Mechanisms of augmented vasoconstriction induced by 5-hydroxytryptamine in aortic rings from spontaneously hypertensive rats.

British Journal of Pharmacology (2008) 155, 210–216 & 2008 Macmillan Publishers Limited All rights reserved 0007– 1188/08 $32.00 www.brjpharmacol.org RESEARCH PAPER Mechanisms of augmented vasoconstriction induced by 5-hydroxytryptamine in aortic rings from spontaneously hypertensive rats K Budzyn1,3, RM Ravi2, AA Miller2 and CG Sobey2 1 Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia and 2Department of Pharmacology, Monash University, Clayton, Victoria, Australia Background and purpose: To test whether development of enhanced vasoconstriction to 5-hydroxytryptamine (5-HT; serotonin) in SHR was temporally related to hypertension, elevated vascular superoxide (O2
) levels, decreased NO bioavailability, or increased contractile effects of cyclooxygenase or rho-kinase and/or PKC. Experimental approach: We examined systolic blood pressure (SBP), vascular O2
, and 5-HT-induced contractile responses of aortic segments from 4- and 8-week-old WKY and SHR. Key results: SBP was 35% higher in SHR than WKY at 4 weeks and 60% higher at 8 weeks. Contractile responses to 5-HT were similar in WKY and SHR at 4 weeks, but were markedly augmented in SHR at 8 weeks. The NO synthase inhibitor, L-NAME, enhanced contractile responses to 5-HT markedly in both strains at 4 weeks and in WKY at 8 weeks, but only very modestly in SHR at 8 weeks. These functional differences were associated with higher O2
levels in SHR versus WKY at 8 weeks, but not at 4 weeks. The rho-kinase inhibitor, Y-27632, and the PKC inhibitor, Ro 31-8220, each only modestly attenuated contractions in WKY and SHR in each age group, and their effects in each strain were more pronounced at 8 weeks. The cyclooxygenase inhibitor, indomethacin, had no effect on contractile responses. Conclusions and implications: Development of augmented vascular contractile responses to 5-HT in SHR is preceded by hypertension. It is associated with increased vascular O2
levels and reduced modulatory effects of NO, and is unlikely to be due to enhanced activity of rho-kinase, PKC or cyclooxygenase. British Journal of Pharmacology (2008) 155, 210–216; doi:10.1038/bjp.2008.247; published online 16 June 2008 Keywords: hypertension; nitric oxide; PKC; reactive oxygen species; rho-kinase Abbreviations: eNOS, endothelial nitric oxide synthase; NO, nitric oxide; O2
, superoxide; PKC, protein kinase C; Ro 31-8220, (3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide); SBP, systolic blood pressure; SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto; Y-27632, (R-( þ )-trans-N-(4-pyridyl)-4-(1aminoethyl)-cyclohexanecarboxamide) Introduction Endothelial dysfunction is a common characteristic of advanced cardiovascular diseases associated with reduced bioavailability of the endogenous vasodilator, nitric oxide (NO), such as chronic hypertension. Endothelial NO is a critical modulator of vascular contractility, and its absence— for example, due to physical removal of the endothelium or endothelial NO synthase (eNOS) gene deletion—leads to enhanced vascular contractile responses, particularly to G Correspondence: Dr CG Sobey, Department of Pharmacology, Monash University, Building 13E, Wellington Road, Clayton, Victoria 3800, Australia. E-mail: 3 Division of Cardiology, Emory University School of Medicine, 101 Woodruff Circle, Atlanta, Georgia, USA. Received 22 April 2008; revised 12 May 2008; accepted 15 May 2008; published online 16 June 2008 protein-coupled receptor agonists such as 5-HT (Lamping et al., 1985; Lamping and Faraci, 2003; Budzyn et al., 2004). Increased levels of reactive oxygen species such as superoxide (O2
), which occurs in the vasculature of chronically hypertensive animals (Zalba et al., 2000; Landmesser et al., 2003; Matsuno et al., 2005), can also contribute to compromised NO bioavailability and consequent vascular dysfunctions (Cai and Harrison, 2000). It is also recognised that ‘Ca2 þ sensitisation’—the mechanism of vascular contraction that occurs independently of increasing intracellular Ca2 þ levels is another major mechanism that regulates vascular contractility, particularly during hypertension (Soloviev and Bershtein, 1992; Shaw et al., 1997). In particular, the role of rhokinase—the predominant mediator of Ca2 þ sensitisation—in Enhanced vasoconstriction to 5-HT in SHR K Budzyn et al 211 the regulation of vascular tone during hypertension has been studied extensively (Uehata et al., 1997; Chrissobolis and Sobey, 2001; Wehrwein et al., 2004; Jin et al., 2006). Furthermore, although early studies suggested that PKC might also contribute to increased vascular contractility during hypertension (Bruschi et al., 1988; Shibata et al., 1990; Secrest et al., 1991; Soloviev and Bershtein, 1992), very few studies have sought to clarify the relative roles of each kinase in hypertension. Although many studies have described abnormalities in vascular function in established chronic hypertension (Shaw et al., 1997; Endemann et al., 2002; Jarajapu and Knot, 2005; Northcott et al., 2005), very little is known about the timing of the occurrence of certain abnormalities during its development. Hence, the main aims of this study were to test whether the development of enhanced vasoconstriction to 5-HT, early in genetic hypertension, is temporally related to increased blood pressure (BP), elevated vascular O2
levels, decreased NO bioavailability, or increased contractile effects of rho-kinase and/or PKC. CaCl2 2.5, bubbled with 5% CO2 in O2 at 37 1C. Tension was continuously recorded on a chart recorder (Model 3721, Yokogawa, Japan). Aortic segments from 8 week-old rats were mounted at 5 mN passive tension in 10 mL organ chambers containing Krebs-bicarbonate solution bubbled with 5% CO2 in O2 at 37 1C. Tension was continuously recorded using a Grass FT03 force transducer and Powerlab Chart computer software (Version 5.2.2). Following 45 min equilibration, arterial segments were exposed to an isotonic high K þ -containing physiological saline solution (KPSS; [K þ ]KPSS ¼ 124 mM). KPSS-induced contraction reached a stable level after 10–20 min. Following washout and return to a stable baseline, segments were precontracted to B50% of their KPSS response with phenylephrine (1–3 mM). Relaxation in response to acetylcholine (10 mM) confirmed the presence of functional endothelium. Following washout and return to stable baseline, cumulative concentration–response curves were established to 5-HT (10 nM–0.3 mM). Methods Effects of NOS, cyclooxygenase, rho-kinase and PKC inhibition on contractile responses We assessed the effect of 30 min pretreatment with the NO synthase (NOS) inhibitor, No-nitro-L-arginine methyl ester (L-NAME, 100 mM) on contractile responses to 5-HT. Similarly, the effects of the cyclooxygenase inhibitor, indomethacin (10 mM), or the rho-kinase inhibitor, Y-27632 (1 mM) and/or the PKC inhibitor, Ro 31-8220 (5 mM), on responses to 5-HT were also (...truncated)