Bradykinin-induced contraction of the rat isolated ileum involves extracellular calcium, protein kinase C and inositol trisphosphate receptor P.L. Johnson, R.I. Prince, W.M. Purcell & R.H. Osborne Centre for Research in Biomedicine, University of the West of England, Coldharbour Lane, Bristol BS16 1QY.

Print abstract Search PubMed for: Johnson PL Prince RIP Purcell WM Osborne RH

Bradykinin (BK) is reported to cause contraction of longitudinal muscle in the rat ileum via activation of B₁ receptors (Meini ., 1996) that stimulate the production of inositol trisphosphate (InsP₃) and diacylglycerol (DAG) (Hall, 1992). The aim of this study was to determine the sources of Ca²⁺ used during BK-induced tissue contraction in the absence and presence of 2-amino-ethoxydiphenylborate (2-APB), a modulator of InsP₃-induced Ca²⁺ release, (Maruyama et al., 1997), the protein kinase C inhibitor H7 (Hidaka et al., 1984), the SERCA (Smooth Endoplasmic Reticulum Calcium) ATP-ase inhibitor BHQ (tert-butylbenzohydro-quinone (Wictome et al., 1992), the L-type Ca²⁺ channel blocker verapamil (Lee & Tsien, 1983) and low external Ca²⁺.

Ileal segments (2 sections taken 15-30 cm from the ileo-caecal junction) were obtained from male Wistar rats (220 - 250g) and mounted in 10ml organ baths containing Tyrode's solution at 35oC (plus 1 µM atropine, gassed with 95% O₂ : 5% CO₂) under a tension of 1g. Tissues were equilibrated for 30 min before non-cumulative dose response curves to BK were obtained using a 4 min cycle with 2 min contact time using an isotonic transducer. In Ca²⁺-free solutions, Ca²⁺ was replaced isosmotically with Mg²⁺. Antagonists were added to the buffer and tissues equilibrated for 30 min before retesting the effects of BK. Data reported are mean ± s.e.m. and statistical significance was determined using Student's t-test of pair differences.

BK (10nM - 1µM)-induced contraction produced a biphasic dose response curve with apparent EC₅₀ values 34 ± 5 nM and 470 ± 70 nM (n = 6). The maximum response occurred at 1µM. Incubation of tissues in low external Ca²⁺ abolished the effects of BK (n = 6, P<0.00001), while maximum response caused by BK was reduced by 75 ± 8% (n = 6; P<0.001) in the presence of 10µM verapamil. At doses of 1µM, 10µM and 50µM, 2-APB decreased the maximum response of BK by 55 ± 15 % (n = 6, P<0.05), 78 ± 18% (n = 6, P<0.001) and 100% respectively (n = 6; P<0.0001), while H7 (10nM) reduced the maximum response of BK by 75 ± 13 % (n = 5, P<0.05). BHQ, 1nM and 10nM, decreased the BK maximum response by 51 ± 16% (n = 6, P<0.05) and 68 ± 14% (n = 6; P<0.002) respectively.

These data suggest that BK receptors in the rat ileum activate the bifurcating phosphoinositide pathway to cause ingress of extracellular Ca²⁺ through L-type channels. Thus, the effects of 2-APB show that BK-induced tissue contraction involves release of Ca²⁺ from InsP₃-activated stores while the action of H7 suggests that BK receptor activation triggers release of DAG which activates PKC. Finally, the inhibitory effect of BHQ suggests that the SERCA ATPase function may be to reduce cytosolic Ca²⁺ levels so as to maintain cell responsiveness.

Hall, J.M. (1992) Pharmacology & Therapeutics, 56, 131-190.
Hidaka, H. et al. (1984) Biochemistry, 23, 5036-5041.
Lee, K.S. & Tsien, R.W. (1983) Nature 302, 790-794.
Maruyama, T. et al. (1997) J. Biochem. 122, 498-505.
Wictome et al., (1992) Fed. Eur. Biochem. Soc. 304, 109-113.