John A. Watts

Comparison of the protective effects of verapamil diltiazem nifedipine and buffer containing low calcium upon global myocardial ischemic injury

This study was designed to compare the effects of the Ca2+ slow channel blocking agents verapamil (2 X 10(-6) M), diltiazem (7.5 X 10(-7) M), and buffer containing reduced Ca2+ content (0.95 mM) on myocardial ischemic injury. These treatments were equiactive, reducing cardiac function to 20% of the control value, and fully reversible in nonischemic, isolated, working rat hearts. Hearts which were reperfused (30 min) following 27 min of global ischemia recovered 17% of control cardiac function and had a markedly reduced ATP and creatine phosphate content and ATP/ADP ratio compared to nonischemic hearts. When verapamil, diltiazem, nifedipine, or low Ca2+ treatments were given before and during ischemia, equal improvement in cardiac function was observed upon reperfusion, and tissue ATP levels, creatine phosphate levels, and ATP/ADP ratio were significantly higher than in hearts which did not receive the treatments or which received the drug vehicle. Large increases in recovery of contractile function were observed with a partial preservation of ATP reserves. These treatments, which were equiactive in nonischemic hearts, provided equivalent preservation of cardiac function, ATP, and creatine phosphate in the reperfused ischemic hearts. When the ischemic period was increased to 33 min and the effective concentrations reduced to depress cardiac function to 40% of the control value (4.5 X 10(-7) M verapamil, 2.5 X 10(-6) M diltiazem, 3 X 10(-7) M Nifedipine, 1.25 mM Ca2+), equal improvement in cardiac function was again observed. Thus, major differences among these Ca2+ slow channel blockers or low Ca2+ treatment were not detected in this experimental system.

Protection by verapamil of globally ischemic rat hearts: Energy preservation, a partial explanation

The relationship between energy preservation and the recovery of heart function was studied in globally ischemic hearts which were treated with verapamil. Isolated working rat hearts reperfused after 27 min of ischemia recovered 17.9 +/- 5.11% of pre-ischemic contractile function and had markedly reduced tissue ATP, total adenine nucleotide, and creatine phosphate levels. The ATP/ADP ratio was also decreased in these hearts. When verapamil (2 X 10(-6) M) was present before and during ischemia, but not during reperfusion, the recovery of cardiac function following reperfusion was improved (82.4 +/- 12.1%). When hearts were treated with 0.0, 7.5 X 10(-8) M, 5 X 10(-7) M, or 2 X 10(-6) M verapamil, the recovery of cardiac function was proportional to the concentration of verapamil present and showed a linear relationship with the depression of cardiac function prior to ischemia. The ATP, total adenine nucleotide and creatine phosphate levels were significantly higher in those hearts which were treated with verapamil, but the increase was not proportional to the recovery of cardiac function. It is possible that a critical pool of ATP may correlate with the recovery of verapamil treated hearts, but a large degree of mechanical recovery occurred with significant loss of high energy phosphate stores. Thus, while high energy compounds were preserved, there was not a good correlation between recovery of cardiac function and the preservation of total tissue energy reserves. A portion of the protection afforded by verapamil to globally ischemic hearts may be due to energy preservation, but additional mechanisms may also be involved in the enhanced recovery of contractile function.

Ultrastructure of the heart of the marine mussel geukensia demissa

The structure of the heart of Geukensia demissa, a common object of physiological and biochemical investigation, is described by scanning, transmission and freeze‐fracture electron microscopy. A single‐cell epithelial layer covers the ventricle, but an endothelium is lacking. Myofibers are small (6–7 μm diam.), mononucleate, and tapered. Glycogen is concentrated peripherally. Mitochondria are particularly concentrated under the sarcolemma, near the ends of the nucleus, and in rows between bundles of myofilaments. The myofilaments (6–8nm thin, 30–35 nm thick filament diam.) are loosely arranged into sarcomeres (2–4 μm) by Z bodies. Many of these Z bodies interconnect, and some anchor to the sarcolemma forming attachment plaques. Cells are joined by intercalated discs consisting of fascia adherentes, spot desmosomes, and gap junctions. The gap junctions include intramembrane particles. T tubules are absent. The sarcolemma is coupled to the junctional sarcoplasmic reticulum (JSR) over 357ndash;40% of the cell surface. Tubules extend from the JSR deep into and throughout the cell as an irregularly dispersed network. The SR occupies 1% of the cell volume. A few, small (0.1–1.0 μm) unmyelinated nerves are present, but no neuromuscular junctions were seen. The auricles have fewer and smaller myocytes than the ventricle. The auricles also contain podocytes with pedicels having 20–35 nm slits and containing sieve‐like projections. The morphology of the Geukensia heart is similar to that of other bivalves.

Effects of diltiazem on lactate, ATP, and cytosolic free calcium levels in ischemic hearts

Changes in tissue lactate, ATP, and cytosolic free calcium (Cai) were examined in isolated, perfused rat hearts receiving 20 min of zero-flow global ischemia (37°C). Addition of diltiazem before ischemia caused a concentration-dependent decrease in lactate accumulation. This effect was not mediated by modulation of nor-epinephrine release since depletion of catecholamines by reserpine did not alter lactate accumulation, and diltiazem treatment reduced lactate accumulation in catecholamine-depleted hearts. Diltiazem-treated hearts showed a concentration-dependent decrease in tissue ATP utilization that was associated with the decrease in tissue lactate during ischemia. Basal time averaged Cai, determined by fluorine NMR using 5FBAPTA, was 620 nM. Diltiazem (0.9 μM) decreased this value to 489 nM and reduced heart rate and maximum pressure developed (81.3 and 53.9% of control, respectively) before ischemia. Ca; increased fourfold between 9 and 15 min of ischemia in hearts receiving no drug, while there was no increase in Cai in diltiazem-treated hearts. These results show that diltiazem reduces the use of ATP and therefore production of lactate during ischemia, and indicate a relationship between preservation of ATP and maintenance of Cai that may be important in the beneficial effects of diltiazem during myocardial ischemia.

Effects of diltiazem upon globally ischemic rat hearts.

Addition of diltiazem (0.0, 0.95, 2.5 or 7.5 microM) to isolated working rat hearts before and during ischemia, produced a concentration-dependent increase in recovery of contractile function. Recovery of post-ischemic pressure-rate product showed a strong relationship with depression of pre-ischemic pressure-rate product, primarily from decreased heart rate before ischemia and increased pressure development following reperfusion. Increased recovery in treated hearts was associated with higher ATP and adenine nucleotide levels (ADN), but no relationship was observed between energy levels and degree of recovery of function or concentration of diltiazem. Hearts made ischemic for 20 min without reperfusion had increased ATP and decreased lactic acid accumulation when treated with 7.5 microM diltiazem. The results indicate contractile-dependent mechanisms of action of diltiazem in global ischemic hearts which can only be partly explained by preservation of ATP and ADN, but also are associated with reduced lactic acid accumulation.

Competitive inhibition by dimethylsulfoxide of molluscan and vertebrate acetylcholinesterase

Anticholinesterase-like effects of dimethylsulfoxide (DMSO) were demonstrated on a variety of invertebrate muscles. The excitatory effects of acetylcholine (ACh) on the isolated preparations of the Geukensia demissa heart and anterior byssus retractor muscle (ABRM), and of the Busycon contrarium radula protractor muscle, were potentiated by DMSO (1-5 microliters/ml; 1 microliter/ml = 14 mM). The negative chronotropic effects of ACh, but not of 4-ketoamyltrimethylammonium, were potentiated by DMSO (1-5 microliters/ml) on the isolated heart of the oyster Crassostrea virginica. These four muscles have acetylcholinesterase enzymes of high activity. In contrast, Mercenaria mercenaria hearts have weak cholinesterase activity, and the effects of ACh on this isolated myocardium were not potentiated by DMSO (2-20 microliters/ml). DMSO (0.1-15 microliters/ml) was a competitive inhibitor of both a crude preparation of oyster heart acetylcholinesterase (AChE) (the Km increased 24-fold with DMSO at 15 microliters/ml; the I50 was 1.3 microliters/ml DMSO when [ACh] = Km) and a purified Electrophorus AChE (the Km increased 4.5-fold when DMSO was 10 microliters/ml; the I50 was 10 microliters/ml DMSO near [ACh] = Km). The same doses of DMSO were needed to potentiate the pharmacological effects of ACh on the oyster heart, as to inhibit the AChE of this tissue.

A low concentration of nisoldipine reduces ischemicheart injury: Enhanced reflow and recovery of contractile function without energy preservation during ischemia

Isolated working rat hearts which received no drug treatment had reduced ATP and creatine phosphate levels and increased lactate content during 20 min of ischemia. When subjected to 33 min of ischemia and 30 min of reperfusion, these hearts recovered low values of cardiac output (9.8 ml/min), heart rate, maximum developed pressure, pressure-rate product (72.9, 32.6, 27.5% of control, respectively), had low levels of tissue ATP, and reduced coronary flow upon reperfusion. Addition of nisoldipine (1 nM) 10 min before ischemia caused no decrease in cardiac output or heart rate, slightly decreased maximum developed pressure and pressure-rate product (93% of control), and did not reduce the degradation of ATP and creatine phosphate or the accumulation of lactate during 20 min of ischemia. When nisoldipine was included 10 min before ischemia, during ischemia (33 min) and reperfusion (30 min), however, the recovery of cardiac function and tissue ATP levels was significantly increased. This protective effect occurred when drug treated ischemic hearts were reperfused with control buffer, indicating residual effects. The beneficial effects of nisoldipine were not due to changes in afterload or preload (isolated perfused heart), collateral flow (zero flow model), energy preservation during ischemia (little contractile depression, ATP not enhanced during ischemia), or reduced lactate accumulation during ischemia. The beneficial effects were associated with increased coronary flow (31% higher than no drug) during reperfusion, indicating a reduction in the no-reflow phenomenon.

Dimethyl sulfoxide: Inhibition of acetylcholinesterase in the mammalian heart

The heart rate of the isolated, perfused, working rat heart was significantly and equally depressed by 1 X 10(-6)M acetylcholine (ACh) and by 6 X 10(-5)M 4-ketoamyltrimethylammonium (4K), a cholinomimetic agonist. Dimethyl sulfoxide (DMSO) (10 microliter/ml, 140 mM) strongly potentiated the effect of ACh but did not alter the effect of 4K. DMSO (10 microliter/ml, 140 mM) strongly potentiated the effect of ACh but did not alter the effect of 4K. DMSO (10 microliter/ml, 140 mM final concentration) alone had no significant effect upon heart rate when added to the perfusate in incremental additions of 1 microliter X (ml perfusate)-1 X min-1 over a 10-min period. The specific activity of atrial homogenate cholinesterase was 48.8 +/- 3.46 nmoles X min-1 X (mg protein)-1 (mean +/- S.E.M.), 38.2 +/- 1.60 for butyrylcholinesterase, and 11.2 +/- 0.86 for acetylcholinesterase (AChE). True AChE activity (measured in the presence of a maximally effective concentration of tetraisopropylpyrophosphoramide) had a Vmax of 13.4 +/- 0.17 nmoles X min-1 X mg protein)-1 and an apparent Km value of 1 X 10(-4)M acetylthiocholine. At this Km substrate concentration, DMSO inhibited atrial AChE activity (I50 = 9 microliter/ml). At the concentration tested, DMSO inhibited atrial AChE and potentiated ACh effects.

Reversal of hypotension induced by Vibrio vulnificus lipopolysaccharide in the rat by inhibition of nitric oxide synthase.

Intravenous infusion of Vibrio vulnificus lipopolysaccharide (LPS) (1 mg/kg body wt) in rats caused a dramatic drop in mean arterial pressure within 10 min and a further decline in mean arterial pressure and heart rate which lead to death between 25 and 70 min. Rats treated with LPS followed 10 min later by the intravenous infusion of NG-monomethyl-L-arginine (L-NMMA, 20 mg/kg body wt) showed an initial drop in mean arterial pressure owing to the LPS infusion, followed by a transient rise in mean arterial pressure which lasted for approximately 40 min after the infusion of L-NMMA. The pressure values then remained level for at least 150 min post-LPS infusion. Control rats treated with equivalent volumes of saline infusion showed stable values of mean arterial pressure and heart rate. Additional control rats receiving L-NMMA alone showed the transient rise in mean arterial pressure, followed by a return to the baseline values. The results indicate that the symptoms of endotoxic shock resulting from V. vulnificus LPS may result in part from the stimulation of the activity of nitric oxide synthase. Inhibition of nitric oxide synthase by L-NMMA is a possible treatment for toxic shock induced by V. vulnificus.

Comparison of the effects of bepridil and diltiazem upon globally ischemic rat hearts

Effects of the Ca2+ antagonists, bepridil (20 microM) and diltiazem (2.5 microM), upon ischemia/reperfusion injury were assessed in perfused, working rat hearts. These treatments were equally cardiodepressant in non-ischemic hearts. A lower concentration (5 microM) of bepridil was also assessed. Hearts which were reperfused following 33 min of global ischemia recovered 12.8% of preischemic pressure-rate product and had markedly reduced ATP, total adenine nucleotides, ATP/ADP ratio, and mitochondrial function. Treatment with bepridil before and during ischemia did not improve recovery of cardiac function, tissue energy reserves, or mitochondrial function upon reperfusion with control buffer. Control hearts treated with bepridil had normal levels of high energy compounds. Treatment with diltiazem significantly improved contractile function, and metabolic parameters. Ischemia/reperfusion injury was associated with a doubling of tissue Ca2+ content. Pretreatment with diltiazem, but not bepridil, reduced Ca2+ overload. Bepridil did not directly protect the myocardium from ischemia/reperfusion injury perhaps due to its inability to prevent Ca2+ overload.