Guinea pig ventricular myocytes in whole cell configuration were treated with tyrosine kinase (TK) inhibitors [genistein (Gst), tyrphostin A23 (T23), and tyrphostin A25 (T25)] and with inactive analogs [daidzein, genistin, and tyrphostin A1 (T1)] to measure effects on L-type Ca2+ current ( I Ca,L). Gst inhibited I Ca,L(IC50 = 47 μM) without affecting its time course or shifting the I Ca,L-voltage relationship. At the highest concentration of isoflavone tested (200 μM), I Ca,L was inhibited by 66 ± 7% (Gst), 22 ± 2% (daidzein), and 1 ± 3% (genistin). Inhibition of I Ca,L by the active tyrphostins was significantly larger than inhibition by T1; at 200 μM the inhibitions were 72 ± 6% (T23), 71 ± 6% (T25), and 27 ± 6% (T1). The phosphotyrosine phosphatase inhibitor orthovanadate (1 mM) had a small stimulatory effect (6 ± 2%) on basal I Ca,L and blocked the inhibition of I Ca,L by TK inhibitors. The data suggest a role for the TK-phosphotyrosine phosphatase system in the regulation of cardiac Ca2+ channels.

1. Kinase-mediated chloride currents (I1c) in guinea-pig ventricular myocytes were activated by application of phorbol ester or forskolin, and compared with currents induced by hyposmotic swelling. Swelling-activated current was identified as Ici from changes in reversal potential, outward rectification and conductance when the Cl-gradient was modified.2. Kinase-stimulated currents were relatively time and voltage independent, whereas hyposmotic swelling-stimulated (hyposmotic-stimulated) currents inactivated during 100 ms puises to positive potentials. Forskolin stimulated time-independent IcI in myocytes with current unresponsive to hyposmotic superfusion, and superimposed a similar pedestal on time-dependent I.c in swollen myocytes.3. Less negative holding potentials depressed hyposmotic-stimulated Ici tested at +80 mV;inhibition was half-maximal at -25 mV. Pulses from -80 to +80 mV inactivated up to 75 % of Ici along a multi-exponential time course; repolarization elicited inwardly developing tail currents whose time courses suggest complex gating. 4. Hyperpolarizations, after strongly-inactivating depolarizations, triggered reactivating tail currents whose amplitude and configuration were dependent on voltage and Cl-gradients; tails were large and inwardly developing at potentials negative to the calculated Clequilibrium potential (Ecl), small and outwardly developing at potentials positive to Ec1, and time independent near Ecl.5. These results suggest that the volume-sensitive Cl-channels investigated here are distinct from other Cl-channels in guinea-pig ventricular myocytes. However, their voltagedependent properties strongly resemble those of volume-sensitive Cl-channels in certain epithelial cells. (Vandenberg et al. 1994), confirmatory biophysical evidence is lacking. The hyposmotic swelling-stimulated ('hyposmoticstimulated') currents recorded in the studies cited above were time independent and had outwardly rectifying current-voltage (I-V) relationships, i.e. they were kinetically indistinguishable from basal (Duan & Nattel, 1994) and kinase-activated (e.g.

Guinea‐pig ventricular myocytes were superfused with hyperosmotic (sucrose) Tyrode solution (1.2–2.8 times (T) normal osmolality) for up to 40 min. Action potentials were recorded with microelectrodes, and membrane currents with the perforated‐ or ruptured‐patch technique. Hyperosmotic treatment for 20 min shrunk cell volume and hyperpolarized the membrane. Moderate (1.2–1.5T) treatment caused biphasic changes in action potential configuration (rapid minor shortening quickly followed by lengthening to a stable 110% control duration). Severe (2.2–2.8T) treatment caused triphasic changes (marked early shortening, strong rebound lengthening and subsequent pronounced shortening). At peak lengthening (6–10 min) action potentials (165% control duration) had a hump near –30 mV and slowed terminal repolarization. In accordance with previous studies, hyperosmotic solution inhibited the delayed rectifier K+current, and enhanced the outward Na+–Ca2+ exchange current (INaCa) at plateau potentials. A novel finding was that hyperosmolality reduced the amplitude of L‐type Ca2+ current (ICa,L) and slowed its rate of inactivation. Experiments on myocytes loaded with indo‐1 suggest that the reduction in ICa,L is due to a rapid elevation of [Ca2+]i. When impaled myocytes were preloaded with EGTA, severe hyperosmotic treatment induced a rapid monotonic shortening of the action potential to a stable 20% of control duration. Addition of external K+ quickly nulled the hyperpolarization and slowly lengthened the action potential. The results suggest that modified electrical activity in osmotically shrunken myocytes is primarily caused by increases in [K+]i, [Na+]i and [Ca2+]i: (i) elevated [K+]i hyperpolarizes the membrane (which may contribute to increased [Na+]i); (ii) elevated [Na+]i shortens all phases of the action potential (increased outward‐directed INaCa); and (iii) elevated [Ca2+]i has antagonistic plateau shortening (inhibition of inward ICa,L) and plateau lengthening (reduced outward INaCa) influences, as well as a strong subplateau lengthening effect (enhanced inward INaCa).

Although the observed L(p) is relatively small in magnitude, the low E(a) and the sulfhydryl reagent-induced modification of L(p) are characteristic of channel-mediated water transport. These data suggest that water flux across the sarcolemma of guinea-pig and rat heart cells occurs through parallel pathways, i.e., the majority passing through water channels and the remainder penetrating the lipid bilayers.

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