Partial purification and characterization of deoxyguanosine kinase from pig skin.

Deoxyguanosine kinase, which catalyses the phosphorylation of deoxyguanosine to form deoxyguanosine 5'-monophosphate, was purified 1024-fold from extracts to newborn-pig skin. This activity requires the presence of a bivalent cation and a nucleoside triphosphate, which functions as a phosphate donor, ATP being twice as effective as CTP or GTP and 4 times as effective as UTP. The enzyme appears to have a molecular weight of 58500 as determined by Sephadex-column chromatography. Optimal enzymic activity was observed at pH 8.0; however, the enzyme remained active over a broad pH range of 5.5-9.0. Several deoxyribonucleoside and ribonucleoside monophosphates and triphosphates were tested as effectors of catalytic activity. Effective inhibitors were dGMP [Ki(app.) = 7.6 x 10(-5) M] and dGTP [Ki(app.) = 2.1 x 10(-5) M]. Both of these inhibitors acted in a competitive manner. A Km(app.) of 3.2 x 10(-7) M was measured for deoxyguanosine and a Km(app.) of 3.3 mM was determined for MgATP. Of the four major deoxynucleosides tested, this catalytic activity appears to phosphorylate only deoxyguanosine; thus the enzyme is a specific deoxyguanosine kinase.

Autonomic regulation of type 1 protein phosphatase in cardiac muscle.

Muscarinic cholinergic agonists such as acetylcholine attenuate phosphorylation of phospholamban induced by agents that activate cAMP-dependent protein kinase. However, cAMP accumulation is variably affected or only slightly reduced; thus, the choline ester might produce effects in addition to inhibition of adenylate cyclase. We hypothesized that acetylcholine might regulate a phosphatase in mammalina myocardium. Exposure of Langendoff-perfused guinea pig ventricles to isoproterenol (10 nM) for 45 s increased phosphatase inhibitor-1 activity 2-fold. Co-administration of acetylcholine (100 nM) antagonized the effect of isoproterenol, and atropine (1 microM) blocked the effect of acetylcholine. Forskolin (1 microM) caused a 3-fold increase in inhibitor-1 activity, and acetylcholine markedly attenuated the effect of forskolin. However, acetylcholine did not lower cAMP levels in the same tissues. Both isoproterenol and forskolin reduced the type 1 phosphatase activity intrinsic to sarcoplasmic reticulum by 25-50%, using [32P]phosphorylase a or 32P-labeled membrane vesicles as a substrate for the phosphatase. Co-administration of acetylcholine markedly attenuated these effects of isoproterenol and forskolin. Acetylcholine alone caused a 50% increase in type 1 phosphatase activity. We concluded that inhibitor-1 and type 1 phosphatase can be regulated in intact cardiac muscle by agents that increase intracellular cAMP and by acetylcholine.

Non-insulin-dependent diabetes-induced defects in cardiac cellular calcium regulation.

Non-insulin-dependent diabetic (NIDD) male Wistar rats develop a cardiomyopathy approximately 9 mo after the onset of the diabetic condition. This cardiomyopathy is characterized by reduced contractility, relaxation, cardiac work, and diastolic compliance. Although the basis for these defects is not completely understood, altered cellular Ca2+ regulation appears to play a major role in their development. In both isolated sarcolemmal membrane and cardiomyocytes, significant diabetes-linked defects in Ca2+ metabolism were observed. A small, but significant, decrease in the rate of sarcolemmal ATP-dependent Ca2+ transport of the diabetic heart was observed. Also evident was a major defect in sarcolemmal Na(+)-Ca2+ exchange as determined by reduced Na(+)-dependent Ca2+ transport into vesicles and Na(+)-dependent Ca2+ efflux from 45Ca(2+)-loaded cardiomyocytes from diabetic rats. In isolated cardiomyocytes, it was observed that the relative fluorescence of fura-2 at 502 nm was higher in cells from NIDD hearts, suggestive of a higher cytosolic free Ca2+. Consistent with diabetes-linked defects in Ca(2+)-transporter activities, the accumulation of Ca2+ after depolarization with KCl was greater in the diabetic. This study demonstrates that diabetes-induced defects in Ca2+ movement by the various Ca2+ transporters lead to abnormal cytosolic Ca2+ regulation by the diabetic cardiomyocytes. This observation supports the notion that abnormal Ca2+ regulation contributes to the development of the NIDD cardiomyopathy.

Management of amikacin overdose.

A woman with subarachnoid hemorrhage inadvertently received 18 g of amikacin over a 4-hr period, 20 times the recommended total daily dose. Intravenous fluids were administered to expedite renal excretion of the amikacin, and a peritoneal dialysis was performed to augment drug elimination. Drug levels were measured sequentially in serum, urine, and peritoneal dialysate. Renal clearance of the drug was increased compared to clearance following a standard dose and the drug was rapidly excreted in the urine. Amikacin was not detected in the peritoneal dialysate. There were no apparent toxic effects from the overdose. A patient with normal renal function who receives a potentially toxic dose of amikacin can be appropriately managed by careful hydration and maintenance of a generous diuresis.

Effect of membrane depolarization on binding of [3H]nitrendipine to rat cardiac myocytes.

Binding of the dihydropyridine calcium antagonist [3H]nitrendipine was studied in cardiac myocytes incubated in normal and high potassium buffer so that we might examine the voltage dependence of dihydropyridine binding. Hearts were obtained from adult male Wistar rats, and isolated calcium tolerant myocytes were dissociated by enzymatic dispersion. Cells in 5.6 mM extracellular potassium showed specific binding of [3H]nitrendipine with KD 587 +/- 50 (mean +/- SE) pM and maximum receptor density, Bmax, 10.8 +/- 1.3 fmol/mg wet weight. Cells depolarized in 50 mM potassium showed no change in KD, 661 +/- 77 pM, but approximate doubling of Bmax 25.6 +/- 3.7 fmol/mg wet weight. Binding equilibrium was reached within 5 minutes at 37 degrees C, and the KD determined by kinetic analysis was in good agreement with KD determined by saturation experiments. Unlabeled nitrendipine and nifedipine completely inhibited [3H]nitrendipine binding with slope factors less than one, whereas verapamil and diltiazem only partially inhibited binding with slope factors substantially less than one. As a function of increasing extracellular potassium concentration from 2.4-54.1 mM, the number of nitrendipine-binding sites increased gradually 115%. Aconitine also produced a 58% increase in binding sites over a concentration range of 1-30 micrograms/ml. The potassium-induced changes in number of binding sites occurred rapidly and were rapidly reversible with changes in extracellular potassium concentration. There was no change in the number of [3H]nitrendipine-binding sites as a function of potassium or aconitine concentration in dead or digitonin-treated myocytes. We conclude that nitrendipine receptor density in viable myocytes is voltage dependent, but we detect no change in KD as a function of voltage.

Denervation supersensitivity of refractoriness in noninfarcted areas apical to transmural myocardial infarction.

Denervation supersensitivity was demonstrated in anesthetized dogs 5 to 10 days after transmural myocardial infarction produced by latex embolization of a diagonal branch of the left anterior descending coronary artery. Sympathetic efferent denervation in noninfarcted myocardium apical to the infarction was demonstrated by a 90% depletion of myocardial norepinephrine content in the apical (45 +/- 15 pg norepinephrine/g tissue) vs basal (437 +/- 76 pg/g tissue) regions and by the lack of effective refractory period (ERP) shortening during bilateral ansae subclaviae stimulation in 34% of sites apical to the infarction. Supersensitivity in the area apical to the infarction was manifested by an exaggerated shortening of the ERP during both norepinephrine and isoproterenol infusions, with an upward and leftward shift in the dose-response curves in the apical vs basal regions (p less than .001). The cellular mechanism for denervation supersensitivity did not involve detectable changes in the beta-adrenergic receptor adenylate cyclase system. There was no difference in the density of beta-adrenergic receptors ([125I]-cyanopindolol) in the apical (268.6 +/- 22.7 fmol/mg protein) vs the basal (253.5 +/- 24.8 fmol/mg protein) regions. Adenylate cyclase activity stimulated by guanosine triphosphate plus isoproterenol was slightly greater in the apical (58.7 +/- 17.4%) than in the basal (49.6 +/- 10.9%) region, but this difference did not reach statistical significance (p = .068). Muscarinic modulation of beta-receptor coupling (oxotremorine attenuation of guanosine triphosphate plus isoproterenol-stimulated adenylate cyclase activity) also was not significantly different at the apical (31.6 +/- 17.5% inhibition) and basal (21.4 +/- 20.9% inhibition) sites. These data show that a transmural myocardial infarction produces denervation supersensitivity in areas apical to the infarction, but in this preparation no differences in the total number or a redistribution of beta-adrenergic receptors or adenylate cyclase activity were detected.