Earl T. Wallick

Effects of vanadate on cardiac contraction and adenylate cyclase

Abstract Vanadate produces a positive inotropic effect on ventricular muscle from rat, rabbit, guinea pig and cat; a positive inotropic effect on the atria of rat and rabbit, but a negative inotropic effect on the atria of guinea pig and cat. The effects of vanadate are completely reversible and occur in a concentration range of 10 −5 M to 10 −3 M. In this same concentration range, vanadate also causes a marked activation of cardiac adenylate cyclase suggesting that the positive inotropic action might be due in part to an elevation of cyclic AMP levels. The effects of vanadate are not influenced by alprenolol, cimetidine, or mepyramine, indicating a lack of involvement of β-adrenergic or histamine H 2 and H 1 receptors.

A study of calcium binding and uptake by isolated cardiac sarcoplasmic reticulum: The use of a new ionophore (X537A)☆

Calcium “binding” (absence of oxalate) and “uptake” (presence of oxalate) were studied in isolated cardiac sarcoplasmic reticulum. X537A (< 5 μg/mg protein) inhibits binding and uptake similarly; the same concentrations induce a highly significant augmented calcium release from binding sites only in the absence of oxalate. At higher concentrations, “uptake” is virtually eliminated while “binding” (I50 = 16 μg/mg) is less inhibited suggesting an additional action of X537A on a step unique to uptake. Data suggest that “binding” and “transport” of calcium may be different but they may initially share sites.

A kinetic comparison of cardiac glycoside interactions with Na+,K+-ATPases from skeletal and cardiac muscle and from kidney.

Abstract The rates of association of [3H]ouabain to Na+,K+-ATPase and the rates of dissociation of the enzyme-ouabain complexes were determined for enzymes isolated from dog skeletal muscle, beef heart muscle, and lamb kidney medulla. The rates of association were strongly influenced by the presence of ligands such as magnesium, sodium, potassium, ATP, and inorganic phosphate. For a particular set of binding ligands, the rates of association did not vary much amongst the three enzymes studied, although enzyme from skeletal muscle was the fastest. In contrast, the rates of dissociation were relatively independent of the ligand conditions. The rates of dissociation also varied greatly amongst the enzyme sources, with skeletal muscle Na+,K+-ATPase being the fastest. Although the major determinant of the affinity of the Na+,K+-ATPase for ouabain is the rate of dissociation, the rate of association also plays a role. Since the binding of ouabain to the Na+,K+-ATPase in the presence of magnesium, ATP, sodium, and potassium is very slow, it is difficult to obtain an I50 (equilibrium) value for the inhibition of hydrolytic activity by ouabain. If measurements of activity are made after a long period of time (3 h), the affinity of the enzyme for ouabain, estimated from inhibition of Na+,K+-ATPase activity, approached the value calculated from [3H]ouabain binding. The ratio of the I50 value for ouabagenin to that for ouabain for the skeletal muscle enzyme was the same as that for cardiac muscle enzyme, indicating that the sugar moiety of ouabain was interacting with the receptor of both enzymes. It is apparent, therefore, that the absence of a sugar binding site in skeletal Na+,K+-ATPase is not the reason for the faster dissociation rate of this enzyme.

Decrease in Na+,K+-ATPase activity and [3H]ouabain binding sites in sarcolemma prepared from hearts of spontaneously hypertensive rats.

Na+,K+-ATPase activity, phosphorylation, and [3H]ouabain binding in sarcolemma isolated from spontaneously hypertensive rat (SHR) hearts were compared to the same parameters in sarcolemma from normotensive rat (WKY) hearts. Sarcolemma prepared from SHR heart contained significantly less ouabain-inhibitable ATPase activity than sarcolemma from WKY heart. No significant differences in sarcolemmal protein content or recovery were noted between the two groups. The numbers of phosphorylation sites and ouabain binding sites were lower for SHR hearts than for WKY hearts. The KD values for ouabain binding were the same (0.30 muM) in cardiac sarcolemma of SHR and WKY. The I50 values for inhibition by ouabain of Na+,K+-ATPase were also the same for both groups (SHR = 49 microM; WKY = 44 microM). These data suggest that the decrease of cardiac sarcolemmal Na+,K+-ATPase activity in SHR hearts is due to a decrease in the number of active sites.

Effects of extracts of rat brain on the digitalis receptor.

There is a possibility that an endogenous substance exists which interacts with a ouabain binding site on Na+, K+-ATPase. Recently, several reports have appeared suggesting the presence of an endogenous digitalis-like substance in acid-acetone extracts of brain. We have demonstrated that in preparing an acid-acetone extract, peroxidized lipids and lysophospholipids are produced, both of which inhibit Na+, K+-ATPase, thereby complicating interpretation. Preliminary evidence suggests, however, that when rat brains are extracted with an aqueous-acetone mixture under nitrogen, a principle is obtained which specifically inhibits Na+, K+-ATPase.

Effect of palmitylcarnitine on ouabain binding to Na, K-ATPase.

Abstract Palmitylcarnitine, an endogenous long-chain fatty acyl ester, inhibited cardiac Na, K-ATPase activity and binding of [ 3 H]ouabain to the enzyme. The inhibitory effects on enzyme hydrolytic activity and drug binding were time and concentration dependent, but also dependent upon the ratio of palmitylcarnitine to protein. Palmitylcarnitine inhibitory effects were irreversible, but could be prevented by bovine serum albumin. In the presence of Mg 2+ + ATP or Mg 2+ + Pi, [ 3 H]ouabain binding was fully inhibited by 100 μ m palmitylcarnitine. The addition of sodium, or sodium plus potassium to the drug-binding medium reduced the inhibitory effect. The protective action of Na + was concentration dependent and was optimal at 75 μ m palmitylcarnitine. Equimolar amounts of choline chloride did not have the same protective effect as sodium chloride. Binding of ouabain to the enzyme in the absence of palmitylcarnitine prevented the protective effect of Na + . Inhibition of Na, K-ATPase functional properties occurred at a concentration range of palmitylcarnitine reported to occur in the cytosol of ischemic cells during episodes of experimental myocardial ischemia. It is suggested that elevated levels of palmitylcarnitine in ischemic myocardium may play a role in altering cellular function as well as the inotropic response of ischemic cardiac muscle to digitalis glycosides.

Canine mesenteric artery Na+,K+-ATPase: vasopressor receptor for digitalis?

Summary Localization and characterization studies of Na+,K+-ATPase in canine superior mesenteric artery were undertaken to examine the role of the Na+-K+ pump in the vasopressor response of cardiac glycosides. The enzymatic component of the membrane-bound Na+-K+ pump, Na+,K+-ATPase, was found by histochemical and cell fractionation techniques to be localized primarily in the sarcolemma of the smooth muscle cell in superior mesenteric artery. The enzyme could be enriched in microsomal and partially purified sarcolemma preparations of superior mesenteric artery and first-order arterial side branches. Binding of [3H]ouabain to arterial microsomes followed pseudo-first-order reaction kinetics. In the presence of magnesium plus ATP, sodium stimulates and potassium inhibits the rate of binding. Scatchard analysis indicated a single class of [3H]ouabain binding sites with a KD of 2–9 nM and a Bmax of 2.3–3.5 pmol/mg protein. Although the characteristics of [3H]ouabain binding to mesenteric artery microsomes resemble the characteristics of [3H]ouabain binding to purified Na+,K+-ATPase, the density or total number of Na+-K+ pump sites in mesenteric artery is small compared with either heart muscle or kidney parenchyma. In isolated mesenteric arterial strips, more than 80% of a 2.5 μM ouabain-induced contraction could be inhibited or reversed by α-adrenoceptor blockade with 1 μM phentolamine. These data indicate that although cardiac glycosides interact with specific receptor sites in smooth muscle of canine superior mesenteric artery, the direct vasoconstrictor effect, which may be related to this digitalis-smooth muscle Na+,K+-ATPase interaction, is meager and may be a reflection of the low density of Na+-K+ pump sites.

Effect of quinidine on the digoxin receptor in vitro.

To investigate the basis for a clinically important digitalis-quinidine interaction that is characterized by increases in serums digoxin concentrations when quinidine is administered to digoxin-treated patients, we have studied in vitro the interaction of quinidine with the digoxin receptor. Evidence has been obtained that quinidine is capable of decreasing the affinity for digoxin of cardiac glycoside receptor sites on purified Na,K-ATPase and on intact human erythrocyte membranes. As others have shown, quinidine is capable of inhibiting Na,K-ATPase activity, and evidence has been obtained in the current study that, while quinidine can reduce the affinity of the enzyme for digoxin, it is also capable of acting together with digoxin in inhibiting enzyme activity to a degree greater than the inhibitory effect of digoxin alone. The concentrations of digoxin and quinidine used in this study were considerably greater than their therapeutic serum concentrations. Nevertheless, these observations are consistent with the hypothesis that the increases in serum digoxin concentrations and the decreases in volumes of digoxin distribution observed clinically when quinidine is administered to digoxin-treated patients may reflect, at least in part, a decrease in the affinity of tissue receptors for digoxin. The possibility must also be considered that enhanced cardiac effects of digoxin may occur clinically as the result of an augmentation, by quinidine, of digoxin effects, which more than compensates for the modest reduction in digoxin binding.

Homology of ATP binding sites from Ca2+ and (Na,K)-ATPases: Comparison of the amino acid sequences of fluorescein isothiocyanate labeled peptides

Ca2+ and (Na,K)-stimulated ATPases from various species and tissues were labeled with fluorescein isothiocyanate (FITC). Labeled peptides were solubilized by tryptic digestion and purified by reverse phase high pressure liquid chromatography. The amino acid sequences of the labeled peptides reveal considerable homology between sarcoplasmic reticulum Ca2+-ATPases from various sources. These Ca2+-ATPases also contain a region of homology with all other ATPases thus far sequenced. A difference was demonstrated between dog skeletal and cardiac Ca2+-ATPases. These results demonstrate homology of the putative ATP binding site of ATPases, which extends over tissue, species, and cation specificity, including the completely conserved amino acid sequence: lys-gly-ala-pro-glu.

Effects of vanadate on myocardial function

SummaryThe influence of vanadate (NH4VO3, Na3VO4) and vanadyl (VOSO4) on myocardial function was studied using a variety ofin vivo andex vivo cardiac preparations. In addition, the influence of vanadate on a variety of cardiac subcellular systems was examinedin vitro.In isolated ventricular and atrial muscle of dog, cat, guinea pig, rabbit and rat, vanadate produces a positive inotropic effect. The effective concentration for positive inotropy in these tissues ranged from 10 to 500 μM. Vanadyl sulfate was less effective than either sodium ortho-vanadate or ammonium meta-vanadate. Vanadate produced negative inotropic effects in guinea pig and cat isolated left atrium, at concentrations lower than those required to produce positive inotropic effects in sensitive species and tissues.In blood perfused papillary muscle or whole heart, or in whole animal (anesthetized or conscious), vanadate generally reduced ventricular performance and produced a marked peripheral and coronary vasoconstriction. Vanadate also contracted isolated poocine coronary artery strips, an effect similar to that produced by ouabain.Vanadate caused a variety of effects on isolated subcellular systems of cardiac muscle. Low concentrations (0.1–100 μM) inhibited sarcolemmal Na+, K+-ATPase and Ca2+-ATPase, sarcoplasmic reticulum Ca2+-ATPase, Ca2+-binding to and uptake by sarcoplasmic reticulum, but stimulated hormone-sensitive adenylate cyclase. Higher concentrations of vanadate (100–1000 μM) inhibited myofibrillar ATPase, phosphorylation of troponin I, and mitochondrial ATPase.On the basis of these effects of vanadate on cardiac subcellular systems, mechanisms by which vanadate acts on intact myocardium are proposed. Furthermore, consideration of anin vivo, regulatory role of vanadate is presented with regard to myocardial function.ZusammenfassungDie Wirkung von Vanadat (NH4VO3, Na3VO4) und Vanadyl (VOSO4) auf die Myokardfunktion wurde an einer Reihe von In-vivo- und In-vitro-Präparationen des Herzens untersucht, ebenso wie der Vanadateffekt auf subzelluläre kardiale Systeme.Vanadat wirkt positiv inotrop in isolierten Ventrikeln und Vorhöfen von Hund, Katze, Meerschweinchen, Kaninchen und Ratte. Die dafür notwendigen Konzentrationen waren 10–500 μM. Vanadyl war weniger wirksam als Na3VO4 oder NH4VO3 Vanadat wirkt negativ inotrop an Meerschweinchen- und Katzenvorhofpräparaten in niedrigeren Konzentrationen als für den positiv inotropen Effekt notwendig sind.An mit Blut perfundierten Papillarmuskeln oder Herzen sowie am narkotisierten oder nicht narkotisierten Tier reduziert Vanadat die linksventrikuläre Funktion und verursacht eine deutliche periphere und koronare Vasokonstriktion. Isolierte Koronararterienstreifen (Schwein) gehen durch Vanadat in Kontraktur über ähnlich der g-Strophanthinwirkung.An isolierten subzellulären Systemen des Herzmuskels hemmen niedrige Vanadatkonzentrationen (0.1–100 μM) die sarkolemmale Na+, K+-ATPase, die Ca++-ATPase des sarkoplasmatischen Retikulums sowie dessen Ca++-Bindung und Ca++-Aufnahme. Andererseits wird die kardiale Adenylatzyklase stimuliert. Höhere Vanadatkonzentrationen (100–1000 μM) hemmen die myofibrilläre ATPase, die Phosphorylation von Troponin I und die mitochondriale ATPase.Auf der Basis der verschiedenen Wirkungen des Vanadats werden die Mechanismen zur Diskussion gestellt, über die Vanadat am intakten Herzen wirken könnte. Außerdem wird die mögliche regulatorische Rolle des Vanadats in vivo in Hinsicht auf die Myokardfunktion beurteilt.