Louis A. Sordahl

Effects of magnesium, ruthenium red and the antibiotic ionophore A-23187 on initial rates of calcium uptake and release by heart mitochondria

Abstract Initial rates of calcium uptake by rabbit heart mitochondria were measured by dual-beam spectroscopy. Mg2+ produced a marked reduction in the rate of Ca2+ uptake. In the absence of Mg2+, Ca2+-stimulated mitochondrial respiration could not be further increased by addition of ADP. After Ca2+ addition with Mg2+ present, ADP produced apparent coupled respiratory stimulation. Both the Ca2+ transport inhibitor, ruthenium red, and the divalent cation-specific, ionophoretic antibiotic, A-23187, inhibited Ca2+ uptake in the presence or absence of Mg2+. Ruthenium red added to mitochondria after Ca2+ accumulation caused a slow efflux of Ca2+ from mitochondria; A-23187 caused a rapid discharge of accumulated Ca2+ from mitochondria in the presence or absence of ruthenium red. The results suggest that Mg2+ exerts a “protective” effect on the mitochondrial phosphorylating mechanism and may modulate the competitive effects of Ca2+ and ADP for electron transport chain-generated energy. Further, the effects of ruthenium red and A-23187 suggest the possibility of two pathways or “channels” for mitochondrial Ca2+ uptake and release.

Correlation of plasma serotonin changes with platelet aggregation in an in vivo dog model of spontaneous occlusive coronary thrombus formation.

The role of platelets in contributing to occlusive coronary artery thrombus formation remains unresolved. A large number of studies have utilized in vitro techniques to study platelet aggregation. This report describes a model of spontaneous in vivo thrombus formation which involves application of current in the left circumflex coronary artery of the dog. Changes in mean coronary blood flow velocity (50% above control) are used to predict the point at which current can be discontinued without interrupting the ongoing process of thrombus formation. Thrombus formation proceeds to total vessel occlusion within 62 ± 18 minutes after discontinuation of current. Coronary sinus plasma serotonin concentrations are used as an in vivo index of platelet aggregation during thrombus formation. Plasma serotonin levels increased only slightly above baseline levels during initial thrombus formation. Coronary sinus serotonin levels rose markedly after cessation of current, reaching a peak just prior to total vessel occlusion. The marked increase in serotonin concentration observed in the latter stages of thrombus formation strongly suggests that platelet aggregation is a significant factor in the evolution of an occlusive coronary thrombus.

Effects of atractyloside and palmitoyl coenzyme a on calcium transport in cardiac mitochondria

Abstract Palmitoyl CoA (PCoA) and the adenine translocase inhibitor atractyloside (ATR) appear to produce a similar effect in discharging accumulated calcium from cardiac mitochondria. Although mitochondrial respiration is stimulated upon addition of either PCoA or ATR to preparations preloaded with calcium, the effect is not the same as that produced by classical uncouplers. PCoA and ATR also do not interfere with respiration-supported calcium uptake by mitochondria. The presence of exogenous ATP can prevent the calcium discharging effects of PCoA or ATR. Carnitine will prevent the PCoA calcium discharging effect, but has no effect on ATR-induced discharge. It is suggested the PCoA may act at a site on or near the adenine translocase, perhaps through allosteric interaction, to produce an efflux of calcium from mitochondria. The results also suggest that the internal adenine nucleotide pool plays a significant role in mitochondrial calcium retention.

Postischemic recovery of mitochondrial adenine nucleotides in the heart.

BackgroundAdenine nucleotides (AdNs) are lost from the mitochondrial fraction of the heart cell during ischemia. It is unknown whether this pool of AdNs can be replenished after reperfusion. The purpose of this study was to evaluate the postischemic recovery of the mitochondrial AdN pool. Methods and ResultsThe left anterior descending coronary artery (LAD) of the canine heart was occluded for 30 minutes followed by either no reflow, 30-minute reflow, 1-day reflow, or 7-day reflow. Systolic shortening in the TAD-supplied region was absent during occlusion but recovered to approximately 30% of preocclusion values during early reperfusion. Mitochondrial and tissue AdNs (ATP, ADP, and AMP) were determined in the LAD-supplied and left circumflex-supplied (control) regions of the heart. The AdN content (expressed as percent of control values) of mitochondria from the LAD region was 55±101% (p<0.002), 64±7% (p<0.001), 81±6% (p<0.03), and 94±8% for the no-reflow, 30-minutereflow, 1-day-reflow, and 7-day-reflow groups, respectively. The AdN content (expressed as percent of control values) of tissue samples from the LAD region was 52±9% (p<0.002), 48±12% (p<0.02), 68±5% (p<0.002), and 70±9% for the no-reflow, 30-minute-reflow, 1-day-reflow, and 7-day-reflow groups, respectively. There was a good correlation between mitochondrial and tissue AdN (r=0.95). Using initial exchange rates, adenine nucleotide translocase activities of mitochondria from the LAD and control regions were not significantly different. State 3 respiration of LAD mitochondria was depressed (approximately 25%, p<0.05) only in the no-reflow group. Acceptor control ratios of the LAD mitochondria were not significantly different from control values in any group. Conclsions. After 30 minutes of regional ischemia, postischemic restoration of the mitochondrial AdN pool occurs between 1 and 7 days; this restoration is preceded by recovery of respiratory and adenine nucleotide translocase functions. Although the abnormally low levels ofAdN persist in the mitochondrial compartment during the early reperfusion period, postischemic contractile dysfunction cannot be explained by depressed mitochondrial respiratory activity.

Temporal changes in adenylate cyclase activity in acutely ischemic dog heart: evidence of functional subunit damage.

Abstract Literature reports have indicated increased tissue levels of adenosine-3′,5′-cyclic phosphoric acid (cAMP) following ischemic insult to the myocardium. Studies were undertaken to determine possible changes in adenylate cyclase activity from ischemic and control heart tissue following complete coronary artery ligation. No changes were found in adenylate cyclase of ischemic tissue compared to controls following 10 min of occlusion. Basal adenylate cyclase velocities were slightly decreased after 20 min of ischemia and exhibited a 39% decrease after 60 min of coronary occlusion. Isoproterenol- and fluoride-stimulated activities exhibit a greater progressive decline in activity after 20 to 60 min of ischemia. The unphysiological substrate MnATP was utilized to produce a receptor-uncoupled state of the adenylate cyclase enzyme complex. Assays utilizing the substrate MnATP, revealed marked decreases in ischemic heart tissue adenylate cyclase activity following 20 min of coronary artery ligation. The data from the receptor-coupled and uncoupled states suggest initial damage in the catalytic subunit of the adenylate cyclase complex following the onset of myocardial ischemia.

Intermittent ischemia produces a cumulative depletion of mitochondrial adenine nucleotides in the isolated perfused rat heart.

The purpose of the present study was to determine if repetitive myocardial ischemia would result in the cumulative loss of mitochondrial adenine nucleotides. Isolated perfused rat hearts were subjected to continuous or intermittent ischemia. A single 5-minute period of continuous ischemia did not result in a significant decrease in the mitochondrial adenine nucleotide pool; a single 10-minute period of ischemia resulted in a decrease of approximately 17%. Next, the adenine nucleotide content of mitochondria from preischemic and 30-minute continuous ischemic hearts was compared with two groups of hearts undergoing intermittent ischemia (both groups receiving a total of 30 minutes of ischemia). One group received three 10-minute episodes of ischemia interrupted by 5-minute periods of reperfusion (3 x 10-minute intermittent ischemia); the other intermittent ischemic group received six 5-minute episodes of ischemia interrupted by 5-minute periods of perfusion (6 x 5-minute intermittent ischemia). The mitochondrial adenine nucleotide content (expressed as nanomoles per nanomole cytochrome a) for the preischemic and 30-minute continuous ischemic hearts was 14.7 +/- 0.6 and 8.0 +/- 0.4, respectively. The mitochondrial adenine nucleotide content of the 3 x 10-minute intermittent ischemia group (8.5 +/- 0.5) was not significantly different from the 30-minute continuous ischemic group. The mitochondrial adenine nucleotide content of the 6 x 5-minute intermittent ischemia group (11.0 +/- 0.6) was significantly larger than that of the 30-minute continuous and the 3 x 10-minute intermittent ischemia groups (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo inhibition of platelet aggregation and occlusive coronary thrombus formation by a new calcium antagonist (R023-6152)

Summary: Reports have shown that all three major classes of calcium antagonists can inhibit platelet aggregation in vitro. In this study, we compared the platelet antiaggregatory effects of R023–6152, a thiazepinone-type calcium antagonist, with diltiazem in an in vivo canine model of spontaneous coronary thrombosis. Plasma serotonin (5-HT) levels measured in the coronary sinus were used as an index of in vivo platelet aggregation and coronary flow measured by a Doppler flow probe. Untreated controls developed total coronary occlusion in 62 × 18 min after the current used to initiate thrombus formation was discontinued. Control 5-HT levels peaked at 213 × 63 ng/ml just before occlusion. Dogs receiving intravenous (i.v.) R023–6152 (200 μg/kg) or diltiazem (50 μg/kg) immediately after the current was discontinued exhibited no significant elevations in 5-HT values (12.3 × 1.4 and 1.84 × 0.42 ng/ml for R023–6152 and diltiazem, respectively) or development of coronary occlusions in the next 3 h. Small, transient decreases in arterial pressure (8–10 mm Hg) and changes in contractility occurred during infusion of both drugs. Gravimetric determinations of thrombus weights showed significantly smaller thrombi in the drug-treated animals as compared with controls. The results indicate that both R023–6152 and diltiazem are effective in suppressing in vivo platelet aggregation associated with occlusive coronary thrombus formation.

In vitro and in vivo effects of R023-6152 on heart mitochondrial calcium and energy metabolism.

Summary: Previous studies have shown that Ca2+ channel antagonists in all chemical classes can inhibit Na+ induced Ca2+ release from mitochondria. The effects of R023–6152, a new thiazepinone Ca2+ channel antagonist, on isolated rabbit heart mitochondrial Ca2+ transport and respiratory activity were compared with those of diltiazem. Heart mitochondria were also isolated and assayed from dogs treated in vivo with either 8023–6152 or diltiazem. The results indicate that R023–6152 produces half-maximal inhibition of Na+-induced Ca2+ release from isolated mitochondria at relatively the same concentrations (10–30 μM) as diltiazem but also produces inhibition of mitochondrial Ca2+ uptake and state 3 respiration at concentrations (25–100 μM), at which diltiazem has no effect. The greater lipophilicity of R023–6152 in gaining access to and inhibiting the phosphate transporter in the mitochondrial membrane as compared with that of diltiazem may explain these results. Heart mitochondria isolated from dogs treated with diltiazem and R023–6152 exhibited lower rates of state 3 respiration as compared with controls. We suggest that this may result from a reduction in transsarcolemmal Ca2+ flux causing a down-regulation in mitochondrial dehydrogenase activity and not from any direct intracellular effects of the two drugs.

The Biochemistry of Myocardial Failure

Almost twenty years ago, Richard Bing and colleagues [1] addressed the question, “What is cardiac failure?” These authors concluded that we had no answer to this question, partly because of limitations in our techniques, but more likely because there is not one single answer to the complex array of changes associated with the heart’s failure to pump blood. Recently, Mall and O’Rourke [2] noted that the relationship between biochemical changes and depressed myocardial contractility associated with failure remain unclear and a subject of continuing investigation. Despite tremendous advances in techniques and knowledge, our understanding of the fundamental biochemical mechanisms underlying myocardial contractile function and its regulation is still limited. These limitations make the understanding of myocardial failure all the more difficult and congestive heart failure continues to be a major clinical problem [3]. Although major deficiencies still exist in our knowledge of cause and effect relationships between biochemical “defects” and myocardial failure, it is clear that prolonged hemodynamic stress leads to decreased myocardial contractility [4]. Sustained hemodynamic stress can be caused by a variety of factors including hypertension, valvular heart disease and loss of functional myocardium following ischemic injury. Clinical congestive heart failure, in the vast majority of cases, results from hypertensive or coronary artery disease (ischemic injury). The focus of this chapter will be on those biochemical changes related to these two general types of hemodynamic stress.