The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein-free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.

In TTX-sensitive nerve and skeletal muscle Na+ channels, selective modification of external carboxyl groups with trimethyloxonium (TMO) or water-soluble carbodiimide (WSC) prevents voltage-dependent Ca2+ block, reduces unitary conductance, and decreases guanidinium toxin affinity. In the case of TMO, it has been suggested that all three effects result from modification of a single carboxyl group, which causes a positive shift in the channel's surface potential. We studied the effect of these reagents on Ca2+ block of adult rabbit ventricular Na+ channels in cell-attached patches. In unmodified channels, unitary conductance (gamma Na) was 18.6 +/- 0.9 pS with 280 mM Na+ and 2 mM Ca2+ in the pipette and was reduced to 5.2 +/- 0.8 pS by 10 mM Ca2+. In contrast to TTX-sensitive Na+ channels, Ca2+ block of cardiac Na+ channels was not prevented by TMO; after TMO pretreatment, gamma Na was 6.1 +/- 1.0 pS in 10 mM Ca2+. Nevertheless, TMO altered cardiac Na+ channel properties. In 2 mM Ca2+, TMO-treated patches exhibited up to three discrete gamma Na levels: 15.3 +/- 1.7, 11.3 +/- 1.5, and 9.8 +/- 1.8 pS. Patch-to-patch variation in which levels were present and the absence of transitions between levels suggests that at least two sites were modified by TMO. An abbreviation of mean open time (MOT) accompanied each decrease in gamma Na. The effects on channel gating of elevating external Ca2+ differed from those of TMO pretreatment. Increasing pipette Ca2+ from 2 to 10 mM prolonged the MOT at potentials positive to approximately -35 mV by decreasing the open to inactivated (O-->I) transition rate constant. On the other hand, even in 10 mM Ca2+ TMO accelerated the O-->I transition rate constant without a change in its voltage dependence. Ensemble averages after TMO showed a shortening of the time to peak current and an acceleration of the rate of current decay. Channel modification with WSC resulted in analogous effects to those of TMO in failing to show relief from block by 10 mM Ca2+. Further, WSC caused a decrease in gamma Na and an abbreviation of MOT at all potentials tested. We conclude that a change in surface potential caused by a single carboxyl modification is inadequate to explain the effects of TMO and WSC in heart. Failure of TMO and WSC to prevent Ca2+ block of the cardiac Na+ channel is a new distinction among isoforms in the Na+ channel multigene family.

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2Cl- cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volume(test)/volume(control)) were determined. Exposure to 10 microM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 was 0.77 +/- 0.33 microM. Activating guanylate cyclase with 100 microM sodium nitroprusside also decreased cell volume to 0.889 +/- 0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 microM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2Cl- cotransport was tested by blocking the cotransporter with 10 microM bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 microM 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2Cl-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 microM 8-Br-cGMP prevented the effect of 1 microM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 microM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100 microM) amplified the volume decrease caused by a low dose of ANF (0.01 microM) approximately fivefold. In contrast, neither 100 microM 8-Br-cAMP nor 50 microM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also appear to modulate cell volume.

Voltage clamp hyperpolarization and depolarization result in currents consistent with depletion and accumulation of potassium in the extracellular clefts o cardiac Purkinje fibers exposed to sodium-free solutions. Upon hyperpolarization, an inward current that decreased with time (id) was observed. The time course of tail currents could not be explained by a conductance exhibiting voltage-dependent kinetics. The effect of exposure to cesium, changes in bathing media potassium concentration and osmolarity, and the behavior of membrane potential after hyperpolarizing pulses are all consistent with depletion of potassium upon hyperpolarization. A declining outward current was observed upon depolarization. Increasing the bathing media potassium concentration reduced the magnitude of this current. After voltage clamp depolarizations, membrane potential transiently became more positive. These findings suggest that accumulation of potassium occurs upon depolarization. The results indicate that changes in ionic driving force may be easily and rapidly induced. Consequently, conclusions based on the assumption that driving force remains constant during the course of a voltage step may be in error.