MECHANISMS OF TOLERANCE TO SODIUM-NITROPRUSSIDE IN RAT CULTURED AORTIC SMOOTH-MUSCLE CELLS

1 While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sG C) activation, the mechanisms responsible for this phenomenon in intac t cells remain unclear. In the present study, possible mechanisms of t olerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 mu M SNP over a 15 min period in the presence of a phosphodiesterase (PD E) inhibitor. 2 Pretreatment of rat aortic smooth muscle cells with 10 -500 mu M SNP led to a dose-dependent downregulation of cyclic GMP acc umulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 mu M SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 mu M oxyhaemoglobin. 3 Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast , resulted in downregulation of the SNP-induced cyclic GMP accumulatio n in a time- and concentration-dependent manner, that was first eviden t after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H8 9. 4 beta(1) sGC steady state mRNA levels of S-nitroso N-acetylpenicil lamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as i ndicated by Northern blot analysis. However, Western blot analysis rev ealed that a, protein levels were decreased in zaprinast, but not in S NP, SNAP or 8Br-cyclic GMP pretreated cells. 5 While thiol depletion d id not prevent the development of tolerance, pretreatment of cells wit h SNP in the presence of reducing agents partially or completely resto red the ability of cells to respond to SNP. 6 We conclude that toleran ce to SNP results from two distinct mechanisms: an early onset, NO-med iated event that is reversed by reducing agents and a more delayed, PK A-sensitive process that is mediated through increases in cyclic GMP a nd a decrease in sGC protein levels.