Kye-Chang Ko

The Effects of Substrates on Contractility of Rat Atria Depressed with Halothane

A concentration of approximately 6 mg/100 ml halothane was necessary to maintain 50 per cent depression of contractility of rat atria suspended in a modified Krebs-Ringer bicarbonate glucose medium, pH 7.4, kept at 30 C for two hours. Sodium pyruvate, sodium acetate and lactic acid partially restored the contractility of the depressed atria. Maximally effective concentrations were 2.5 mM for pyruvate and acetate and 5 niM for lactic acid. Neither 5 nor 20 mM of additional glucose restored the force of contraction of halothane- depressed atria, although these concentrations markedly increased the contractility of normal atria not exposed to halothane. Contractility of normal atria was not increased by acetate or lactic acid and was increased only slightly by pyruvate. The results arc consistent with the hypothesis that at least part of the negative inotropic action of halothane is the result of inhibition of glucose uptake or utilization in the glycalytic pathway. Blockade must occur prior to the conversion of pyruvate to acetyl CoA.

Effect of Starvation on Contractile Response of Isolated Rat Atria to Citrate and Bicarbonate-Free Medium

Summary Isolated atria from 24-hr starved rats show a smaller rate of decline of contractility with time, when incubated in the absence of exogenous substrate than atria from fed rats. During starvation, endogenous substrate apparently accumulates and can be used by the heart to maintain contractility in vitro. The nature of this endogenous substrate was elucidated by the use of bicarbonate-free medium and 1.5 mM citrate. These agents apparently depress contractility by an inhibition of the phosphofructokinase (PFK) reaction in the glycolytic cycle. Both inhibitors produced marked, but not complete, contractile depression of atria from starved or fed rats incubated in the absence of exogenous substrate. This depression is likely due to an interference with the utilization of endogenous substrate, probably glycogen, normally metabolized via PFK. The maintenance of some degree of contractility in the presence of these inhibitors indicates that endogenous substrates, not metabolized via PFK, are utilized. Addition of 20 mM glucose to atria from fed or starved rats incubated in substrate-free medium resulted in a marked positive inotropic effect in both cases. This may indicate that glycolytic enzymes are present and functional after 24 hr of starvation.

Contractile Depression of Rat Atria by Halothane in the Absence of Glucose

Halothane (approximately G mg/100 ml) administered to isolated rat atria produced about the same degree of contractile depression whether or not glucose was present in the bathing medium. Thus, the depressant action of halothane is independent of exogenous glucose and cannot be simply attributed to a block in glucose uptake or phosphorylation to glucose-G-phosphate. The rate of depression of contractility induced by talothane in the presence of glucose is much faster than that induced when glucose is omitted from the medium, further suggesting that the site of action is not glucose uptake or phosphorylation. The rate of depression induced by halothane is similar to those seen with the known inhibitors of glycolysis, citrate and bicarbonate-free medium. The action probably takes place at the glucose phosphate isomerase step responsible for converting glucose-G-phosphate to fructose-G-phosphate. Previous reports have ruled out actions after this step. The possibility of multiple sites of action cannot be ruled out.

The Effect of Fructose on Halothane-depressed Rat Atria

Fructose produced dose-dependent increases in the force of contraction of isolated rat atria depressed by substrate-free Krebs-Ringer bicarbonate medium. The maximally effective concentration of fructose was 30 mM. The nonmetabolized sugar sucrose, also administered at 30 mM, was without effect when administered under the same conditions. Neither fructose (30 mM) nor glucose (20 mM) was effective in restoring force of contraction of atria depressed by Krebs-Ringer medium containing 5.5 mM glucose without bicarbonate; pyruvate (5 mM), however, produced a marked positive inotropic effect in this medium. Since bicarbonate is necessary for phospho-fructokinase activity, these results are taken as evidence that fructose is metabolized via this step to serve as an energy-yielding fuel for atrial contractility. In another experiment atria suspended in Krebs-Ringer bicarbonate glucose medium were depressed 50 per cent by approximately 6 mg/100 ml of halothane. Addition of 30 mM fructose to these depressed atria resulted in a marked increase in contractile force. The results are consistent with a previous report suggesting blockade by halothane of the uptake or utilization of glucose in the glycolytic pathway, and further pinpoint the blockade as an early step in the glycolytic sequence prior to the phospho-fructokinase step.

Multiple Mechanisms of Action of Halothane and Methoxyflurane on Force of Contraction of Isolated Rat Atria

The positive inotropic actions of 5 mM pyruvate and 30 mM fructose were determined in isolated rat atria bathed in normal medium (Krebs-Ringer bicarbonate with 5.5 mM glucose) and in atria depressed by exposure to substrate-free, bicarbonate-free, halothane, methoxyflurane, or low (½ normal) calcium containing medium. Pyruvate produced the maximum increase in force of contraction in atria depressed by substrate-free or bicarbonate-free medium, had less effect (about half the maximum effect) on atria depressed by halothane or methoxyflurane, and had no effect on atria exposed to normal or low-calcium medium. Fructose produced similar results except that it was without effect in atria depressed by bicarbonate-free medium. These results are interpreted as indicating that halothane and methoxyflurane depress the force of contraction by two or more mechanisms: one involving a block in glycolysis as previously demonstrated; another involving another mechanism(s).

Rate of Depression of Atrial Contractility Inducted by Citrate, Bicarbonate-Free Medium, Hydrochloric Acid, and Halothane

Summary Citrate, bicarbonate-free medium, and halothane all produced rapid depressions of atrial contractility, depression occurring much more quickly than that seen by merely omitting glucose from the bathing medium. This suggests a site of action for these agents which is different from the glucose uptake step or the hexokinase step involving phosphorylation of glucose to glucoses-6-phosphate. The results are consistent with previous reports suggesting the phos-phofructokinase step as the site of action of citrate and bicarbonate-free medium and also suggests the glucose phosphate isomerase step as the site of halothane action. Hydrochloric acid produced a slow decline in contractility, similar to that seen with glucose-free medium. This can be interpreted as either a rapid effect on glucose uptake or phosphorylation or a slowly developing effect at the phosphofructokinase step. Addition of 20 mM glucose partially overcame the depression induced by citrate but had little or no effect on that produced by bicarbonate-free medium, halothane, or hydrochloric acid. This suggests that citrate depression is surmountable and probably competitive in nature while the other depressants may be of the noncompetitive type.

The Effect of Halothane on the Contractility of Atria from Starved Rats

The purpose of this experiment was to determine whether halothane interferes with the utilization of lipid as a source of fuel for the contractile process by the isolated rat atria. Rats were starved 24 hours in order to increase the lipid content of the heart. Atria from starved rats were better able to maintain their contractility in the absence of exogenous substrate and also were more resistant to depression by halothane than atria from fed rats. Approximately 11 mg/100 ml (1.41 vol per cent) halothane were necessary to achieve 50 per cent depression of the force of contraction of atria from starved rats whether glucose was present in the bathing medium or not. To achieve the same degree of depression in the presence or absence of glucose in atria from fed rats required about 6.5 mg/100 ml (0.83 vol per cent). Atria from starved rats behaved like atria from fed rats in that the depressant action of halothane was independent of external glucose. The site of action of halothane in either case cannot be related to the uptake or phosphorylation of glucose, but may involve the glucose phosphate isomerase step, as previously suggested. The greater halothane requirement for atria from starved rats suggests that endogenous lipid accumulates during starvation and is used as an energy source for the contractile process in the face of a halothane-induced block in glycolysis. Other possible reasons for the resistance of atria from starved rats to halothanes depressant action are discussed.

The effects of substrates on rat atria depressed with bicarbonate-free medium, citrate, or low calcium.

Summary Atria, incubated in bicarbonate-free medium containing 5.5 mM glucose at pH 7.4, demonstrated a progressive decline in contractility not seen in bicarbonate-containing normal medium. Addition of 5 ml pyruvate, lactate, or acetate to atria depressed by bicarbonate-free medium produced a marked increase in contractility. Such an effect was not seen by addition of glucose (30 mM) or fructose (20 mM) indicating a block in the Embden-Meyerhof pathway produced by the bicarbonate-free medium at some point between fructose and pyruvate and most probably at the phos-phofructokinase step. Addition of citrate to atria incubated in the normal bicarbonate-containing medium produced dose-dependent decreases in contractility. The effects of substrates on citrate-depressed atria were in some respects similar to their effects on atria depressed by bicarbonate-free medium. Pyruvate (5 mM) and lactic acid (5 mM) increased the force of contraction of citrate-depressed atria whereas fructose (30 mM) was without effect. These results are consistent with an inhibitory effect of citrate at some point in the Embden-Meyerhof pathway such as the phosphofructokinase step. Addition of acetate (5 mM) to citrate-depressed atria produced little, if any, effect. Additional glucose (20 mM) produced a marked positive inotropic effect in citrate-depressed atria, This is not in accord with a citrate-induced block of phos-phofructokinase unless one postulates an effect of glucose on contractility which is not dependent on its metabolism via the phos-phofructokinase step. The mechanism of the depressant action of citrate cannot be solely attributed to a lowering of ionized medium calcium by chelation with citrate since the action of substrates on atria depressed by low calcium was quite different from their action on citrate-depressed atria. Pyruvate and lactic acid, which produced marked increases in contractility of citrate-depressed atria, were without effect on atria depressed by low calcium. Addition of glucose had less effect on atrial contraction when added to the low-calcium medium than when added to the citrate medium. Indeed, the marked positive inotropic action of 20 mM additional glucose seen in normal or citrate-depressed atria was virtually abolished in atria depressed by removal of 3/4 of the calcium from the medium.

Effect of Prolonged Starvation on the Functional Status of the Isolated Rat Atria

Summary Starvation results in a marked loss of body weight in rats, e.g., a 30% loss in 4 days after which the animals begin to die. By the sixth day only 5 of the 20 were still living. Despite these drastic effects on the rat as a whole, cardiac function, as determined by developed tension of isolated atria in glucose-containing medium, was as good or better than that from fed rats. When placed in substrate-free medium, atria from fed rats showed a marked decline in developed tension, e.g., a 44% decline in 40 min. In contrast, the developed tension of atria from rats starved 1 to 6 days showed a smaller rate of decline with the least decline seen in atria from rats starved for 2 days. These results suggest (a) that prolonged starvation has no deleterious effect on cardiac function and (b) starvation increases the storage of readily metabolizable substrate useful for the functional activity of the atria.

The Mechanism of the Negative Inotropic Effect of Methoxyflurane on Isolated Rat Atria

The force of contraction of isolated rat atria suspended in Krebs-Ringer bicarbonate medium containing 5.5 mM glucose was depressed 55 per cent by approximately 9 mg/100 ml of methoxyflurane in the medium bathing the atria. This corresponds to a partial pressure of 1.84 torr (0.24 vol per cent). Addition of fructose (30 mM) or pyruvate (5 mM) to atria depressed by methoxyflurane resulted in a marked positive inotropic action. Additional glucose (20 mM) was relatively ineffective in atria similarly treated. These results suggest an interference with glucose utilization at an early step in glycolysis above the phosphofructokinase step. Methoxyflurane had its usual depressant action in the absence of exogenous glucose (substrate-free medium). Thus, glucose uptake and phosphorylation were ruled out as the site of action of methoxyflurane. These results suggest that methoxyfluranes depressant action results from a block at the glucose phosphate isomerase step, the step responsible for converting glucose-6-phosphate to fructose-6-phosphate. Multiple sites of action are not ruled out by these results.