Myotonic muscular dystrophy: altered calcium transport in erythrocytes.

Erythrocytes from patients with myotonic muscular dystrophy accumulate calcium at a significantly higher rate than normals do. This increased rate of net accumulation appears related to an enhanced permeability of the membrane to calcium, rather than to an impairment in its active outward transport.

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyteghosts.

Incubation of etythrocyte ghosts with carbonylcyanide m-chlorophenyl-hydrazone (CCCP) plus Ca-2+ resulted in inactivation of the Ca-2+ -stimulated ATPase activity. Omission of Ca-2+ or lowering of the temperature below 25 degrees C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of beta-mercaptoethanol did not influence the Ca-2+ -dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca-2+ -containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca-2+ -stimulated, Mg-2+ -ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg-2+ -ATPase slightly. CCCP was much more specific for the Ca-2+ -stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca-2+ for inactivation was also quite specific.

Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ +K+, and 5'-adenylylimidodiphosphate.

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate, (AMP-P(NH)P). This compound, in which the oxygen connecting the beta and gamma phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg2+-ATPase activities. The K1 of AMP-P(NH)P for Mg2+ ATPase activity was 2.0 - 10-4 M and, while the Km of ATP for this activity was also 2.0 - 10-4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na+, K+ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 - 10-3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-free porcine erythrocyte ghosts were studied in order to characterize the system more adequately.

Phenothiazine inhibition of calmodulin stimulates calcium-dependent potassium efflux in human red blood cells.

Elevation of red blood cell calcium increases the efflux of potassium. The active extrusion of calcium from the red cell is regulated by calmodulin. Phenothiazines bind to calmodulin in a calcium-dependent manner preventing the calmodulin from activating a wide variety of cellular processes. The present study shows that phenothiazines increase the efflux of potassium from red cells incubated with the calcium ionophore A23187. The dose dependent effect of trifluoperazine on potassium efflux correlates with its inhibition of Ca-ATPase activity. The phenothiazine effects are dependent upon ATP in that increases in potassium efflux are not observed in energy depleted cells. In calcium buffered ghosts no direct effect of calmodulin or an antibody to calmodulin can be shown. These data suggest that phenothiazines stimulate calcium-dependent potassium loss indirectly by a drug-induced blockage of the calmodulin-activated Ca-ATPase.

Insulin resistance in patients with myotonic dystrophy.

Evaluation of insulin sensitivity in 12 patients with myotonic dystrophy gave results different from those found in other insulin-resistant conditions. Nine of our subjects were insensitive to exogenous insulin, but only three had elevated fasting insulin concentrations. Eight had an excessive insulin response to a glucose challenge. Monocyte insulin receptor affinity was decreased (myotonics, 1.21 +/- 0.74 X 10(9) liters per mole; controls, 2.62 +/- 1.28 X 10(9)), and this parameter correlated best with the insulin resistance. No circulating receptor antibody or insulin binding inhibitor was found. Our studies suggest that the insulin resistance seen in patients with myotonic dystrophy is related to decreased insulin receptor affinity.

Bio-effects of high magnetic fields: a study using a simple animal model.

The desire to do clinical imaging and spectroscopy at magnetic field strengths greater than 2 Tesla (T) necessitates investigation of possible bioeffects at these high fields. A simple T-maze was utilized to evaluate the aversive effects of exposure to three levels of static magnetic field (0, 1.5, and 4 T). The right arm of the maze extended into the center of a 30-cm horizontal bore magnet, while the left arm extended into a mock magnet bore with the same dimensions. The self-shielded design of the magnet reduces the fringe field to zero within 1 m of the bore, placing the start box of the maze outside the 5-G line of the magnet. Each rat performed a total of ten trials at each level of magnetic field strength. A follow-up subset was run at 4 T with the maze reversed. At 0 T, the rats entered the magnet freely. No significant differences from the control were observed at 1.5 T. At 4 T, however, in 97% of the trials the rats would not enter the magnet. In the maze-reversed subset a majority of the rats turned toward the magnet, indicating that they had learned an aversive response from the previous trials at 4 T. However, in only 4 decisions out of 58 did the rats actually enter the magnet. Eighteen decisions to turn around were made at the edge of the magnet in a region of strong field gradients (up to 13 T/m) and a field strength up to 1.75 T.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium concentration in saliva along the time course of experimental Coriolis sickness.

Procedures designed to evaluate the severity of motion sickness have included subjective reporting of changes in salivation. In order to increase objectivity, we studied the sodium concentration of saliva, which is directly related to the flow rate. Healthy adults with normal vestibular function underwent a modified Coriolis Sickness Susceptibility Index (CSSI) test, utilizing a staircase profile. Saliva was collected without interrupting the stimulus by means of cotton placed beneath the subject's tongue for one minute. Samples were obtained 5 min prior to stimulation, 30 and 45 min following stimulus onset, and/or upon reaching the "nausea II" endpoint. Saliva for analysis by atomic absorption spectrophotometry was obtained by centrifugation of the cotton. A significant difference in sodium concentration was found between the baseline and 30-min sample (p less than 0.01). Although the amount of salivation was rather variable, the pattern of changes in sodium concentration was similar in all experimental cases.

Effects of cadmium and zinc on calcium uptake in human red blood cells.

Cadmium and zinc increased the accumulation of calcium in human red blood cells by increasing passive influx without enhancing the permeability to other ions. The effect of cadmium and zinc appeared specific to these metals, because barium, magnesium, cobalt, strontium, manganese, and nickel had no effect. Changes in calcium uptake by extracellular sodium, potassium, and pH were not altered by zinc and cadmium. Inhibition of calcium uptake by quinine, oligomycin, and iodoacetate was not affected by cadmium or zinc. These results suggest that cadmium and zinc increase calcium movement through normal influx pathways. Cadmium and zinc acted synergistically apparently by different mechanisms. Zinc and cadmium differentially affected calcium uptake in different extracellular calcium concentrations. The cadmium effect was increased by low concentrations of 2-mercaptoethanol and above pH 8.0, while the zinc effect was less sensitive to these factors. These findings suggest that the cadmium effect involves a disulfide bond between cysteinyl residues and the zinc effect involves a different site.

Iodoacetic acid inhibition of calcium-dependent potassium efflux in red blood cells.

The metabolic inhibitor, iodoacetic acid (IAA), has commonly been used to increase Ca-dependent K efflux in red blood cells. It is thought that this effect of IAA involves the irreversible inhibition of glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), resulting in the energy depletion of the cell. Without energy, active transport stops, and the K loss is enhanced both by increasing cellular Ca and by preventing K reuptake. The present study shows that in addition to this metabolic effect, which increases Ca-dependent K efflux, IAA also inhibits this efflux. This inhibition is irreversible and is not related to the ATP or Ca concentrations of the cells. The carboxymethylation of a specific protein band correlates with IAA inhibition of K efflux.

Calcium-dependent association of glutathione S-transferase with the human erythrocyte membrane.

Elevations in intracellular calcium increase the adsorption of a cytoplasmic protein to human red blood cell membrane. This protein migrates on SDS polyacrylamide gels at 23,000 daltons and has been called band 8. The association of this protein with the membrane is increased in sickle cell anemia. This protein is extracted from the membrane with EGTA, a calcium chelator. Enzymatic and immunological studies identify band 8 as a glutathione S-transferase.

Red blood cell alterations in muscular dystrophy: the role of lipids.

Biochemical, morphologic, and biophysical studies support the concept that the red blood cell (RBC) membrane is altered in both myotonic muscular dystrophy (MyD) and Duchenne muscular dystrophy (DMD). These studies have not identified a primary metabolic defect that would explain the various alterations of membrane properties. Since the lipid milieu of the membrane affects most membrane properties, it has been extensively investigated in MyD and DMD. Although some studies have suggested specific lipid abnormalities, no reproducible alterations have been reported in the major lipid constituents of the RBC membrane in these disorders. These findings suggest that major alterations of the predominant membrane lipids are not involved in these diseases. Furthermore, studies of the RBC membrane do not provide definitive statements as to the inborn error of metabolism, whether proteins or lipid constituents are primarily affected, or even whether the described alterations are intrinsic to the membrane or are secondary to some circulating factors. Nevertheless, RBCs have proved useful in demonstrating the involvement of the plasma membrane in muscle disorders and should be important in defining how such membrane perturbations affect transport mechanisms.

Calcium transport in human red blood cells under hypertonic conditions.

Calcium accumulation in intact human erythrocytes is enhanced by incubation in hypertonic solutions. Hypertonicity produces an increased permeability of the membrane to calcium that is time-dependent and occurs in the presence or absence of calcium. When hypertonically treated cells are incubated for more than 30 min in 300 mosmol/kg solutions the permeability of the membrane to calcium returns to basal values. Oligomycin inhibits the effect of hypertonicity on calcium uptake. The inhibitory action of oligomycin diminishes as the external sodium increases and can only be observed when the external concentration of potassium is at or below 3 mM. Low intracellular sodium and high intracellular potassium concentrations increase the uptake of calcium. It is concluded for human erythrocytes that 1) the increased permeability of the membrane to calcium produced by hypertonicity is a time-dependent, reversible phenomenon and is independent of calcium, 2) the increase in intracellular potassium concentration associated with hypertonic exposure is an important factor contributing to this response, and 3) interactions between calcium and components of the sodium-potassium transport system may account for the enhanced uptake of calcium produced by hypertonicity.

Calcium dependent ATP losses in intact red blood cells without cellular accumulations of calcium.

Intact human red blood cells incubated with ionophore A23187 and calcium develop a depletion of ATP that is dependent upon the concentrations of both A23187 and Ca. Incubations of fresh cells with 0.5 micrometer A23187 and concentrations of Ca at or below 70 micrometer produce a depletion of ATP without a net cellular uptake of Ca. In contrast, ATP-depleted cells display an ionophore-dependent cellular uptake of Ca, under identical conditions. A hypothesis is proposed that relates these ionophore-produced ATP depletions to active Ca extrusion by the Ca ATPase.

Involvement of a cytoplasmic protein in calcium-dependent potassium efflux in red blood cells.

The potassium permeability of the human red blood cell increases with the free intracellular calcium concentration. The efflux of potassium can be inhibited by iodoacetic acid. This inhibitory effect correlates directly with the carboxymethylation of a protein band found in both the hemolysate and membrane fractions. The present study provides two additional lines of evidence that this protein is involved directly with the calcium-dependent changes in potassium permeability: its association with the membrane is calcium dependent; and calcium-dependent potassium efflux from resealed ghost is inhibited by the incorporation of antibodies raised against this cytoplasmic protein.

Myotonic dystrophy: calcium-dependent phosphatidic acid synthesis in erythrocytes.

Recently it was reported that calcium-dependent phosphatidic acid synthesis in erythrocyte of patients with myotonic muscular dystrophy (MyD) is markedly impaired when compared to that in control subjects. Using 32P-loaded erythrocytes, we found no significant difference in the levels of 32P-phosphatidic acid synthesized after exposure to calcium and its ionophore A23187 between patients with MyD and controls. In a batch experiment typical of the experiments with 32P, and a twofold increase of phosphatidic acid in both groups was determined by inorganic phosphate measurements. Thus, the specific activity of the 32P-phosphatidic acid increased four- to five-fold in response to calcium Analyses of 32P-polyphosphoinositide breakdown in ghosts and in adenosine triphosphate-depleted erythrocytes also appeared normal for patients with myotonic muscular dystrophy. Possible discrepancies between the results presented here and those reported previously are discussed.

Purification and measurement of calpromotin, the cytoplasmic protein which activates calcium-dependent potassium transport.

A simple procedure is described for the purification of calpromotin, a protein from the cytoplasm of red blood cells which is capable of activating calcium-dependent potassium transport. The purification steps involve a salt gradient elution from an anion exchange column (Whatman DE-52) followed by a potassium phosphate gradient elution from a column of hydroxyapatite (HA Ultrogel). These steps result in a 54% yield with a 161 fold purification. The calpromotin is estimated to be 99% pure as determined by densitometry of the protein profile on an SDS polyacrylamide gel. A competitive enzyme-linked immunosorbent assay (ELISA) using rabbit anti-human calpromotin antibodies, is described for measuring levels of calpromotin in the 5 to 100 ng range.

13C NMR spectroscopic analysis of phospholipid metabolism in adrenal chromaffin cells.

Phosphatidylcholine (PC) metabolism in bovine adrenal chromaffin cells has been studied in vivo with 100 MHz 13C NMR spectroscopy. The incorporation of 13[CH3]choline was examined in cells embedded in 1% agarose strands and perfused at 37 degrees C. The pattern of 13C NMR spectroscopy. The incorporation of [13CH3]choline incorporation into PC of suspension cultures of chromaffin cells. The chemical shifts of labelled carbons in [13CH3]choline before and after their incorporation into PC are identical (ca 56.4 ppm). Incorporated [13CH3]choline can be released from the cells by phospholipase D which reduces the cellular PC signal by greater than 85%, indicating that a major fraction of the labelled PC is on the external membrane surface. This application of NMR spectroscopy provides a basis for direct measurement of the in vivo metabolism of PC in functioning adrenal chromaffin cells.

Calcium-activated potassium transport and high molecular weight forms of calpromotin.

Investigations of human red blood cells show that a cytoplasmic protein called calpromotin is involved in the regulation of calcium-activated potassium transport. Calpromotin associates with the membrane in the presence of calcium and undergoes a chemical transformation. High performance gel filtration and gel electrophoresis show that the cytoplasmic and membrane-bound calpromotin can exist in both low and high molecular weight forms. The biochemical properties of the high molecular weight membrane-bound calpromotin are not the same as the high molecular weight cytoplasmic calpromotin. The high molecular weight membrane forms of calpromotin are increased by leupeptin and diminished by iodoacetic acid. Therefore, the leupeptin enhancement and iodoacetic inhibition of calcium-activated potassium transport may involve the high molecular weight forms of membrane-bound calpromotin.

Phosphoglyceride biosynthesis in bovine adrenal chromaffin cells.

Cultured adrenal chromaffin cells, representing a virtually homogeneous population of neuronal elements, have been utilized to examine the final enzymes in the formation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), namely, choline phosphotransferase, ethanolaminephosphotransferase, and the N-methyltransferases in the sequential methylation of PE to PC. Each enzyme has been characterized extensively in terms of substrate requirements, pH optima, detergent and cation effects, and response to inhibitors revealing properties very similar to those in other neural preparations. The respective activities are stable for up to two weeks of adrenal chromaffin cell culture suggesting that this system is a suitable model for examining the relative roles and the regulation of each pathway in PC formation.

Reconstitution of Ca(2+)-dependent K+ transport in erythrocyte membrane vesicles requires a cytoplasmic protein.

We have demonstrated that calcium-dependent potassium transport in erythrocytes requires the participation of a cytoplasmic protein. Activation of calcium-dependent potassium transport causes an increase in the membrane-bound levels of this protein which is dependent on the calcium concentration and which is highly correlated (r = 0.791, p less than 0.0001) with the loss of potassium. Reconstitution of this transport pathway in sonicated erythrocyte membrane vesicles was achieved only in vesicles containing the cytoplasmic protein indicating a causal relationship in this transport system. The protein is found in high levels within the cytoplasm of erythrocytes (5.6 mg/ml red blood cells) and yet less than 1% of the protein located in the cytoplasm is required to bind to the membrane in order to initiate the potassium efflux. The analysis of rat organ homogenates demonstrated that this protein is located in most tissues with particular enrichment in adrenal glands, brain, lung, and blood. These results demonstrate that there is a cytoplasmic protein, herein named calpromotin, which is a necessary and sufficient cytoplasmic component of calcium-dependent potassium transport in erythrocytes and perhaps other tissues.