Homogenates Of Slices Research Articles

Catecholamine-induced phosphorylation of cardiac muscle proteins

The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing 32P to partially label intracellular ATP. Subsequent addition of 10 −5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in 32P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in 32P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of 32P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on 32P incorporation, it prevented the isoproterenol-induced incorporation of 32P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in 32P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10 −5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of 32P into proteins of the 100 000 × g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the 32P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 10 3 and decreased the incorporation into a 30 · 10 3 dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility.

Transient formation of superoxide radicals in polyunsaturated fatty acid-induced brain swelling.

The involvement of superoxide free radicals and lipid peroxidation in brain swelling induced by free fatty acids has been studied in brain slices and homogenates. The polyunsaturated fatty acids linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), and docosahexaenoic acid (22:6) caused brain swelling concomitant with increases in superoxide and membrane lipid peroxidation. Palmitic acid (16:0) and oleic acid (18:1) had no such effect. Furthermore, superoxide formation was stimulated by NADPH and scavenged by the addition of exogenous superoxide dismutase in cortical slice homogenates. These in vitro data support the hypothesis that both superoxide radicals and lipid peroxidation are involved in the mechanism of polyunsaturated fatty acid-induced brain edema.

Relationship between PAH transport and Na-K-ATPase activity in the rabbit kidney

The relationship between Na-K-ATPase activity and p-aminohippurate (PAH) transport was examined in renal cortical slices of mixed-breed rabbits by using ouabain to vary the level of Na-K-ATPase activity. Ouabain increased passive PAH uptake, measured in the presence of probenecid. Slice homogenate Na-K-ATPase activity was inhibited by 50% with about 2 X 10(-6) M ouabain. When the concentration was 10(-5) M or above, active PAH uptake was inhibited. Lower concentrations (10(-7) and 10(-6) M) of ouabain stimulated active PAH uptake. Acetate stimulated PAH uptake when medium ouabain was less than 10(-4) M. Ouabain, 10(-5) M and higher, caused intracellular K+ to fall and Na+ to rise. The changes in active PAH uptake observed with ouabain correlated much better with changes in intracellular K+ or Na+ levels (P less than 0.01) than with changes in slice homogenate Na-K-ATPase activity. Vmax for active PAH uptake decreased with 5 X 10(-5) M ouabain, and tended to increase with 10(-7) M ouabain. Ouabain did not alter Km for active PAH uptake. The data support the hypothesis that there is a functional link between active PAH transport and Na-K-ATPase activity. This linkage may be an indirect one mediated by changes in intracellular cation concentrations.

Hormonal modulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity in rat renal cortex. Specificity of enzyme translocation.

Many of the intracellular actions of cyclic adenosine 3',5'-monophosphate are expressed through phosphorylation reactions mediated by cAMP-dependent protein kinases, but little is known about hormonal control of endogenous protein kinase activity (PK) in kidney. In the present study, we examined the effects of parathyroid hormone, glucagon, and isoproterenol on cAMP and PK in slices of rat renal cortex. In the presence of 0.5 mM 1-methyl, 3-isobutyl xanthine, all three hormones activated PK in slices, as reflected by an increase in the ratio of enzyme activity assayable in homogenates of the slices without addition of cAMP to the kinase reaction mixture (cAMP-independent activity) over total enzyme activity (+2 uM cAMP in the reaction mixture). When enzyme activity was assayed in whole homogenates prepared from slices, the increase in the enzyme activity ratio (- cAMP/+cAMP) which followed hormonal stimulation was due entirely to an increase in cAMP-independent activity, with no change in total activity. In general, a good correlation existed between the alterations in tissue cAMP levels mediated by the hormones and/or 1-methyl, 3-isobutyl xanthine and concomitant alterations in PK. All three hormones increased PK activity ratios to near unity, suggesting complete enzyme activation. However, the concentrations of parathyroid hormone and glucagon which produced maximal activation of PK were much lower than those required for maximal cAMP responses. Studies with charcoal indicated that these hormonal actions on PK reflected intracellular events rather than representing activation of the enzyme during tissue homogenization, due to release of sequestered cAMP. Thus, homogenization of tissue in charcoal prevented activation of PK by subsequent addition of exogenous cAMP, but did not lower enzyme activity ratios in homogenates of hormone-stimulated cortical slices. When PK was determined in the 20,000 g supernatant fraction of renal cortical slices incubated with the hormones, enzyme activity ratios also increased, but total enzyme activity declined. Lost activity was recovered by extraction of particulate fractions with 500 mM KCl or NaCl, results which implied particulate binding of activated PK. Activated soluble PK from renal cortex was bound equally well by intact, heat- and trypsin-treated renal cortical pellets and by intact and heated hepatic pellets. Accordingly, the apparent translocation of enzyme in hormone stimulated cortex does not necessarily represent binding of the activated PK to specific acceptor sites in the particulate cell fractions or constitute a physiologic hormonal action. Activation of renal cortical PK by increasing concentrations of salts suggests that the enzyme in this tissue resembles the predominant type found in heart.

Cyclic AMP and intestinal glycoprotein synthesis: the effect of -adrenergic agents, theophylline, and dibutyryl cyclic AMP.

Incorporation of [1-14C]glucosamine by acid-precipitable protein was enhanced in intestinal slices by isoproterenol, epinephrine, theophylline, and dibutyryl cyclic AMP. Moreover, the effect of either isoproterenol or epinephrine on [1-14C]glucosamine incorporation was markedly potentiated by the addition of theophylline. Uptake of [1-14C]glucosamine into the acid-soluble compartment of intestinal slices was not significantly affected by isoproterenol, epinephrine, or theophylline.The stimulatory effect of isoproterenol and theophylline was completely inhibited by cycloheximide. Subfractionation of intestinal slice homogenates indicated that increased synthesis was not confined to one glycoprotein class but appeared to effect mucins and glycocalyx glycoproteins equally. These results indicate that β-adrenergic hormones and cyclic AMP stimulate intestinal glycoprotein synthesis and may control the production of glycoproteins located at the intestinal surface.

Studies on a Na + + K +-dependent, ouabain-sensitive adenosine triphosphatase in the avian salt gland

1. 1. A Na + + K +-dependent, ouabain-sensitive ATPase was found in homogenates of the avian salt gland. Some of its properties have been investigated. 2. 2. The levels of ouabain-sensitive and ouabain-insensitive ATPase activities were the same in homogenates of tissue from gulls maintained on fresh water and from gulls maintained on 1.5% salt water. 3. 3. The levels of ouabain-sensitive and ouabain-insensitive ATPase activities were the same in homogenates of slices which had been incubated in the absence of acetylcholine and slices which had been incubated in the presence of acetylcholine. (Acetylcholine stimulates NaCl secretion in this tissue.) 4. 4. The oxygen uptake in unstimulated salt-gland slices was reduced about 25% in the presence of 10 −4 M ouabain. The increase in oxygen uptake which occurs in response to acetylcholine did not occur if ouabain was also present. 5. 5. The level of 7-min acid-hydrolyzable phosphate esters in salt-gland slices fell in the presence of acetylcholine but this fall did not occur if ouabain was also present. 6. 6. These results, and the relationship between the rates of Na + transport and ATPase activity in the salt gland, are discussed.