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K D Philipson

Publications and source records attributed to K D Philipson.

At least 91 records · Page 5Linked to original sources
PubMed1990

Influence of phospholipid fatty acyl composition on sarcolemmal and sarcoplasmic reticular cation transporters.

Using solubilization/reconstitution techniques, we have investigated the influence of membrane fatty acyl composition on the activities of sarcolemmal and sarcoplasmic reticular transporters. The sarcolemmal Na(+)-Ca2+ exchanger and Na+, K(+)-ATPase and the sarcoplasmic reticular Ca2(+)-ATPase were reconstituted into phosphatidylcholine:phosphatidylserine:cholesterol (30:50:20% by weight) proteoliposomes of defined fatty acyl composition. Transport activities varied considerably with phospholipid fatty acyl composition. Quite strikingly, the dependence on membrane fatty acyl composition for all three transporters was identical.

Animals↗
PubMed1990

Identification of the cardiac sarcolemmal Na(+)-Ca2+ exchanger using monoclonal antibodies.

We have previously partially purified the sarcolemmal Na(+)-Ca2+ exchange protein and produced rabbit polyclonal antibodies to the exchanger (Philipson, K.D., Longoni, S., Ward, R. 1988. Biochim. Biophys. Acta 945:298-306). We now describe the generation of three stable murine hybridoma lines which secrete monoclonal antibodies (MAb's) to the exchanger. These MAb's immunoprecipitate 50-75% of solubilized Na(+)-Ca2+ exchange activity. The MAb's appear to be reactive with native conformation-dependent epitopes on the Na(+)-Ca2+ exchanger since they do not react on immunoblots. An indirect method was used to identify Na(+)-Ca2+ exchange proteins. A column containing Na(+)-Ca2+ exchanger immobilized by MAb's was used to affinity purify the rabbit polyclonal antibody. The affinity-purified polyclonal antibody reacted with proteins of apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our previous association of Na(+)-Ca2+ exchange with these proteins.

Animals↗
PubMed1989

Influence of sterols and phospholipids on sarcolemmal and sarcoplasmic reticular cation transporters.

We have examined the influence of different sterols and phospholipids on the activities of the cardiac sarcolemmal Na+-Ca2+ exchanger and Na+,K+-ATPase and the sarcoplasmic reticular Ca2+-ATPase in reconstituted proteoliposomes. When either the solubilized Na+-Ca2+ exchanger or the Na+,K+-ATPase is reconstituted into phosphatidylcholine (PC):phosphatidylserine (30:50 by weight) vesicles, high cholesterol levels (20% by weight) are required for activity to be expressed. This sterol requirement is highly specific for cholesterol. Several cholesterol analogues with minor structural changes are unable to support Na+-Ca2+ exchange or Na+,K+-ATPase activities. When solubilized sarcolemma is reconstituted into PC:cardiolipin vesicles, however, the requirement for cholesterol is lost. Substantial activity can be obtained in the complete absence of cholesterol or in the presence of several cholesterol analogues. Thus, sterol/protein interactions can be highly dependent on the phospholipid environment. In contrast, the skeletal muscle sarcoplasmic reticular Ca2+-ATPase functions equally well in the presence or absence of cholesterol after reconstitution into either PC:phosphatidylserine or PC:cardiolipin proteoliposomes. Phospholipid requirements of the transporters were also examined. The sarcolemmal Na+-Ca2+ exchanger, Na+,K+-ATPase, and the sarcoplasmic reticular Ca2+-ATPase all function optimally in the presence of phosphatidylserine or cardiolipin after reconstitution. Thus, the sarcolemmal cation transporters have similar sterol and phospholipid requirements and may have structural similarities in their hydrophobic regions. The sarcoplasmic reticular Ca2+ pump evolved in a low cholesterol membrane and has different lipid interactions. These findings may have general applicability to other plasma membrane and endoplasmic reticular enzymes.

Animals↗
PubMed1989

Functional characteristics of the cardiac sarcolemmal monocarboxylate transporter.

We have previously shown that a mechanism for transporting L-lactate is located in cardiac sarcolemmal membranes (Am. J. Physiol. 252:C483-C489, 1987). This mechanism has now been shown to transport pyruvate also. The transporter recognizes a wide range of monocarboxylic acids with chain lengths of three to six carbons, as evidenced by their ability to inhibit L-lactate uptake into sarcolemmal vesicles. The ability of the monocarboxylate analogues to inhibit depends strongly on the nature of substituents, particularly at the second carbon. L-lactate and pyruvate transport are not affected by dicarboxylates other than oxaloacetate. The transporter is inhibited by the protein modifiers diethylpyrocarbonate, dinitrofluorobenzene, and phenylisothiocyanate. Diethylpyrocarbonate inhibition is not reversed by hydroxylamine, nor is dinitrofluorobenzene inhibition reversed by thiol reagents, suggesting that the target residues are not histidine, or tyrosine or cysteine, respectively. Several monocarboxylates effectively protect the transporter from inhibition by the modifying reagents, suggesting that the modified residue(s) may be at or near the binding site. Alternatively, the target amino acid(s) in the transport protein may become inaccessible due to a conformation change triggered by the substrate analogues. Overall, the results suggest that a sensitive free amino group, associated with substrate binding, is attacked by the protein-modifying reagents.

Animals↗
PubMed1989

Developmental changes of sarcolemmal Na+-H+ exchange.

We previously demonstrated that the effect of respiratory acidosis on cardiac contractility in the newborn was less than in the adult rabbit, and these data suggested a higher [Na+]i and [Na+]i-[Ca2+]o exchange in the newborn as compared to the adult. In this study, we investigated developmental changes of Na+-H+ exchange in isolated sarcolemmal vesicles. Sarcolemmal purification for Na+-K ATPase was 61.9 and 67.1 fold in the newborn and the adult rabbit heart, respectively. In the presence of an outwardly directed proton gradient across the vesicular membrane, sarcolemmal 22Na uptake rate in the newborn (0.22 +/- 0.01 nmol Na+/mg prot/s) was significantly higher than than in the adult (0.16 +/- 0.01 nmol Na+/mg prot/s). 1.0 mM amiloride inhibited 22Na uptake by 75% and 80% in the newborn and the adult, respectively. In the absence of a pH gradient, vesicular 22Na uptake in the newborn and the adult were not significantly different. In conclusion, the higher Na+-H+ exchange in the newborn may lead to a higher [Na+]i and subsequent calcium influx via Na+-Ca2+ exchange as compared with the adult during acidosis. This may explain the greater recovery of mechanical function in the newborn heart as compared to the adult heart during acidosis.

4-Nitrophenylphosphatase↗
PubMed1989

Ouabain treatment of cardiac cells induces enhanced Na+-Ca2+ exchange activity.

Inhibition of the cardiac Na+-K+-ATPase with cardiac glycosides causes a rise in internal Na+ and a subsequent increase in cellular Ca2+ due to the sarcolemmal Na+-Ca2+ exchange mechanism. We investigated the adaptation of cultured cardiac cells to prolonged elevation of internal Ca2+ after exposure to ouabain. Cultured neonatal rat heart cells were treated with 100 microM ouabain for 4-48 h. This ouabain concentration inhibited Na+-K+-ATPase activity by approximately 45% and caused modest cellular Ca2+ loading. We found that cells adapted to ouabain treatment by increasing the amount of sarcolemmal Na+-Ca2+ exchange activity by 50-90% over a 24-h period. Kinetic and immunological data indicate that the increase was due to increased numbers of functional exchangers. Neither total cellular nor total sarcolemmal protein content was affected by the ouabain treatment. These results may be relevant toward understanding the effects of therapeutic use of cardiac glycosides.

Animals↗
PubMed1989

Effect of ischemia on sarcolemmal Na+-Ca2+ exchange in neonatal hearts.

We studied the effect of cardiac ischemia on sarcolemmal enzymes, Na+-Ca2+ exchange, Ca2+ binding, and Ca2+ efflux in the newborn and adult rabbit. Rabbit ventricle was made ischemic by incubation in hypoxic, glucose-free Tyrode solution at 37 degrees C for 60-120 min. Ischemia inhibited Na+-K+-ATPase and K+-p-nitrophenylphosphatase (PNPPase) activity in the adult myocardium more than in the newborn. In the oxygenated (control) hearts, Na+-Ca2+-exchange activity in the newborn sarcolemma [Michaelis constant (Km) 18 microM; maximum velocity (Vmax) 33] was similar to that in the adult (Km = 16 microM, Vmax = 32). After 60 min ischemia, however, Na+-Ca2+ exchange in the newborn (Km = 16 microM, Vmax = 18) was inhibited less than in the adult (Km = 25 microM, Vmax = 18). In the two age groups, Ca2+ binding and efflux rate were not increased after ischemia, which suggested that Ca2+ permeability did not increase during ischemia. In conclusion, ischemia inhibited sarcolemmal enzymes and Na+-Ca2+ exchange in the newborn less than in the adult, and this lesser inhibition might contribute to or be caused by the greater tolerance of the newborn heart to ischemia.

Adenosine Triphosphatases↗
PubMed1988

Purification of the cardiac Na+-Ca2+ exchange protein.

We have used fractionation procedures to enrich solubilized cardiac sarcolemma in the Na+-Ca2+ exchange protein. Sarcolemma is extracted with an alkaline medium to remove peripheral proteins and is then solubilized with decylmaltoside. Next, the exchanger is applied to DEAE-Sepharose and eluted with high salt. The DEAE fraction is applied to WGA-agarose, and a small fraction of protein, enriched in the exchanger, can be eluted by changing the detergent to Triton X-100. This fraction is reconstituted into asolectin proteoliposomes for measurement of Na+-Ca2+ exchange activity and gel electrophoresis. The purified fraction has a Na+-Ca2+ exchange activity of 600 nmol Ca2+/mg of protein per s at 10 microM Ca2+ and a purification factor of about 30 as compared with control reconstituted sarcolemmal vesicles. Ca2+-Ca2+ exchange and Na+-Ca2+ exchange activities were both present in the same final reconstituted vesicles indicating that the same protein is responsible for both transport activities. SDS-PAGE reveals two prominent protein bands at 70 and 120 kDa. After mild chymotrypsin treatment (1 microgram/ml), there is no loss of exchange activity, but the 120 kDa band disappears and the 70 kDa band becomes more dense. This suggests that the 70 kDa band is due to an active proteolytic fragment of the 120 kDa protein. Under non-reducing gel conditions, only a single protein band is seen with an apparent molecular weight of 160 kDa. Antibodies to the purified exchanger preparation are able to immunoprecipitate exchange activity and confirm that the 70 kDa protein derives from the 120 kDa protein. We propose that both the 70 and 120 kDa proteins are associated with the Na+-Ca2+ exchanger.

Animals↗
PubMed1988

Protein methylation inhibits Na+-Ca2+ exchange activity in cardiac sarcolemmal vesicles.

We have examined the effect of membrane methylation on the Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles using S-adenosyl-L-methionine as methyl donor. Methylation leads to approximately 40% inhibition of the initial rate of Nai+-dependent Ca2+ uptake. The inhibition is due to a lowering of the Vmax for the reaction. The inhibition is not due to an effect on membrane permeability and is blocked by S-adenosyl-L-homocysteine, an inhibitor of methylation reactions. The following experiments indicated that inhibition of Na+-Ca2+ exchange was due to methylation of membrane protein and not due to methylated phosphatidylethanolamine (PE) compounds (i.e., phosphatidyl-N-monomethylethanolamine (PMME) or phosphatidyl-N,N'-dimethylethanolamine (PDME]: (1) We solubilized sarcolemma and reconstituted activity into vesicles containing no PE. The inhibition by S-adenosyl-L-methionine was not diminished in this environment. (2) We reconstituted sarcolemma into vesicles containing PMME or PDME. These methylated lipid components had no effect on Na+-Ca2+ exchange activity. (3) We verified that many membrane proteins, probably including the exchanger, become methylated.

Animals↗
PubMed1988

Phospholipid composition modulates the Na+-Ca2+ exchange activity of cardiac sarcolemma in reconstituted vesicles.

Na+-Ca2+ exchange activity in cardiac sarcolemmal vesicles is known to be sensitive to charged, membrane lipid components. To examine the interactions between membrane components and the exchanger in more detail, we have solubilized and reconstituted the Na+-Ca2+ exchanger into membranes of defined lipid composition. Our results indicate that optimal Na+-Ca2+ exchange activity requires the presence of certain anionic phospholipids. In particular, phosphatidylserine (PS), cardiolipin, or phosphatidic acid at 50% by weight results in high Na+-Ca2+ exchange activity, whereas phosphatidylinositol and phosphatidylglycerol provide a poor environment for exchange. In addition, incorporation of cholesterol at 20% by weight greatly facilitates Na+-Ca2+ exchange activity. Thus, for example, an optimal lipid environment for Na+-Ca2+ exchange is phosphatidylcholine (PC, 30%)/PS (50%)/cholesterol (20%). Na+-Ca2+ exchange activity is also high when cardiac sarcolemma is solubilized and then reconstituted into asolectin liposomes. We fractionated the lipids of asolectin into subclasses for further reconstitution studies. When sarcolemma is reconstituted into vesicles formed from the phospholipid component of asolectin, Na+-Ca2+ exchange activity is low. When the neutral lipid fraction of asolectin (including sterols) is also included in the reconstitution medium, Na+-Ca2+ exchange activity is greatly stimulated. This result is consistent with the requirement for cholesterol described above. Proteinase treatment, high pH, intravesicular Ca2+ and dodecyl sulfate all stimulate Na+-Ca2+ exchange in native sarcolemmal vesicles. We examined the effects of these interventions on exchange activity in reconstituted vesicles of varying lipid composition. In general, Na+-Ca2+ exchange could be stimulated only when reconstituted into vesicles of a suboptimal lipid composition. That is, when reconstituted into asolectin or PC/PS/cholesterol (30:50:20), the exchanger is already in an activated state and can no longer be stimulated. The one exception was that the Na+-Ca2+ exchanger responded to altered pH in an identical manner, independent of vesicle lipid composition. The mechanism of action of altered pH on the exchanger thus appears to be different from other interventions.

Animals↗
PubMed1988

Sarcolemmal calcium kinetics in the neonatal heart.

We characterized sarcolemmal enzymes, Na+-Ca2+ exchange, Ca2+ efflux, and Ca2+ binding in the neonatal rabbit heart. Sarcolemmal vesicles were isolated by differential and sucrose gradient centrifugation. The sarcolemmal purification factor for K+-p-nitro-phenylphosphatase (pNPPase) was 38.8 and 34.5 in the newborn and the adult, respectively. Contamination by mitochondria and sarcoplasmic reticulum was minimal. Specific activities of Na+-K+ ATPase and K+-pNPPase in the newborn were significantly lower than those in the adult. In the newborn, maximal Na+-Ca2+ exchange was 24.6 +/- 1.1 nmol Ca2+/mg/1.5 s, passive Ca2+ efflux rate was 14.6 +/- 2.2 nmol Ca2+/mg/min and Ca2+ binding was 69.4 +/- 3.3 nmol Ca2+/mg/4 min. These values were not significantly different from the adult values. At pH 6.0, Na+-Ca2+ exchange and Ca2+ binding were approximately 60% of control values (pH = 7.4) in two age groups. These values were stimulated at pH 8.0 and 9.0. In conclusion, Na+-Ca2+ exchange, Ca2+ efflux, passive Ca2+ binding and the effect of pH on Na+-Ca2+ exchange and Ca2+ binding in the newborn were similar to those in the adult.

Aging↗
PubMed1988

Expression of cardiac sarcolemmal Na+-Ca2+ exchange activity in Xenopus laevis oocytes.

Injection of Xenopus laevis oocytes with rabbit heart poly(A)+RNA results in expression of Na+ inside (Nai+)-dependent Ca2+ uptake activity. The activity was measured by first loading the oocytes with Na+ using nystatin and then incubating the oocytes in K+ or Na+ medium containing 45Ca. The expressed Na+ gradient-dependent Ca2+ uptake was five to eight times that observed with water-injected oocytes or with poly(A)+RNA-injected oocytes for which the Na+ load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive to nifedipine. Fractionation of the poly(A)+RNA on a sucrose gradient determined that the active message had a size range between 3 and 5 kb. The properties of the Na+ gradient-dependent Ca2+ uptake indicated that Na+-Ca2+ exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na+-Ca2+ exchange.

Animals↗
PubMed1987

Enrichment of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by alkaline extraction.

Exposure of canine cardiac sarcolemmal vesicles to alkaline media (greater than or equal to pH 12) results in the extraction of 33% of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that specific proteins are being solubilized. Most of the phospholipid and sialic acid remains with the pellet after centrifugation. Electron microscopy reveals that alkaline treatment does not cause gross morphological damage to the vesicles, although freeze-fracture demonstrates some aggregation of intramembrane particles. The data indicate that high pH probably removes peripheral proteins and leaves the integral proteins in place. We find complete recovery of Na+-Ca2+ exchange activity in alkaline-extracted membranes after solubilization and reconstitution. These vesicles contain only 50% of the protein of vesicles reconstituted from control sarcolemma. Thus, the specific activity of Na+-Ca2+ exchange is doubled. Alkaline extraction is a useful and reproducible procedure for enrichment of the Na+-Ca2+ exchange protein. (Na+ + K+)-ATPase is completely inactivated by exposure to pH 12 medium though immunodetection shows that the (Na+ + K+)-ATPase proteins are not extracted. We detect both alpha and alpha + forms of (Na+ + K+)-ATPase and deduce that the Na+ pump proteins do not comprise a major fraction of sarcolemmal protein.

Animals↗
PubMed1987

Modulation of Na+-Ca2+ exchange and Ca2+ permeability in cardiac sarcolemmal vesicles by doxylstearic acids.

We examine the effects of 5-, 12- and 16-doxylstearic acids on the Na+-Ca2+ exchange and passive Ca2+ permeability of cardiac sarcolemmal vesicles. Stearic acid is a weak stimulator of Na+-Ca2+ exchange. A doxyl moiety potentiates stimulation with the order of increasing potency being 5-, 12- and then 16-doxylstearic acid. Stearic acid has little effect on vesicle Ca2+ permeability but again the doxylstearates are more effective. The sequence of potency is reversed, however, from that for increasing Na+-Ca2+ exchange. 5-Doxylstearic acid most markedly exchanges passive Ca2+ flux followed by the 12-, and then 16-doxylstearic acids. Methyl esters of the doxylstearates have no effect on either Na+-Ca2+ exchange or Ca2+ permeability. We model the results as follows. For a fatty acid to stimulate Na+-Ca2+ exchange activity, an anionic charge is required to interact with the exchanger protein at the membrane surface. Stimulation is potentiated by a perturbation (such as provided by a doxyl group) within the lipid bilayer. The perturbation is most effective at a location towards the center of the bilayer. To increase passive Ca2+ permeability an anionic charge is again essential. Disorder within the bilayer is also important, but now the most important site is near the membrane surface. Results of experiments with linolenic and gamma-linolenic acid and previous studies with other fatty acids also support this model.

Animals↗
PubMed1987

Lactate transport by cardiac sarcolemmal vesicles.

L-lactate is taken up by cardiac sarcolemmal vesicles in a process that is saturable with respect to L-lactate, stereospecific, associated specifically with the sarcolemmal membrane, and inhibited by other monocarboxylic acids and by the protein modifiers p-chloromercuriphenyl-sulfonate and N-ethylmaleimide. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of the inorganic anion transporter, is without effect. The L-lactate transport is very sensitive to pH. Uptake is stimulated by a proton gradient directed inward and decreased when internal pH is lower than external pH. Passive diffusion of nonionized lactic acid into the vesicles is negligible at physiological pH and appears to remain minor even when external pH is lowered by more than one unit. Also, the mechanism does not require specific Na+-L-lactate contransport. The properties of the L-lactate transporting system in cardiac sarcolemmal vesicles appear similar to those of the monocarboxylate transporter in erythrocytes, hepatocytes, and Ehrlich ascites cells. The present results do not allow a distinction to be made between stepwise interaction of lactate- and H+ or association of nonionized lactic acid with the carrier.

Alkalies↗
PubMed1986

Role for sulfur-containing groups in the Na+-Ca2+ exchange of cardiac sarcolemmal vesicles.

Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na+-Ca2+ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na+-Ca2+ exchange. The treatment with dithiothreitol resulted in a decrease in Vmax values but did not alter the Km for Ca2+ for the Na2+-Ca2+ exchange reaction. If Na+ replaced K+ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na+-Ca2+ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N'-ethylmaleimide, were capable of altering Na+-Ca2+ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na+-Ca2+ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na+-Ca2+ exchange, depending on whether Na+ or K+ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfur-containing residues interact with a Na+-binding site for Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.

Animals↗