perfused with Na-free buffer, (Cl)c slowly falls to a value that does not differ significantly from that predicted for an equilibrium distribution, and psimc depolarizes significantly. Finally, when the tissue is again bathed in the Na-containing buffer, (Cl)c rapidly returns to a value well above equilibrium. These results, together with those of Frizzell et al. (J. Membrane Biol. 46:27, 1979), provide direct evidence that: (1) Cl is accumulated against its electrochemical potential difference (32 mV) by this tissue, and (2) this accumulation is coupled to and energized by the entry of Na down its steep electrochemical potential difference.

Animals↗

PubMed1979

Sodium-coupled chloride transport by epithelial tissues.

There is compelling evidence that active Cl absorption by a variety of epithelia, widely distributed throughout the animal kingdom, is the result of an electrically neutral Na-coupled transport process at the luminal membrane and that the energy for transcellular Cl movement is derived from the Na gradient across that barrier. These co-transport processes are found predominantly in "leaky" or "moderately leaky" epithelia and permit these tissues to absorb Na and Cl with high degrees of efficacy. In addition, there is a growing body of evidence that cyclic AMP and Ca-induced electrogenic Cl secretion by a wide variety of epithelia may involve electrically neutral, Na-coupled Cl entry across the contraluminal membrane and that the energy for these secretory processes is derived from the Na-gradient across that barrier. A model for electrogenic Cl secretion that accounts for the available data is presented.

Absorption↗

PubMed1979

Models of electrolyte absorption and secretion by gastrointestinal epithelia.

An attempt has been made to account for the electrolyte transport properties of several gastrointestinal epithelia in terms of relatively simple cell models. In doing so, it becomes apparent that the complex patterns of electrolyte absorption and secretion by tissues such as in vitro rabbit ileum may actually represent a combination or superposition of several basic transport processes that can be more readily identified in other epithelia. Thus, attempts to explain the effects of agents such as cyclic AMP in terms of a single mechanism of action may prove to be unproductive for such tissues. Clearly, our approach has involved certain speculations and, undoubtedly, oversimplifications. Only those processes which appear to be responsible for sodium and chloride transport have been dealt with in any detail; much remains to be learned about the processes responsible for the absorptive and secretory movements of other ions, principally HCO3. However, this analysis may be useful in bringing a degree of uniformity to a complex area and, hopefully, has identified certain important gaps in our understanding of electrolyte transport at the cellular level which require further investigation.

Animals↗