MODULATION OF RABBIT VENTRICULAR CELL-VOLUME AND NA+/K+/2CL- COTRANSPORT BY CGMP AND ATRIAL-NATRIURETIC-FACTOR

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-mu-M 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 Was 0.77 +/- 0.33-mu-M. Activating guanylate cyclase with 100-mu-M 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-mu-M) 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-mu-M bumetanide (BUM) and removing the transported ions. After BUM treatment, 10-mu-M 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methane-sulfonate 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-mu-M 8-Br-cGMP prevented the effect of 1-mu-M ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10-mu-M) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100-mu-M) amplified the volume decrease caused by a low dose of ANF (0.01-mu-M) approximately fivefold. In contrast, neither 100-mu-M 8-Br-cAMP nor 50-mu-M forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by I-125-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also ap ear to modulate cell volume.