Amar K. Sen

Impairment of endothelium‐dependent relaxation in aortae from spontaneously diabetic rats

1 Diabetes mellitus is known to produce alterations in vascular reactivity. The present study examined the effects of endothelium‐dependent and endothelium‐independent relaxing substances on thoracic aorta from control and spontaneously diabetic rats. 2 Endothelium‐dependent relaxation produced by acetylcholine or the calcium ionophore, A23817, in aortic rings precontracted with phenylephrine was significantly attenuated in diabetic vessels. 3 Relaxations produced by sodium nitroprusside or adenosine in diabetic preparations were comparable to those in control vessels. 4 The results show that diabetes leads to a specific impairment of endothelial‐dependent relaxation.

Stability and ligand sensitivity of [3H]ouabain binding to (Na+ + K+)-ATPase

Abstract Specific [3H]ouabain binding to guinea pig kidney ( Na + + K + )- ATPase (ATP phosphohydrolase, EC 3.6.1.3) at 37° was determined. At less than 1·10−6 M ouabain specific binding to the native enzyme was negligible. Specific binding occurred in two ways. Binding was stimulated by Mg2+ or Mg2+ and Pi which greatly increased the affinity of the enzyme for ouabain. This binding was saturated at 10 μM ouabain and reduced by Na+ or EDTA. The Na+ inhibition showed cooperative effects, apparent K i values between 2.5 and 70 mM and appeared to be indirect. K+ at low concentrations stimulated, then at higher concentrations inhibited this binding. The inhibition appeared to be competitive. Binding in the presence of 200 mM Na+ required ATP and Mg2+. In the presence of ATP and Mg2+, binding was stimulated by 0–16 mM Na+ and further increased by 200 mM Na+. SrCl2 or ADP did not support binding in the presence of Na+. The half-life of the enzyme-ouabain complex was 9 h at 0°. On warming, the rate of dissociation increased exponentially with temperature to t 1 2 = 3 min at 37°. The experiments suggest that there is one type of specific binding site and that the binding pathways are reciprocally related with respect to Na+. They suggest different binding sites and conformations of maximum affinity for Na+ and ouabain. The Na+-stimulated pathway requires phosphorylation. Phosphorylation and/or ouabain binding appear to alter the affinity of the Na+ sites. Any single non-covalent bond type appears insufficient to explain the enzyme-ouabain interaction.

Species and tissue differences in the rate of dissociation of ouabain from (Na+ + K+)-ATPase

The rates of the association and dissociation reactions of ouabain with (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) were compared. The enzymes were prepared from kidney, heart and brain tissues obtained from guinea pig, dog and cat. The rate of dissociation of ouabain from (Na+ + K+)-ATPase varied about 60-fold between different species. The dissociation rate also differs between tissues within a given species. The rate of association of ouabain with these enzyme preparations did nog differ appreciably. The results suggest that the species differences in sensitivity to the cardiac glycosides are principally related to the different rates of dissociation of the drug-enzyme complex.

Induction of the catalytic protein of (Na++K+)-ATPase in the salt gland of the duck

The (Na+ plus K+)-ATPase activities in salt gland homogenates increased 3- to 4-fold after saline treatment of ducks for 3 weeks. The ATPase was purified to a specific activity of 460 and 1015 mumol Pi/mg protein per h, respectively, in control and saline-treated ducks. The catalytic protein was identified on polyacrylamide electrophoresis gels by phosphorylating the enzyme with (32P)ATP. The molecular weight of the protein was estimated to be 98 000. The amount of catalytic unit increased commensurately with the enzyme activity after saline treatment. It is therefore concluded that the increased enzyme activity is due to a de novo enzyme synthesis and is not an activation effect. Phospholipid concentration in the salt gland tissue increased 1.7-fold after the saline treatment. Significant increases occurred in the percentage of the total phospholipids as phosphatidylserine and sphingomyelin. In the partially purified (Na+ plus K+)-ATPase preparation, the percentage composition of phosphatidylserine and phosphatidylethanolamine increased after saline treatment.

Solubilization of guinea pig kidney (Na+ + K+)-ATPase with Lubrol W and Triton X-100

Abstract 1. 1. A comparison has been made of two non-ionic detergents, Lubrol W and Triton X-100 for the solubilization of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3). 2. 2. The optimum concentrations for maximum solubilization with Triton X-100 and Lubrol W were 0.05 and 0.1%, respectively. 3. 3.|Monovalent cations, such as Na+ and K+, and ATP increased the specific activity of the enzyme solubilized by Triton X-100 and decreased that by Lubrol W. 4. 4. Mg2+ inhibited solubilization with either of the two detergents. 5. 5. Triton X-100 was found superior to LUbrol W in solubilization of (Na+ + K+)-ATPase. 6. 6. A procedure for obtaining soluble enzyme preparation with specific activity ranging between 140 and 200 μmoles Pi per mg protein per h is described. 7. 7. The soluble enzyme was more stable in the presence of Na+, K+ or ATP, but ATP was inferior to Na+ or K+.

Inhibition of sodium- and potassium-dependent adenosine triphosphatase by ethacrynic acid: Ligand-induced modifications

Abstract Inhibition of phosphorylation and ATP hydrolyzing activity of(Na + + K + )-ATPase (ATP phosphohydrolase, EC 3.6.1.3) by ethacrynic acid was facilitated by Na + or Mg 2+ plus inorganic phosphate or Na + plus Mg 2+ and ATP. The inhibition of enzyme activity was antagonized by ATP, ADP and K + . ATP with or without ouabain did not reverse the acceleration or retardation of the rate of inhibition of enzyme activity by ethacrynic acid in the presence of Na + or K + . Ouabain did not significantly alter the rate of inhibition of ATP hydrolysis and phosphorylation by ethacrynic acid, indicating that the binding sites of ouabain and ethacrynic acid are probably different. Results show ligand-induced conformational changes in (Na + + K + )-ATPase and give further support to the allosteric model for cation transport involving the formation of a phosphorylated intermediate.

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.

The induction of (Na++K+)-ATPase in the salt gland of the duck

Abstract Hypertonic saline was administered to ducks for 24 h. Protein synthesis was measured in salt-gland slices by a double-isotope technique. Salt-gland slices from saline-treated animals incorporated more radioactivity than those from control animals. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins in the heavy microsomal fraction of the salt glands revealed that the greatest radioactive amino acid incorporation occurred in the catalytic subunit of ( Na + + K + )- ATPase . The results support the tenet that saline treatment increases ( Na + + K + )- ATPase synthesis.

Alterations in atrial reactivity in a strain of spontaneously diabetic rats

1 The present study examined the reactivity of atria from control and spontaneously diabetic rats to various adrenoceptor agonists and to adenosine. 2 Isoprenaline (1.5 nM‐1500 nm) produced concentration‐dependent increases in inotropy which were unchanged in diabetic atria. However, the sensitivity to isoprenaline‐induced changes in chronotropy was reduced in diabetic preparations. 3 In the presence of propranolol (2 μm), phenylephrine (0.2 μm‐100 μm) produced concentration‐dependent increases in both inotropy and chronotropy; however, atria from diabetic rats exhibited a much greater maximal response. The diabetic state did not alter the sensitivity to phenylephrine. 4 Adenosine (0.15 μm‐300 μm) produced concentration‐dependent decreases in both inotropy and chronotropy which were unchanged in diabetic atria. 5 Radioligand binding studies revealed that both α1‐and β‐adrenoceptor populations were substantially reduced in atria from diabetic rats. However, there was no change in receptor affinity for either adrenoceptor. 6 These results show that diabetes leads to an alteration in atrial reactivity to adrenoceptor stimulation. Future studies examining steps following hormone‐receptor coupling are required in order to characterize this defect.

Purification and characterization of protein kinase C isozymes from rat heart

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca2+ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca2− and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca2+.