Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma.

Ca2+ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 . 10(-5) and 1.2 . 10(-3) M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca2+ binding were studied. All cations tested inhibited Ca2+ binding with the following order of potency: trivalent greater than divalent greater than monovalent cations. The order of potency for the monovalent ions was: Na greater than K+ greater than Li+ greater than or equal to Cs+ and for the divalent and trivalent ions: La3+ greater than or equal to Mn2+ greater than Sr2+ greater than or equal to Ba2+ greater than Mg2+. 1 . 10(-3) M caffeine and 1 . 10(-8) M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 . 10(-7) M verapamil, acidosis (pH 6.4), 1 -10(-5) M Mn2+ and 1 . 10(-4) M ouabain depressed the capacity of the low-affinity sites to a range of 154--291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.

Cardiac sarcolemma of the hamster. Enrichment of the (Na+ + K+)-ATPase.

Cardiac sarcolemma was prepared from normal hamsters using gentle homogenization, extraction with 0.6 M KCl and continuous density gradient centrifugation. The final fraction exhibited high (Na+ + K+)-ATPase activity (24 micromol/mg per h) and contained minimal contamination from mitochondria, myofibrils and lysosomes.

Uptake of calcium antagonistic drugs into muscles as related to their lipid solubilities.

Calcium antagonists, e.g. bepridil and verapamil, block the Ca2+-dependent slow action potentials in frog skeletal muscle [L.M. Kerr and N. Sperelakis, J. Pharmac. exp. Ther. 222, 80 (1982)]. To determine whether the calcium antagonistic drugs may enter the fibers and exert an internal action as well, uptake of tritiated bepridil, verapamil, nitredipine, nifedipine, and diltiazem into rat extensor digitorum longus (EDL) muscles was examined. It was found that the uptake values of verapamil, nitrendipine, and bepridil were much higher than those of nifedipine and diltiazem. The order of uptake was: bepridil greater than nitrendipine greater than verapamil much greater than nifedipine greater than diltiazem. The small uptake values of nifedipine and diltiazem may represent primarily binding to the surface membrane. In frog skeletal muscle (sartorius) also, the uptake of bepridil was greater than that of verapamil, and disruption of the T-tubules by the glycerol method did not change them. The same order of drug uptake values was found for monolayer cultures of vascular smooth muscle cells (rat aorta). The order of uptake in isolated sarcoplasmic reticulum (SR) from rat skeletal muscles was: verapamil greater than nitrendipine greater than bepridil greater than nifedipine greater than diltiazem. The lipid solubility values of the calcium antagonists were measured by their partition coefficients in oil/Ringer, octanol/Ringer, and chloroform/Ringer systems. The order of lipid solubility was: bepridil greater than verapamil greater than nitrendipine greater than nifedipine much greater than diltiazem. Thus, the calcium antagonists with the highest lipid solubilities were taken up more by the muscle cells and SR. It is concluded that verapamil, bepridil, and nitrendipine enter and accumulate inside the muscle cells, whereas nifedipine and diltiazem do not permeate readily.

Behavior of vital and killed autologous pericardium in the descending aorta of sheep.

OBJECTIVES: Cardiovascular implants of fresh autologous pericardium produced mixed results including fibrosis with retraction or thinning and dilatation. The reasons for these differences are unknown but may involve activation of cells intrinsic to the tissue implant. To better understand the behavior of autologous pericardial implants, we studied the outcomes of vital pericardium (fresh) versus ethanol-killed pericardium. METHODS: Fresh and ethanol-killed autologous pericardium was transplanted as a patch, a conduit, or a rectangular flap bisecting the lumen in the descending aorta of sheep. The implants, recovered at 1, 5, 10, 15, and 30 days, were evaluated macroscopically and microscopically and by immunohistologic studies. RESULTS: Fresh implants showed good preservation with fibrin deposition on day 15. Microscopically, cells positive for alpha-actin and von Willebrand-related antigen appeared in the fibrin by day 10. By day 30 the flap was fibrotic and retracted whereas the patch and conduit retained their original appearance on the luminal aspect. An endothelium-like layer expressing von Willebrand-related antigen was present in the patch and conduit but absent in the flap. In contrast, the ethanol-killed implants were free of fibrin by day 10. By day 30, there were no signs of fibrosis or retraction, and a surface layer of cells expressing von Willebrand-related antigen, characteristic of endothelial cells, was present on all implants. All ethanol-killed implants were repopulated by host cells. CONCLUSION: The transluminal flap is an interesting model for studying the behavior of intraluminal autologous pericardial cardiovascular implants. Killing of the pericardial implants alleviated the fibrosis and tissue retraction observed with fresh flap implants.

Nifedipine, diltiazem, bepridil and verapamil uptakes into cardiac and smooth muscles.

Vogel et al. (1979; J. Pharmacol. Exp. Ther. 210, 378) reported that one calcium antagonist, bepridil, exerted an effect internally as well as its effect on blocking Ca2+ entry in cardiac muscle. Therefore, the uptakes of tritiated nifedipine, diltiazem, bepridil, and verapamil by cat ileal smooth muscle, chick embryonic ventricular muscle, and rabbit papillary muscle were investigated. It was found that the uptakes of verapamil and bepridil by the muscles were much higher than those of nifedipine and diltiazem. The uptake of bepridil was substantially greater than that of verapamil; thus, the order of uptake was: bepridil greater than verapamil much greater than nifedipine greater than diltiazem. The cardiac muscles accumulated at least 2-fold greater amount of calcium antagonists than the smooth muscle. The amount of a given calcium antagonist accumulated by a muscle was not a function of the ability of that calcium antagonist to inhibit Ca2+ uptake into the muscle, since nifedipine and diltiazem were more potent in depressing Ca2+ uptake, but had the smallest uptakes. The calcium antagonists were more effective in depressing Ca2+ uptake into smooth muscle than into cardiac muscle. Calculation indicates that internal drug concentration at steady state for both cardiac and smooth muscles was either equal to (diltiazem) or much higher than the drug concentration in the medium (bepridil and verapamil). It is concluded that bepridil and verapamil enter and accumulate in the muscle cells, whereas nifedipine and diltiazem permeate more slowly into the muscles. The ability of all four drugs to enter the muscle cells confers the possibility that these calcium antagonists may exert secondary actions on internal sites of the muscle, such as the sarcoplasmic reticulum.

Differential actions of calcium antagonists on calcium binding to cardiac sarcolemma.

The action of four calcium antagonistic drugs, including verapamil, bepridil, nifedipine, and diltiazem, on calcium binding to cardiac sarcolemma from guniea pig was tested. It was found that verapamil (10(-6) to 10(-5) M) inhibited calcium binding to a great extent. Bepridil at the same concentrations was less potent than verapamil in the depression of calcium binding. Nifedipine and diltiazem did not affect sarcolemmal calcium binding. The differential action of the calcium antagonistic drugs was discussed.

Significant increase of aortic root volume and commissural area occurs prior to aortic valve opening.

BACKGROUND AND AIM OF THE STUDY: The increased use of autologous, homologous or heterologous aortic root demands a detailed knowledge of its anatomy and function. The advent of 3-D digital sonomicrometry offered the opportunity to acquire precise information on the root and leaflet movements during the cardiac cycle. METHODS: Under cardiopulmonary bypass, sonomicrometry crystals were implanted in the aortic root and valve of eight sheep. Crystals were sutured at each commissure (n = 3), the top of the sinotubular junction (n = 3), lowest point of the annulus (n = 3), and leaflet tip (n = 3). 3-D coordinates of each crystal were recorded, together with left ventricular and aortic root pressures and ECG. When the animal had returned to a stable hemodynamic condition, the maximum and minimum distances between two crystals, and areas between three crystals, were calculated. Changes in root volume and leaflet position were time-related to the pressure changes. RESULTS: The most significant change between maximum and minimum distance between crystals during the cardiac cycle occurred at the commissural level. Similarly, the triangle defined by the three commissural crystals showed the greatest change in area (47%). The root volume increased by an average of 22%; about 40% of this increase occurred during the isovolumic phase. The aortic leaflets began to open before ejection. CONCLUSION: We postulate that aortic valve opening is initiated by the outward pull of the commissures. These findings should impact on aortic root surgery.

Lipids of myocardial membranes: susceptibility of a fraction enriched in sarcolemma to hydrolysis by an exogenous mammalian phospholipase A2.

A myocardial membrane fraction enriched in sarcolemma was used to determine the susceptibility of the lipids to hydrolysis by a phospholipase A2 from granulocytes. After incubation (37 C, pH 7.0, 5 mM Ca2+) with the phospholipase A2 for 30 min, a more than 3-fold increase in unesterified fatty acids was found (up to 47 nmol/mg protein; P < 0.001) relative to a control incubated without phospholipase A2 or Ca2+. This included a 10-fold increase in the arachidonic acid content (up to 42 mol%) and at least a 7-fold increase in lysophosphatidylethanolamine (up to 7.4 mol% total phospholipid-P). However, the exogenous phospholipase did not augment the quantity of lysophosphatidylcholine produced by endogenous phospholipases in the presence of Ca2+ (5 mM). These results demonstrate the in vitro susceptibility of phospholipids of myocardial membranes, particularly phosphatidylethanolamine, to the neutral-active, Ca2+-dependent phospholipase A2 from granulocytes. This work may be relevant to myocardial inflammation and tissue damage during ischemia, where heterolytic injury of the myocardium may occur subsequent to the accumulation of granulocytes.

Influence of cations and agents on sarcolemmal calcium binding.

Calcium binding to fragmented sarcolemma isolated from dog heart was measured by an ultracentrifugation technique. Two classes of binding sites with dissociation constants of 4.2 x 10(-5) M and 1.2 x 10(-3) M were identified. The maximum number of high and low affinity sites were 15 and 452 nmol/mg, respectively. The effects of various cations and drugs on calcium binding were studied in the presence of 0.1 mM total calcium. All cations tested inhibited calcium binding to a certain degree. La3+ (1 mM) and Mn2+ (1 mM) abolished calcium binding to the sarcolemma. The other divalent cation, Mg2+, used at a concentration of 1 mM, produced a partial inhibition Na+ and K+ also depressed calcium binding, with Na+ being the more potent inhibitor. Verapamil produced a depression at concentrations as low as 10(-7) M and abolished calcium binding at 10(-3) M. The effects of these cations and drugs on fragmented sarcolemma appear different from those reported for the isolated sarcoplasmic reticulum. Thus it is unlikely that the calcium binding observed was caused by a contamination of the sarcolemmal preparation by the fragmented sarcoplasmic reticulum. In addition, the calcium binding seems to be on the sarcolemma itself, rather than to the remaining fragments of the basement membrane, because the calcium binding was not significantly modified by pretreatment with purified neuraminidase (0.25 U/g of sarcolemma).

The mechanism of chlorpromazine and quinidine inhibition of cardiac sarcotubular ATPase.

Although chlorpromazine and quinidine have dissimilar chemical properties, both are negative inotropic agents and both depress the ATPase activity of cardiac sarcotubular fractions. With the aid of gamma-AT32 P we have identified the step in ATP hydrolysis which is inhibited by each of these agents. By optimizing the conditions for formation of a phosphorylated intermediate (EP) by addition of 5 mM calcium to the incubation medium we found that chlorpromazine (0.25--1 mM) severely depressed EP formation, whereas quinidine (0.25--1 mM) had no effect. After the isolation of EP we showed that chlorpromazine had no effect on the magnesium-facilitated hydrolysis of EP, whereas quinidine was a potent inhibitor of this hydrolytic step. The effects of both quinidine and chlorpromazine on the E + ATP equilibrium EP + ADP reaction were studied. Quinidine had no effect on this reaction, whereas chlorpromazine, at 1 mM, shifted the reaction toward ATP formation.

Effect of calcium and magnesium on binding of beta, gamma-methylene ATP to sarcoplasmic reticulum.

Isolated sarcotubular membranes (SR) from skeletal muscle bound 3.7 nmol of beta, gamma-methylene [8-3H]ATP (AMP-PCP) per mg of membrane protein. Only one class of binding site was identified and the dissociation constant (K) for this site was 1.5 X 10(-5) M. Addition of 0.05% Triton X-100 increased the number of binding sites to 5.7 nmol/mg. ATP and ADP competitively inhibited AMP-PCP binding. The dissociation constants for ATP and ADP were 3.5 X 10(-5) M and 3.3 X 10(-6) M, respectively. Since this data was obtained in the presence of 5 mM EDTA, it was established that the sarcoplasmic reticulum has a high affinity for the metal free forms of ATP, ADP, and AMP-PCP. Magnesium concentrations in excess of 1 X 10(-4) M inhibited AMP-PCP binding. Lower concentrations of magnesium had little effect on AMP-PCP binding. The effect of calcium on AMP-PCP binding was biphasic. Calcium concentration between 1 X 10(-6) and 1 X 10(-4) M inhibited AMP-PCP binding. Inhibition was maximal at 1 X 10(-5) M. Calcium concentration above 1 X 10(-4) M facilitated analogue binding. Possible sites of magnesium and calcium actions are discussed.

Veratridine stimulation of calcium uptake by chick embryonic heart cells in culture.

Veratridine (2 X 10(-5) to 1 X 10(-4)M) stimulated Ca uptake into monolayer cultures of chick embryonic heart cells in a dose-dependent manner. The stimulation of veratridine was independent of the age of the chick embryos from which the heart cells were taken, and was inhibited by tetrodotoxin (3 microM). The half-maximal effect of veratridine was 37 microM. Since young embryonic hearts have few or no functional fast Na channels (Sperelakis, N. (1981) Cardiac Toxicol 1, 39-108), the present results indicate that the action of veratridine is not dependent solely on fast Na channels, but could be related to other properties of the surface membrane, such as increasing the resting Na permeability and/or opening of the slow Na channels. The increased Ca influx could then be a consequence of the Ca0:Nai exchange reaction.

Endothelial cell-derived vasoconstrictors: mechanisms of action in vascular smooth muscle.

Intercellular signaling between the endothelial cell (EC) and vascular smooth muscle (VSM) is an important determinant of vasomotor tone. We evaluated mechanisms of action of EC-derived constrictors on VSM using conditioned medium from bovine aortic ECs in culture (EC-CM) or endothelin-1 (ET-1), and isolated coronary arteries or cultured VSM cells. EC-CM enhanced Ca2+ uptake into monolayers of rat aortic VSM and elicited sustained contractions in isolated coronary vessels in a time- and dose-dependent manner. The enhanced Ca2+ uptake and contractions were markedly attenuated by the Ca2+ channel antagonists bepridil, verapamil, and nitrendipine. EC-CM and ET-1 resulted in VSM membrane depolarization and increased excitability to electrical stimulation that was blocked by verapamil. ET-1 and EC-CM induced a dose-dependent increase in steady-state [Ca2+]i in Fura-2-loaded rat VSM cells. Most VSM responded with a rapid and transient increase in [Ca2+]i while others lacked only the transient phase. The elevated poststimulus [Ca2+]i level appeared to precede the influx of extracellular Ca2+ and contraction. EC-CM and ET-1 also resulted in time- and concentration-dependent increases in inositol monophosphate (IP) formation in rat aorta that paralleled the development of isometric force. We propose a biphasic mechanism in which the stable constrictors present in EC-CM elicit a rapid, phospholipase C-mediated mobilization of intracellular Ca2+ accompanied by or coupled to a sustained influx of extracellular Ca2+ through voltage-dependent channels.

Endothelin-1 stimulates phosphatidylinositol hydrolysis and calcium uptake in isolated canine coronary arteries.

The effects of synthetic endothelin-1 (ET-1) (10(-10)-3 x 10(-7) M) on isometric force, 45Ca2+ uptake, and phosphatidylinositol (PI) hydrolysis were determined in isolated canine coronary artery rings. ET-1 caused contraction and stimulated 45Ca2+ uptake and PI hydrolysis (determined as inositol monophosphate accumulation) in a concentration-dependent manner with EC50 values of 6.3 x 10(-9), 2 x 10(-9), and 3 x 10(-9) M, respectively. Maximal responses were obtained with 3 x 10(-8) M ET-1 for all three parameters. At the maximally effective concentration, ET-1 caused a 1.8-fold increase in the rate of 45Ca2+ uptake following a 1-min exposure (the shortest time point tested) while the contractile response reached maximum only after 6 min. ET-1 (3 x 10(-8) M) stimulated a biphasic accumulation of inositol monophosphate with an initial rapid 1.4-fold increase detectable between 30 and 60 s followed by a secondary 11.9-fold increase at 30 min. These data show that PI hydrolysis and Ca2+ uptake are early events in the action of ET-1 on coronary artery vascular smooth muscle that precede the maximal contractile response. It is suggested that all of these responses are triggered by the interaction of ET-1 with a cell-surface receptor.