$12.00~~~~~~~~~~~~~~~ Inhibition by zinc protoporphyrin-IX of receptor-mediated relaxation of the rat aorta in a manner distinct from inhibition of haem oxygenase Lars Ny, Karl-Erik Andersson & 'Lars Grundemar Department of Clinical Pharmacology, Lund University Hospital, S-221 85 Lund, Sweden 1 Carbon monoxide (CO), produced by haem oxygenase through degradation of haem, has been claimed to be a neuromessenger and a possible regulator of vascular tone. We examined whether the haem oxygenase inhibitor, zinc protoporphyrin-IX (ZnPP) and other porphyrins affect the relaxation evoked by various agents in the rat isolated aorta. 2 Pretreatment with ZnPP (0.1 mM) virtually abolished the relaxation evoked by vasoactive intestinal peptide (VIP) and atrial natriuretic peptide (ANP). ZnPP also evoked a rightward shift of the concentration-response curve for the relaxation induced by acetylcholine. 3 In contrast, ZnPP did not affect the relaxation evoked by forskolin and 3-morpholino-sydnonimine, agents which directly activate adenylate and guanylate cyclase, respectively. 4 Although, less effective than ZnPP, tin protoporphyrin-IX (SnPP; 0.1 mM) and protoporphyrin-IX (PP; 0.1 mM) also attenuated the VIP-evoked relaxation. 5 The elevation of cyclic AMP and cyclic GMP levels evoked by VIP and ANP, respectively, were abolished by pretreatment with ZnPP (0.1 mM). 6 ZnPP, SnPP and PP did not affect the contraction evoked by phenylephrine. 7 The results show that ZnPP inhibits relaxation induced by VIP, ANP and acetylcholine, probably by interfering with membrane receptor-coupled signal transduction pathways. This inhibition does not seem to be dependent upon inhibition of haem oxygenase. The lack of specificity of the haem oxygenase inhibiting metalloporphyrins makes them less suitable as pharmacological tools in the investigation of a messenger role for CO. Keywords: Metalloporphyrins; carbon monoxide; blood vessel; relaxation; cyclic nucleotides; second messenger; vasoactive intestinal peptide; atrial natriuretic peptide; acetylcholine Introduction Substantial evidence has indicated that nitric oxide (NO) is an endogenous mediator of various physiological processes in the brain and periphery (e.g. Moncada et al., 1991). It was recently suggested that another gas, carbon monoxide (CO), which is produced by microsomal haem oxygenase, also may be a neuronal messenger in the brain (Maines et al., 1993; Verma et al., 1993). Both NO and CO are known to increase guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in various tissues (Brune & Ullrich, 1987; Furchgott & Jothianandan, 1991). Haem oxygenase is the rate limiting step in the degradation of haem-containing compounds, re- sulting in the formation of CO and biliverdin (e.g. Maines et al., 1993). Haem oxygenase exists in two isoforms haem oxygenase-1 and haem oxygenase-2 (Maines et al., 1993). High levels of haem oxygenase or haem oxygenase-2 mRNA have been found in the rat liver, spleen and brain (Vreman & Stevenson, 1988; Verma et al., 1993). It has been speculated that CO, which shares many proper- ties with NO, may be a regulator of vascular tone (Marks et al., 1991; Schmidt, 1992). The evidence for such a view is, however, circumstantial. Like NO, exogenously applied CO has been shown to relax various isolated blood vessels and to increase cyclic GMP levels (Furchgott & Jothianandan, 1991; Moncada et al., 1991; Lefer et al., 1993; Zygmunt et al., 1994). We have recently demonstrated haem oxygenase activity in various blood vessel homogenates, including rat aorta, by measurement of CO production using a gas chromatographic method (Grundemar et al., 1995). Certain metalloporphyrins, like zinc protoporphyrin-IX (ZnPP) and ' Author for correspondence. tin protoporphyrin-IX (SnPP), are haem oxygenase inhibitors, of which primarily ZnPP has been used in studies suggesting a messenger role for CO in the brain and periphery (e.g. Verma et al., 1993; Rattan & Chakder, 1993). However, little is known about the specificity of these metalloporphyrins. The aim of the study was to examine whether ZnPP and other porphyrins affect relaxation evoked by agents with different modes of action in the rat isolated aorta. Methods Mechanical activity Female Sprague-Dawley rats (250-300 g) were killed by CO2 asphyxia and the thoracic aorta was dissected out. The aorta was placed in an ice-cold Krebs solution and cut into 2 mm long ring segments. The preparations were transferred to thermostatically controlled (370C) 5 ml tissue baths contain- ing Krebs solution bubbled with 5% CO2 and 95% 02, resulting in a pH of 7.4, and mounted between two L-formed hooks, one of which was attached to a force transducer (Grass FT03) for measurement of mechanical activity, and the other was connected to a sledge, which allowed adjust- ment of the passive tension of the vessel. The recordings were made on a Grass polygraph, 7D or 7E. The vessels were repeatedly stretched for 1 h until a stable resting tension of 8 mN was obtained. The contractile capacity was examined by adding an isotonic 60 mm potassium Krebs solution (for composition see below). In order to construct concentration- BrRish Joumal of Pharmacology (1995) 1159 186-190 . 1995 Stockton Press All rights reserved 0007-1188/95 $12.00 00