The effect of the vasodilator nicotinamide on cyclic nucleotide ...

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The effect of the vasodilator nicotinamide on cyclic nucleotide pathways in vascular smooth muscle. DEIRDRE M. BURNS, MARK W. RUDDOCK, JOHN C.W..
132s Biochemical Society Transactions (1997) 25

The effect of the vasodilator nicotinamide on cyclic nucleotide pathways in vascular smooth muscle. DEIRDRE M. BURNS, MARK W. RUDDOCK, JOHN C.W. BROWN, GORDON D. KENNOVIN*, EMMA L. DYKES*, FREDRICK W. FLJTNEY* and DAVID G.HIRST. Radiation Science Group, School of Biomedical Sciences, University of Ulster, Shore Rd., Newtownabbey, Co. Antrim, N. Ireland BT37 OQB and *Cancer Biology Research Group, University of St. Andrews, St Andrews, Scotland. Elevation of cAMP or cGMP in vascular smooth muscle causes vasodilation by activation of protein kinase A or protein kinase G, leading to phosphorylation of intracellular target proteins (1,2). A putative role in the lowering of intracellular Ca2+ has also also been suggested for these agents but the mechanism by which it does this remains unclear (3.4). Nicotinamide (NA), the amide derivative of Vitamin B3 has been shown to be a potent vasodilator of rat tail arteries in ex vivo perfusion studies (5). In vivo, numerous reports have demontrated that it is a potent radiosensitizer in animal tumours (reviewed by 6). This activity is believed to be the result of increased perfusion and thus oxygenation of tumours (7). NA is effective against contractile agonists such as phenylephrine, vasopressin and high KC1 concentrations which activate various pathways in vascular smooth muscle. Therefore we have examined the effect of NA on the second messengers cAMP and cGMP and on phosphodiesterase activity. Approximately 1 cm sections of tail artery from normotensive Wistar rats were mounted in a perfusion apparatus as previously described (Hirst et al., 1994). Nicotinamide and cyclic nucleotide inhibitors MDL12,330A and [lH-L1.2.41 oxadiazole[4,3-a] quinoxalin-1-one] (ODQ were incorporated into Krebs' solution as internal and external perfusates. Bolus injections of increasing concentrations of phenylephrine (PE) which were introduced through a resealable rubber septum produced transient peaks of constriction. Log dose response curves to phenylephrine in the absence and presence of NA, MDL12,330A or ODQ were consmcted. Mean and standard emor of replicates were calculated and significant differences identified using ANOVA (Statview program for Mackintosh) If NA was enhancing CAMP-mediated pathways, the presence of the irreversible inhibitor, MDL12,330A would block the vasodilation caused by NA. Figure 1 shows the effect of MDL12,330A on NA-mediated inhibition of PE-induced constriction in rat tail artery. Continuous perfusion of NA reduced the dose response to PE by 77%. MDL also reduced this response by 69%. In combination NA and MDL were markedly additive, reducing the response to 2.5% of that in PE alone ( p < 0.001). Similarily we used ODQ, a reversible inhibitor of cGMP in an attemut to antarronise NA activitv. Figure 2 show the effect of this inhibitor on Nr\-induced vad3atio;. In this experiment, O D 0

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Log [PEI FIG 1. EFFECT OF MDL 12.330A ON NA-1NDUCF.D ODILATION, Dose response to PE in Krebs' +, in Krebs' with 8.3 mM NA --)- ,in Krebs' w ith (10 pM) MDL 1 2 , 3 3 0 A e ,and in Krebs w ith NA (8.2 mM) and MDL 12,330A (10 M) + .

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Log [PEI FIGURE. 2. EFFECT OF OD0 0N NA-INDUCED

VASODILATION, Dose response to PE in Krebs +, Krebs' with NA and (8.2mM) +,Krebs' with ODQ (1pM) +, and Krebs' with ODQ ( ~ F M and ) NA (8.2mM) +, final recovery in Krebs' +. alone had no significant effect on the PE dose response and it did not antagonise the effect of NA. Alternatively, NA may be enhancing cyclic nucleotide action by inhibiting phosphodiesterase and thus prolong cAMP or cGMP activity. Repeating the above experiments with the broad spectrum phosphodiesterase inhibitor, (IBMX) did not block the effect of NA (data not shown). Previous studies by this group demonstrated that NA is not acting by producing cAMP or cGMP via endothelial activation of preceptors or nitric oxide stimulation of guanylate cyclase respectively (Hirst et al.,1994). The possibility however, that NA could be. acting directly on adenylate and guanylate cyclase within the smooth muscle cells could not be ruled out. ODQ is a recently documented specific inhibitor of soluble guanylate cyclase and in our study, sodium nitroprusside, a known activator of the soluble form was inactive in its presence (8). Thus, any possible action of NA on the particulate form of guanylate cyclase activity are as yet undetermined. Similarily MDL is a specific inhibitor of adenylate cyclase and the finding that the dose response to PE is reduced in the presence of MDL is unexpected. There is some experimental evidence that in smooth muscle cAMP has differing physiological activities depending on whether cGMP is functional. cAMP is thought to mediate it vasodilatory activity indirectly via activation of cGMP but in the absence of cGMP it acts as a vasoconstrictor (9). Thus, blockade of both nucleotides simultaneously may be necessary. In conclusion, these results suggest that NA is unlikely to exert its main vasodilatory effect through enhancement of adenylate or soluble guanylate cyclase mediated pathways. Given the above considerations, however, further studies including direct measurement of the intracellular messengers will be necessary. 1. Conti, M.A. & Adelstein (1981) J. Biol. Chem 256, 31783181. 2. Johnson, R.M. & Lincoln, T.M. (1985) Mol. Pharmacol. 27, 333-342 3. Lincoln, T.M. (1989) Pharmacol. Ther. 41,479-502. 4. Felbel, J.B. Tmker, T., Ecken, W. Landgraf & Hofmann (1988) J.Bio1. Chem. 263,16764-16771. 5. Hirst, D.G., Kennovin, G.D. & Flitney, F.W. (1994) Br. I. Radio]. 67,795-799. 6. Roias, A. (1992) Br. J. Radio]. 24. 174-178. 7. Horsman, M.R., Chaplin, D.J. & Brown, J.N (1989) Radiat. Res. 118,139-150. 8. Cellek, S. Kasakov, L. & Moncada. S. (1996) Br. J. Pharmacol. 118, 137-140. 9. Lincoln, T.M. Comwell, T.L. &Taylor, A.E. (1990) Am. 1. Physiol. 258, C399-C407.