Incubation time: A key factor for halothane to attenuate no-induced cgmp formation in vascular smooth muscle

Halothane (HAL) has been shown to inhibit nitric oxide (NO)-induced vascular relaxation and cGMP formation. It has been postulated that HAL competes with NO for Fe2' binding site on soluble guanylyl cyclase (sGC). Although well characterized after HAL preincubation with sGC, this inhibition was not detected when sGC and NO were simultaneously incubated with HAL. Previously, we demonstrated that NO rapidly equilibrated with sGC in tissue. In comparison, HAL may have a slower equilibration rate in tissue with sGC. We therefore determined if the incubation time of HAL with sGC is a factor in the HAL effect. Six rat aortic rings without endothelium were placed in Krebs-filled organ baths for 60 min. All rings were treated with phosphodiesterase inhibitor IBMX ( 1 mM) 10 min before HAL (2.3%) was delivered to baths via a calibrated vaporizer. NO solution (1 M) was added to the bath (20 sec exposure) at either 0, 1,2,5, 10, 15 min after HAL delivery began. Tissues cGMP content was measured by radioimmunoassay. NOinuueed cGMP formation was significantly inhibited by preincubation with HAL at 5. 10, and 15 min (-50.4± 12%, -64.9 ± 9%, and -66.8 ± 9% respectively), but not at 1 and 2 min. These results demonstrate that HAL inhibits NO-induced cGMP formation in vascular tissue, but sufficient preincubation time to allow HAL to equilibrate with sGC is essential to observe cGMP inhibition. This incubation time dependence may be due tu different tissue equilibrium rates for NO and HAL.