Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Calmodulin activates adenylate cyclase from rabbit heart plasma membranes

It was shown that calmodulin (CM) activates the adenylate cyclase (AC) of rabbit heart light sarcolemma in the presence of micromolar free Ca2+ concentrations and this effect is blocked by trifluoroperazine and troponin I. GTP (in the presence of isoproterenol) and Gpp(NH)p are able to increase the CM-dependent activity of enzyme. It was concluded that there is no special CM-dependent "form' of AC in the heart and the common catalytic component of AC can be regulated both by CM and guanine nucleotide-binding regulatory component (N-protein). In the presence of Ca2+ and guanine nucleotide heart AC exists as a complex: CM-catalytic component-N-protein.     

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.     

Influence of global ischemia on the sarcolemmal ATPases in the rat heart

To elucidate the effect of global ischemia on the energy utilizing processes, regarding the molecular principles, the kinetic and thermodynamic properties of the sarcolemmal ATPases were investigated in the rat heart. The activation energy for hydrolysis of ATP during ischemia was higher when the reaction was catalyzed by Ca-ATPase or Mg-ATPase. For the Na,K-ATPase reaction, no changes in the activation energy were observed. With respect to the enzyme kinetics, ischemia in a timedependent manner induced important alterations in K_M and V_max values of Na,K-ATPase, Ca-ATPase and Mg-ATPase. The V_max value decreased significantly already after 15 min of ischemia, and it also remained low after 30, 45 and 60 min for all 3 enzymes. The significant diminution of K_M values occurred later in the 30th min for Ca-ATPase, in the 45th min for Na,K-ATPase. The observed drop in K_M indicates the increase in the affinity of the enzymes to substrate, suggesting thus the adaptation to ischemic conditions on the molecular level. This effect could be attributed to some conformational changes of the protein molecule in the vicinity of the ATP-binding site developing after longer duration of ischemia.     

Effects of membrane potential on sodium-dependent calcium uptake by sarcolemma-enriched preparations from canine ventricle

Summary The effect of membrane potential on sodium-dependent calcium uptake by vesicles in an isolated cardiac sarcolemma preparation was examined. Initial time course studies showed that the reaction deviated from initial velocity conditions within minutes. This appeared to be due, in part, to loss of the sodium gradient. Assays carried out to 10 sec revealed a linear component of uptake (2 to 10 sec) and a faster component (complete by 2 sec). The latter was eliminated by loading the preparation with ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid (EGTA). This maneuver did not affect the slow component, and subsequent studies used preparations containing EGTA. Potassium Nernst potentials (E _K), established by potassium gradients in the presence of valinomycin, were varied from –100 to +30 mV by changing [K⁺] _o from 1.18 to 153.7mM ([K⁺] _i =50mM). The initial velocity of sodium-dependent calcium uptake was stimulated twofold by changingE _K from –100 to 0 mV and another twofold by raisingE _K from 0 to +30 mV. For the total range ofE _K and [K⁺] _o , 32 to 36% of the increase appeared to reflect stimulation by extravesicular potassium. The remainder appeared to be due to membrane potential. The profile of sodium-dependent calcium uptake versusE _K suggested that calcium influx through electrogenic sodium/calcium exchange may be much more affected by the positive region of the cardiac action potential than by the negative region.     

Modulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by Ca2+ antagonists

Summary The purpose of this study was to examine the effect of three classes of Ca²⁺ antagonists, diltiazem, verapamil and nifedipine on Na⁺-Ca²⁺ exchange mechanism in the sarcolemmal vesicles isolated from canine heart. Na⁺-Ca²⁺ exchange and Ca²⁺ pump (ATP-dependent Ca²⁺ uptake) activities were assessed using the Millipore filtration technique. sarcolemmal vesicles used in this study are estimated to consist of several subpopulations wherein 23% are inside-out and 55% are right side-out sealed vesicles in orientation. The affect of each Ca²⁺ antagonist on the Na⁺-dependent Ca²⁺ uptake was studied in the total population of sarcolemmal vesicles, in which none of the agents depressed the initial rate of Ca²⁺ uptake until concentrations of 10 M were incubated in the incubation medium. However, when sarcolemmal vesicles were preloaded with Ca²⁺ via ATP-dependent Ca²⁺ uptake, cellular Ca²⁺ influx was depressed only by verapamil (28%) at 1 M in the efflux medium with 8 mM Na⁺. Furthermore, inhibition of Ca²⁺ efflux by verapamil was more pronounced in the presence of 16 mM Na⁺ in the efflux medium. The order of inhibition was; verapamil > diltiazem > nifedipine. These results indicate that same forms of Ca²⁺-antagonist drugs may affect the Na⁺-Ca²⁺ exchange mechanism in the cardiac sarcolemmal vesicles and therefore we suggest this site of action may contribute to their effects on the myocardium.     

Studies on oxygen and volume restriction in cultured cardiac cell: possible rearrangement of sarcolemmal lipid moieties during anoxia and ischemia-like states

Summary Cultured heart cells have been shown useful for investigating states of oxygen and volume restrictions, simulating anoxia and ischemia-like states at cellular levels. The sarcolemma has been implicated as one of the early sites of ischemic damage; therefore, lactoperoxidase catalyzed radioiodination was used to study accessibility of the sarcolemmal lipid moieties to this enzymatic probe, reflecting their exposure to the extracellular environment, hence the biophysical state of the sarcolemma. These studies have shown that within one hour of ischemic injuries: (1) The degree of labelling in the total phospholipid fraction is consideraly increased; and (2) Profound changes in the relative extent of labelling of different phospholipid classes were observed. The PE/PC labelling ratio increases dramatically with the progress of ischemia-like state. We suggest that early during ischemic injury, reorganization of the cell surface phospholipids occurs and discuss possible relations to the energy charge of the cell.     

Role of the sodium pump and the background K+ channel in passive K+(Rb+) uptake by isolated cardiac sarcolemmal vesicles

Summary A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of⁸⁶Rb⁺ by the preparation was measured in the presence of an opposing K⁺ gradient using a rapid ion exchange technique. At low extravesicular Rb⁺ concentrations, at least 50% of the uptake was blocked by addition of 1mm ouabain to the assay medium. Orthovanadate (50 m), ADP (2.5mm), or Mg (1mm) were also partial inhibitors of Rb⁺ uptake under these conditions, and produced a complete block of Rb⁺ influx in the presence of 1mm ouabain. When⁸⁶Rb⁺ was used as a tracer of extravesicular K⁺ (Rb ₀ ⁺ 40 m K ₀ ⁺ =0.1–5mm) a distinct uptake pathway emerged, as detected by its inhibition by 1mm Ba²⁺ (K _0.5=20 m). At a constant internal K⁺ concentration (K _in ⁺ =50mm) the magnitude of the Ba²⁺-sensitive K⁺ uptake was found to depend on K ₀ ⁺ in a manner that closely resembles the K⁺ concentration dependence of the background K⁺ conductance (I _Kl) observed electrophysiologically in intact cardiac cells. We conclude that K⁺ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.     

A calmodulin dependent protein kinase activity associated with rabbit heart sarcolemma

Summary Sarcolemmal vesicles prepared from rabbit heart muscle by differential and discontinuous sucrose density gradient centrifugation, exhibited high marker enzyme activities: Na⁺ + K⁺ ATPase 22 µMol Pi × mg^–1 × h^–1. Adenylate cyclase 500 pmole CAMP × mg^–1 × min^–1, calcium antagonist receptors 0.7 pmoles × mg^–1. Calmodulin in the presence of calcium and -ATP³² stimulated rapidly and specifically the ³²P incorporation into two membrane proteins of 54 and 44 kDa. Calmodulin stimulated the phosphorylation of the 44 kDa to a greater extent (17.9 pmol ³²P × mg^–1 protein) than the 54 kDa protein (1.3 pmoles ³²P x mg^–1 protein). Removal of endogenous calmodulin from the membrane by EGTA extraction resulted in a 2.5 fold increase in calmodulin dependent ³²P incorporation into the two proteins in the presence of exogenous calmodulin. It is suggested that the calmodulin dependent protein kinase activity in heart sarcolemma may mediate the effects of calmodulin in the regulation of Ca²⁺ transport across the membrane.     

Subcellular distribution and isolation of the Ca2+ antagonist receptor associated with the voltage regulated Ca2+ channel from rabbit heart muscle

The Ca²⁺ antagonist binding sites associated with the voltage dependent calcium channel in rabbit myocardium were found to distribute with the sarcolemmal Na^– + K⁺ ATPase and adenylate cyclase activities during subcellular fractionation on sucrose-density gradients. The equilibrium dissociation constants (K_D) for the binding of [³H]nitrendipine and [³H]verapamil were 0.31 ± 0.04 nM and 4.1 ± 0.5 nM respectively, and displayed an average density of 0.55 ± 0.05 pmol/mg and 0.4 ± 0.03 pmol/mg protein respectively for the most enriched membrane fraction. The Ca²⁺2 antagonist binding sites were solubilized from the membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and specific binding sites for [³H]PN200-110, [³H]verapamil and [³H]diltiazem were isolated on a wheat-germ lectin column. The binding sites for [³H]PN200-110 were enriched about 2500 fold as compared with the original homogenate and displayed a density of 28.5 ± 8 pmole/mg protein in the isolated fraction. Sodium dodecyl sulfate gel electrophoresis of the isolated drug binding proteins indicated enrichment of proteins of Mr 170000, 140000, 130000, 100 000 and 53000. The isolated receptor contained an intrinsic kinase activity that phosphorylated glycoproteins of Mr 170 000 and 53000. Exogenously added cAMP-kinase stimulated phosphorylation of the 170000, 100000, 53 000 and 28000 Mr glycoproteins in the receptor fraction. The results of this study indicate that the binding sites for [³H]nitrendipine, [³H]PN200-110, [³H]verapamil and [³H]diltiazem residue on glycoprotein(s) which are of sarcolemmal origin, and co-purify together on wheat germ lectin columns. The polypeptide composition of the Ca²⁺ antagonist binding sites from cardiac muscle appears to be very similar to that of the dihydropyridine receptor in skeletal muscle.Abbreviations CHAPS 3-[-(3-cholamidopropyl) dimethylammonio]-propanesulphonate - SDS sodium dodecyl sulphate Scholar of the Ontario Heart and Stroke Foundation.