| Summary: | Investigations were made into the role of blood-borne substances affecting myocardial contractility. Isolated left and right guinea-pig au -ia were used as bioassay for the detection of endogenous cardiotonic substances in bovine serum. Results show that serum that had been buffer exchanged to Krebs-Henseleit by dialysis or Sephadex G-25, produced positive inotropic and positive chronotropic effects on the isolated guinea-pig au-ia. The hypodynamic state was not a prerequisite for the detection of cardioactivity. The cardioactive effectswere not blocked by the combined presence of propranolol and methysergide (both 10 6M) and were also dissimilar in time course from other known cardiotonic agents such as noradrénaline, isoprenaline, 5-hydroxytryptamine and cardiac glycosides. After ultrafiltration on Amicon diaflo membranes (XM100A and PM30A) activity was found solely in the retentate fractions. The ultrafiltrates were devoid of positive inotropic and chronotropic actions. Since the molecular weight cut-off of the XM100A membranes was 100 Kilodaltons, it is probable that the active principle is greater than 100 kilodaltons. Alternatively, a smaller active substance may be bound to a large protein. The cardioaciive factor in whole bovine serum was heat labile and lost after 30 minutes incubation at 70 0C or boiling for 10 minutes. Activity of whole bovine serum was not cold labile during a 30 minute incubation at 0°C. Activity was also present after dialysis at 4-8 °C for up to 48 hours. Cardioactivity of whole serum was lost following acidification to pH 5.0 at 4-8 0C for 24 hours. This experiment was extended to include a wider range of pH values between 4.5 and 75. The positive inotropic and chronotropic activities of serum were markedly reduced at acidic pH values. The cardioactive factor was also lost after equilibration of serum to physiological buffers of a low ionic strength. The loss of activity was independent of the nature of the buffer and was prevented by raising the ionic strength with the addition of NaCl. The cardiotonic principle was also sensitive to the addition of KBr but not equimolar (or higher) concentrations of KCI or NaCl. The above results taken together would be consistent with a proteinaceous nature of the active substance. The major purification methods therefore employed included precipitation techniques involving (NH4)2SO4 and polyethyleneglycol-8000, ion exchange, hydrophobic interaction chromatography and gel filtration. Attempts to isolate and purify the cardiotonic agent were largely unsuccessful due, particularly, to the labile nature of the active molecule when exposed to non-physiological experimental conditions. Investigations in to the cellular mechanism of action of the cardioactive factor in buffer exchanged serum suggested that the effects may perhaps be mediated by increases in the level of endogenous cyclic AMP and not via inhibition of the N a+,Kt ATPase. In conclusion, the present study has demonstrated the existence of a factor in bovine serum that produced positive inotropic and chronotropic effects on the isolated guinea-pig au-ia. Properties of the cardioactive factor suggest the existence of a novel proteinaceous substance with some previously unidentified characteristics.
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