Inhibition of Rho-kinase by 5-HT in 5HT-induced constriction of isolated Bovine Pulmonary Arteries

Angus Shaw, Ross McCallum, Allan MacDonald, Alistair Corbett. Glasgow Caledonian University, Glasgow, UK.

The Rho-A/Rho-kinase pathway plays an important role in agonist–induced constriction of vascular smooth muscle. Indeed inhibition of Rho-kinase has been promoted as a putative management therapy in pulmonary hypertension (Abe et al, 2004). Rho-kinase can be activated by agonist receptors that couple to G12/13 (Fukata et al, 2001) and/or by elevated intracellular calcium (Ayeman et al, 2003). In bovine pulmonary arteries 5-HT induced constriction is mediated by the 5-HT2A and 5-HT1B receptors (McKenzie et al, 2010). The present study investigated the role of Rho-kinase in 5-HT-induce constriction of bovine pulmonary arteries.

Bovine lungs from animals under twenty months were obtained fresh from the local abattoir. Ring segments (0.3-0.5cm in diameter), dissected from the 5th. Arterial generation were mounted in 5 ml organ baths suspended between stainless steel hooks in Krebs-Henseleit buffer (37oC) under a tension of 2 g and gassed with a mixture of O2:CO2 95%/5% v/v. Tissues were allowed to equilibrate for 1 hour before the addition of drugs. All tissues were first contracted with 60 mM KCl. After washing, cumulative concentration response curves (CRCs) to 5HT or a single constriction to the 5-HT2A agonists DOI (10nM) or TCB (1µM) were constructed in the absence or presence of the Rho kinase inhibitors Y27632 (30µM) or H1152 (10µM). CRC to 5-HT were further examined in presence of the 5-HT1B (SB216641, 3μM) and 5-HT1D (BRL15572, 10nM) receptor antagonists or the selective serotonin re-uptake inhibitor paroxetine (10nM) alone or in the presence of Y27632. Contractile responses are expressed as % of initial KCl contraction. Results are means ± s.e. mean. The significance of differences was determined using Student’s t-test.

The concentration response curve for 5-HT-induced constriction was unaffected by the Rho-kinase inhibitors Y27632 or H1152. In contrast constriction induced by the 5-HT2A agonists DOI was reduced from 95.28±6.62 to 39.88±4.02 (n = 4) by Y27632. The 5-HT1B receptor antagonist SB216641 shifted the CRC for 5-HT to the right (control pEC50 = 5.915±0.096, SB216641 pEC50 = 4.771±0.102 (n = 7) and reduced the maximum response by 20% (from 188.2±7.90 to 149.8±9.83, n = 7) and this response was unaffected by the further addition of Y27632. The presence of the 5-HT1D receptor antagonist (BRL15572) produced a 26% increase in the maximum contraction to 5-HT (from 190.4±9.44 to 239±14.76, n = 7), the selective serotonin re-uptake inhibitor (paroxetine) produced a similar potentiation of 24% (from 174.4±3.79 to 217.1±8.6, n = 4). The further addition of Y27632 reduced the maximum response by 36% (from 239±14.76 to 153±9.77, n = 7, in the presence of BRL15572 and 42% (from 217.1±8.6 to 125.1±7.45, n = 4) in the presence of paroxetine.

These observations suggest that 5-HT acting through the 5-HT1D receptor and/or the selective serotonin reuptake mechanism may inhibit the involvement of Rho-kinase in 5-HT-induced constriction of BPA.

Abe et al, 2004, Circulation Research 2004, 94:385-393.

Ayeman et al, 2003, BJP 139:1532–1538.

Fukata et al, 2001, TRENDS in Pharmacological Sciences 22:32-39.

McKenzie et al, 2010, BJP, 159:188–200.