Differentiation by hydroquinone of relaxations induced by exogenous and endogenous nitrates in non‐vascular smooth muscle: role of superoxide anions

Abstract

1 The influence of hydroquinone on relaxations induced by nitric oxide (NO), nitrovasodilator drugs, and non-adrenergic, non-cholinergic (NANC) field stimulation has been investigated in three tissues in which endogenous nitrates have been implicated in the NANC response; the mechanism of action of hydroquinone was also studied. 2 In mouse anococcygeus, hydroquinone (10-1OOpM) produced a concentration-dependent inhibition of relaxations induced by 1S5UM NO. Hydroquinone, 100pMm, which reduced responses to NO by 85%, had no effect on relaxations induced by NANC field stimulation (1OHz; 20s trains), hydroxylamine (1OpM), sodium nitroprusside (1 gM) or sodium azide (20juM).3 In guinea-pig trachea, 100pM hydroquinone reduced relaxations to 150pM NO by 75%, but had no effect on those to NANC stimulation (1OHz; 30s trains) or sodium azide (5 pM).4 In rat gastric fundus, 100pUM hydroquinone reduced relaxations to 1 ,M NO by 85%, but had no effect on those to NANC stimulation (0.5 Hz; 15 s trains) or sodium azide (2pM). 5 Superoxide dismutase (SOD; 50uml-1) had no effect on relaxations of the mouse anococcygeus in response to 15,UM NO or 10Hz NANC stimulation. Further, the inhibition of responses to NO by hydroquinone was unaffected in the presence of SOD. 6 Hydroquinone (10-1OOuM) failed to generate superoxide anions, as detected by a chemiluminescent assay. However, 100piM hydroquinone, like SOD (50uml-1), produced almost complete inhibition of superoxide anion chemiluminescence induced by xanthine (500pgM): xanthine oxidase (0.07 u ml-1). 7 It is concluded that, in our system, hydroquinone inhibits NO by acting as a free radical scavenger rather than by generating superoxide anions. The ability of hydroquinone to block relaxations to NO, but not NANC stimulation, may suggest that the endogenous nitrate substance released by these NANC nerves may not be free NO, but may be an NO-containing, or NO-generating, molecule.

Inhibition of relaxations to nitrergic stimulation of the mouse anococcygeus by duroquinone

“…Both by either HQ and BQ at this concentration (data not given), confirming previous observations with HQ (Hobbs et al, 1991 anion generation by xanthine: xanthine oxidase was investigated. Addition of increasing concentrations of xanthine (50-200 pM) to 70 mu ml-' xanthine oxidase resulted in increasing generation of superoxide anions, as represented by the chemiluminescence signal (Figure 6a).…”

“…The object of the present study was to determine whether this explanation might also hold true in the mouse anococcygeus, and might explain the differential effect of HQ on NO-and nerve-induced relaxations of this tissue, since there is some uncertainty whether HQ acts as a superoxide anion generator or via some other 'free radical scavenger' mechanism (Hobbs et al, 1991). To investigate the interaction of superoxide anions and Cu/Zn SOD with nitrergic relaxations in the anococcygeus, we used duroquinone (DQ,2,3,5,, a compound structurally related to HQ, but which is known to generate superoxide anions within tissues via chemical reduction by several flavoprotein enzymes (Powis & Appel, 1980;Boersma et al, 1994).…”

“…Superoxide anions were detected by chemiluminescence (Hobbs et al, 1991). Two ml of Krebs solution was placed in clear plastic test tubes, and to this was added 250 gM lucigenin.…”

“…However, several aspects of this novel (nitrergic) neurotransmission system await full elucidation, including the observation that a number of drugs are potent inhibitors of relaxations induced by exogenous NO, but have little effect on relaxation due to nitrergic nerve stimulation; these drugs include hydroquinone (HQ, Hobbs et al, 1991;Gibson et al, 1992), hydroxocobalamin (Rand & Li, 1993), pyrogallol and LY83583 (Gillespie & Sheng, 1990;Barbier & Lefebvre, 1992;Knudsen et al, 1992;Liu et al, 1994). Such results have inspired a number of possible explanations (for reviews see Gibson et al, 1995;Rand & Li, 1995): these are: (1) the NO produced in the nerve terminal may be released in a modified form, perhaps attached to a carrier molecule; (2) the rapid rate of diffusion of NO released from a point source (Wood & Garthwaite, 1994) may render it resistant to the actions of scavenger molecules, at least over short distances; and (3) NO may indeed be released as a free radical, but may be protected by other substances which preferentially interact with potential NO-scavengers.…”

Effects of hydroxocobalamin and carboxy‐PTIO on nitrergic transmission in porcine anococcygeus and retractor penis muscles

“…These agents generally block the responses to exogenous NO but have little e ect on responses to nitrergic nerve stimulation (Hobbs et al, 1991;Rand & Li, 1995c;Lefebvre, 1996).…”