Effect of temperature on muscarinic cholinoceptor-mediated phosphoinositide metabolism and tension generation in bovine tracheal smooth muscle

The effect of decreased temperature on phosphoinositide metabolism was studied in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM) by investigating the consequences of cooling on muscarinic-cholinoceptor-mediated [³H]inositol phosphate ([³H]InsP) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) accumulation, basal phosphoinositidase C (PIC) activity and airways smooth muscle (ASM) tone. Cooling of [³H]Ins labelled BTSM slices from 37°C to 27°C for 20 min prior to the addition of agonist caused a substantial (73.0±2.5%) inhibition of carbachol (100 M, 30 min)-stimulated [³H]InsP accumulation compared to values measured at 37°C. The degree of inhibition of [³H]InsP accumulation was similar at all agonist time points (2–30 min) studied. In parallel experiments, cooling of unlabelled BTSM slices from 37°C to 27°C resulted in a 34% reduction in basal Ins(1,4,5)P₃ mass (37°C, 13.1±0.6 pmol mg^– protein; 27°C, 8.9±0.9 pmol mg^–1 protein; P<0.02) and markedly attenuated carbachol (100 M)-stimulated increases in Ins(1,4,5)P₃ accumulation. Basal PIC activity in the soluble fraction of BTSM homogenates, measured using a [³H]phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) /deoxycholate assay system, was also significantly lower at 27°C compared to 37°C (initial velocities of PtdIns(4,5)P₂ hydrolysis of 853±167 (37°C) and 418±119 (27°C) pmol min^–1 ml^–1 (1/400 diluted) BTSM cytosol; p<0.02). Cooling of BTSM strips from 37°C to 27°C for 20 min affected neither the lag period prior to the onset of contraction, the rate of force development, nor the final magnitude of the tension generated by carbachol (100 M). However, a significant attenuation of the contractile response by cooling to 27°C was observed using a submaximal (EC₂₀) concentration of carbachol. Also, the contractile response to 1 mM McN-A-343, a partial agonist at M₃-cholinoceptors was significantly attenuated at 27°C with mean increases in the lag time and the t_1/2 to achieve maximal contraction of 558% and 369% respectively and a mean decrease in the maximum force generated of 37%. Despite previous reports indicating that cooling can enhance agonist-stimulated [³H]InsP₃ accumulation in certain tissues, modest degrees of cooling clearly inhibit basal and carbachol-stimulated phosphoinositide hydrolysis in bovine tracheal smooth muscle and reduces the muscarinic receptor reserve in tracheal smooth muscle contraction.