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1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca 2 þ current (I Ca ) observed in the absence of exogenous b-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell I Ca in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 mmol l À1) increased basal I Ca (Po0.05), whereas H-89 inhibited I Ca in a concentration-dependent manner with an IC 50 B5 mmol l À1. H-89 also inhibited the response to 1.0 mmol l À1 isoprenaline, although relatively high concentrations (30 mmol l À1) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 mmol l À1 H-89 on time-dependent recovery of I Ca from voltage-dependent inactivation as well as the steady-state gating of I Ca . T 0.5 (time for I Ca to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (Po0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of I Ca were unaffected by 10 mmol l À1 H-89 or 1 mmol l À1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V 0.5 for activation and inactivation became more negative. 5 Data show that basal I Ca is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied b-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to b-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.

1 Voltage clamp was used to investigate the effects of N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a potent inhibitor of PKA, on transient outward K þ current (I to ) and inward rectifying K þ current (I K1 ) in rat cardiac muscle. 2 Initial experiments, performed using descending voltage ramps, showed that H-89 inhibited both the outward and inward ramp currents in a concentration-dependent manner at concentrations between 5 and 60 mmol l À1 . A similar degree of inhibition was observed when I to and I K1 were recorded using square wave depolarising and hyperpolarising voltage steps, respectively. 3 The IC 50 was 35.8 mmol l À1 for I to and 27.8 mmol l À1 for I K1 compared to 5.4 mmol l À1 for L-type Ca 2 þ current (I Ca ). The Hill coefficients for I to , I K1 and I Ca were À1.97, À1.60 and À1.21, respectively. In addition to inhibiting I to amplitude, H-89 also accelerated the time to peak and the rate of voltagedependent inactivation so that the time course of I to was abbreviated. 4 Paired-pulse protocols were performed to study the effects of H-89 on steady-state activation and inactivation as well as recovery from voltage-dependent inactivation. H-89 produced a concentrationdependent rightward shift in voltage-dependent activation but had no significant effect on steady-state inactivation. Recovery from voltage-dependent inactivation was delayed, although this was only visible at the highest concentration (60 mmol l À1 ) used. 5 In experiments investigating the effects of elevated cyclic AMP, the b-adrenergic agonist isoprenaline and the phosphatase inhibitor calyculin A had no major effects on I to or I K1 . 6 Data suggest that the effects of H-89 on K þ currents are more complex than simple inhibition of PKA-mediated phosphorylation.

General anaesthetics have previously been shown to have profound effects on myocardial function. Moreover, many patients suffering from diabetes mellitus are anaesthetised during surgery. This study investigated compromised functioning of cardiac myocytes from streptozotocin (STZ)-induced diabetic rats and the additive effects of halothane on these dysfunctions. Ventricular myocytes were isolated from 8 to 12 weeks STZ-treated rats. Contraction and intracellular free calcium concentration ([Ca2+]i) were measured in electrically field-stimulated (1 Hz) fura-2-AM-loaded cells using a video-edge detection system and a fluorescence photometry system, respectively. L-type Ca2+ current was measured in whole cell, voltage-clamp mode. Halothane significantly (p < 0.01) depressed the amplitude and the time course of the Ca2+ transients in a similar manner in myocytes from control and STZ-treated rats. However, the effect of halothane on the amplitude of shortening and L-type Ca2+ current was more pronounced in myocytes from STZ-treated animals compared to age-matched controls. Myofilament sensitivity to Ca2+ was significantly (p < 0.01) increased in myocytes from STZ-treated rats compared to control. However, in the presence of halothane the myofilament sensitivity to Ca2+ was significantly (p < 0.05) reduced to a greater extent in myocytes from STZ-treated rats compared to controls. In conclusion, these results show that contractility, Ca2+ transport and myofilament sensitivity were all altered in myocytes from STZ-treated rats and these processes were further altered in the presence of halothane suggesting that hearts from STZ-induced diabetic rats are sensitive to halothane.

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