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The relationship between inhibitory factor, EDRF, nitrite and nitric oxide
Suppl. VII,
THROMBOSIS RESEARCH
1987
5
THE RELATIONSHIP NITRIC OXIDE W. Martin, Department University
BETWEEN
INHIBITORY
FACTOR,
EDRF,
NITRITE
AND
M.J. Lewis and A.H. Henderson, J.A. Smith, of Cardiology and Pharmacology & Therapeutics, of Wales College of Medicine, Heath Park, Cardiff
Inhibitory factor (IF) extracted from bovine retractor penis 1980) has many properties in (BRP) (Gillespie and Martin, Both IF and common with EDRF (Furchgott and Zawadzki, 1980). hydrophilic substances whose ability EDRF are labile, anionic, to induce vasodilatation and elevate cyclic GMP levels is blocked by haemoglobin and methylene blue. Recently, it has been proposed that EDRF is nitric oxide (NO) and that the inactive form of IF is nitrite from which the active species, We have further by acid (Furchgott, 1987). NO, is generated investigated the possibility that the biological activity of IF is derived from nitrite. The nitrite content of measured by diazo formation (Benngtt unactivate3 'IF extracts, to 18.7 pmoles/lO et al, 19861, was 5.7+0.4 PM (equivalent cells). Following acid-activation the nitrite content fell by 30.1+7.2%. Taking extracts to pH 2 with intermittent vortexing for 60 min (to remove the NO generated) led to a loss of nitrite b,elow detectable limits (CO.3 PM) as well as a loss of vasodilator activity. Adding further nitrite (15 or 60 PM) to extracts followed by acid-activation and neutralisation led to a proportionate increase in vasodilator activity that was reduced by boiling. By contrast, acidactivation and neutralisation of 15 PM or 60 PM solutions of nitrite in water had no vasodilator activity. Extracts of bovine aortic endothelial cells (BAEC) contained nitrite at a level of 39.9 pmoles/106 cells, and 85+31% of the vasodilator activity found in extracts from an equivalent number of cells of BAEC for 60 min with A23187 Following stimulation of BRP. of superoxide dismutase (60 u/ml), (3 )'M) in the presence cells of nitrite was recovered from the 145215 pmoles/106 bathing medium (formed probably by the interaction of NO with Stimulating the cells with an additional 3 PM A23187 for 02). a further 60 min increased nitrite production to 258~35 content of BAEC. pmoles/106 cells - i.e. > x 6 the nitrite Our results are consistent with the idea that the inactive the active form is NO, the form of IF is nitrite and that activity of which is stabilized by an unidentified component of the extract. We cannot, however, exclude the possibility that acid may also generate nitric oxide from other compounds The ability of A23187 to stimulate BAEC present in extracts. to release amounts of nitrite far in excess of their resting content would suggest that the precursor for EDRF (NO) production is not nitrite. This
work
was
supported
Bennett et al., Furchgott, R.F. P.M. Vanhoutte, Furchgott, R.F. Gillespie, J.S.
by
the
British
Heart
Foundation.
1986. J. Pharmac. Exp. Ther. 237, 629-635. In Mechanism of Vasodilatation. ed. (1987). Raven Press, in press, and Zawadzki, J.V. (1980). Nature 285, 373-376. J. Physiol. 302, 55-64. and Martin, W. (1980).
THROMBOSIS RESEARCH
1987
5
THE RELATIONSHIP NITRIC OXIDE W. Martin, Department University
BETWEEN
INHIBITORY
FACTOR,
EDRF,
NITRITE
AND
M.J. Lewis and A.H. Henderson, J.A. Smith, of Cardiology and Pharmacology & Therapeutics, of Wales College of Medicine, Heath Park, Cardiff
Inhibitory factor (IF) extracted from bovine retractor penis 1980) has many properties in (BRP) (Gillespie and Martin, Both IF and common with EDRF (Furchgott and Zawadzki, 1980). hydrophilic substances whose ability EDRF are labile, anionic, to induce vasodilatation and elevate cyclic GMP levels is blocked by haemoglobin and methylene blue. Recently, it has been proposed that EDRF is nitric oxide (NO) and that the inactive form of IF is nitrite from which the active species, We have further by acid (Furchgott, 1987). NO, is generated investigated the possibility that the biological activity of IF is derived from nitrite. The nitrite content of measured by diazo formation (Benngtt unactivate3 'IF extracts, to 18.7 pmoles/lO et al, 19861, was 5.7+0.4 PM (equivalent cells). Following acid-activation the nitrite content fell by 30.1+7.2%. Taking extracts to pH 2 with intermittent vortexing for 60 min (to remove the NO generated) led to a loss of nitrite b,elow detectable limits (CO.3 PM) as well as a loss of vasodilator activity. Adding further nitrite (15 or 60 PM) to extracts followed by acid-activation and neutralisation led to a proportionate increase in vasodilator activity that was reduced by boiling. By contrast, acidactivation and neutralisation of 15 PM or 60 PM solutions of nitrite in water had no vasodilator activity. Extracts of bovine aortic endothelial cells (BAEC) contained nitrite at a level of 39.9 pmoles/106 cells, and 85+31% of the vasodilator activity found in extracts from an equivalent number of cells of BAEC for 60 min with A23187 Following stimulation of BRP. of superoxide dismutase (60 u/ml), (3 )'M) in the presence cells of nitrite was recovered from the 145215 pmoles/106 bathing medium (formed probably by the interaction of NO with Stimulating the cells with an additional 3 PM A23187 for 02). a further 60 min increased nitrite production to 258~35 content of BAEC. pmoles/106 cells - i.e. > x 6 the nitrite Our results are consistent with the idea that the inactive the active form is NO, the form of IF is nitrite and that activity of which is stabilized by an unidentified component of the extract. We cannot, however, exclude the possibility that acid may also generate nitric oxide from other compounds The ability of A23187 to stimulate BAEC present in extracts. to release amounts of nitrite far in excess of their resting content would suggest that the precursor for EDRF (NO) production is not nitrite. This
work
was
supported
Bennett et al., Furchgott, R.F. P.M. Vanhoutte, Furchgott, R.F. Gillespie, J.S.
by
the
British
Heart
Foundation.
1986. J. Pharmac. Exp. Ther. 237, 629-635. In Mechanism of Vasodilatation. ed. (1987). Raven Press, in press, and Zawadzki, J.V. (1980). Nature 285, 373-376. J. Physiol. 302, 55-64. and Martin, W. (1980).