Evidence exists for a specific diabetic cardiomyopathy independent of concurrent vascular disease. We tested the hypothesis that chronic hyperglycaemia found in streptozotocin- (STZ) induced diabetic rats leads to an altered response to and contractile effects of hyperosmotic shrinkage in ventricular myocytes. Analysis confirmed significant hyperglycaemia and revealed significant blood hyperosmolarity in STZ-treated rats. Myocyte volume changes, shortening and intracellular Ca(2+) ([Ca(2+)](i)) transients were measured in cells superfused with normal Tyrode (NT, 300 mmol/kg) and then hyperosmotic Tyrode (HT, 440 mmol/kg) at 35-36 degrees C. Shrinking significantly reduced the amplitude of shortening, whilst the [Ca(2+)](i) transient was significantly increased. The time course of both shortening and the [Ca(2+)](i) transient were prolonged in myocytes from STZ-treated compared to control rats. Time to peak shortening was 130.3 ms in STZ compared to 100.2 ms in control myocytes. Time to peak [Ca(2+)](i) transient was 70.8 ms in STZ compared to 44.6 ms in control myocytes and the time from peak to half recovery was 191.0 ms in STZ compared to 169.1 ms in control myocytes. Fractional SR Ca(2+) release, assessed by the application of caffeine, was increased by shrinking. However, the effects of raised extracellular osmolarity on volume changes, contractility and [Ca(2+)](i) were not altered by the chronic hyperglycaemia found in STZ-treated rats.

Microtubules form part of the cytoskeleton. Their role in adult ventricular myocytes is not well understood although microtubule proliferation has previously been linked with reduced contractile function. We investigated the effect of the anti‐tumour drug taxol, a known microtubule polymerizing agent, on Ca2+ handling in adult rat ventricular myocytes. Treatment of cells with taxol caused proliferation of microtubules. In taxol‐treated cells there was a reduction in the amplitude of contraction, no significant effect on the amplitude of L‐type Ca2+ current, but a significant reduction in the amplitude of the Ca2+ transient. Caffeine was used to release Ca2+ from the sarcoplasmic reticulum (SR). There was a significant reduction in the ratio of electrically stimulated : caffeine‐induced Ca2+ transients in taxol‐treated cells. This observation is consistent with the hypothesis that taxol reduces fractional SR Ca2+ release. We suggest that the negative inotropic effect of taxol may, at least in part, be the result of reduced release of Ca2+ from the SR. Microtubules may be important regulators of Ca2+ handling in the heart.

It is widely believed that Ca release from the sarcoplasmic reticulum (SR) in heart muscle is due to "Ca-induced Ca-release" (CICR), triggered by transmembrane Ca entry. However, in intact guinea-pig cells or cells dialysed with cAMP there may be an additional mechanism - SR release may be activated directly by membrane depolarisation without Ca entry. The first objective of the present study was to investigate whether this "voltage-activated Ca release" (VACR) mechanism is present across species such as rabbit, rat and guinea-pig. The second objective was to characterise the dependence of a VACR mechanism on internal [cAMP]. Membrane current was measured with the whole-cell patch-clamp technique, intracellular [Ca] was monitored with Fura-2 (or a combination of Fluo-3/SNARF-1). Rapid changes of superfusate (within 100 ms) were made using a system which maintained cell temperature at 37 degrees C. We used a train of conditioning pulses to ensure a standard SR load before each test pulse. In rabbit myocytes dialysed with 100 microM cAMP, 89.6 +/- 7.0% of the control intracellular Ca (Cai) transient was still elicited by depolarisation during a switch to 5 mM Ni, which blocked pathways for Ca entry. This suggested that rabbit myocytes possess a VACR mechanism. The percentage of control Cai transient elicited by depolarisation in the presence of 5 mM Ni (i.e. magnitude of VACR) increased in a graded fashion with the pipette [cAMP] between zero and 100 microM. In rat myocytes dialysed with 50 microM cAMP, 64.4 +/- 6.2% of SR release was activated by depolarisation in the presence of 5 mM Ni, suggesting the presence of a VACR mechanism. The extent to which VACR triggered SR release increased with the pipette [cAMP] between zero and 50 microM. In guinea-pig myocytes dialysed with 100 microM cAMP, 74.6 +/- 3.6% of the control Cai transient was elicited by depolarisation in the presence of 5 mM Ni. The degree to which VACR triggered SR release was also graded with the pipette [cAMP] between zero and 100 microM. It therefore appears that each of the three species might possess a VACR mechanism which can be modulated by the internal [cAMP]. This may reflect an effect of cAMP to phosphorylate key proteins involved in excitation-contraction coupling. Under normal physiological conditions with a basal [cAMP] between 2 and 20 microM, VACR may play a role in triggering SR release. The role of VACR may increase under conditions which increase internal [cAMP].

We have developed a technique for making a rapid solution change, whilst at the same time maintaining the temperature of the preparation at 37 degrees C. It is technically difficult to use rapid solution changes when experiments are performed at normal mammalian body temperature. As a solution is heated from room temperature to 37 degrees C, gas bubbles form in the rapid-flowing solution streams, and these disturb a cell or attached recording pipettes. We describe a system that has been developed to eliminate these problems. We show how to construct the different components of the system, and we have designed an electronic circuit to control solution changes. We have performed tests to characterise the function of this system. Solution flow out of the nozzle of the device (0.88 ml min-1, linear flow velocity 11.6 cm s-1) caused a fall in the steady-state temperature at the experimental preparation of only 0.3 degrees C. The device which takes between 0.5 and 1 s to completely change the superfusate of a single cell, was used routinely with five different experimental solutions. This system may be valuable in studies which require rapid solution changes to be performed at a normal mammalian body temperature.

We investigated the effect of external Cd2+ on the Na/Ca exchange and the L-type Ca channel current (ICa,L) in whole cell patch-clamped rabbit ventricular myocytes at 36 degrees C. After the interfering ion channels and the Na/K pump were blocked, the exchange current was measured as the membrane current that was inhibited by 5 mM nickel. External Cd2+ inhibited Na/Ca exchange with a dissociation constant (KD) of 320.6 +/- 12.4 microM and a Hill coefficient of 1.5 +/- 0.09 (n = 13 cells) and ICa,L with a KD of 2.14 +/- 0.15 microM and a Hill coefficient of 0.74 +/- 0.03 (n = 11 cells). We observed some overlap in the Cd2+ concentration that blocked each mechanism. Cd2+ (100-500 microM) is used commonly to block ICa,L completely. However, 100 microM Cd2+ also inhibits 20% of the Na/Ca exchange activity, whereas 500 microM Cd2+ inhibits 60%.