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Signal transduction following M3 receptor stimulation in the porcine detrusor
M3 receptor stimulation causes phospholipase activation with a consequent production of IP3 and DAG, which ultimately results in intracellular calcium release and an influx of extracellular calcium respectively. M3 receptors can also increase DAG via phospholipase D (Schneider T.,et al 2003). We previously reported that the α1-adrenoreceptor exhibits agonist specificity (Scott R., et al 2004). The aim of this study was to identify the signal transduction mechanism involved in the carbachol or acetylcholine (Ach) induced contraction of the porcine detrusor smooth muscle. Strips of female pig detrusor muscle were set up under 1g tension in gassed Krebs-bicarbonate solution at 37°C. Concentration-response curves were obtained to carbachol or Ach in the absence and presence of drugs that interfere with the second messenger pathways or calcium movements. Results are expressed as the mean ± SEM for pEC50 values and maximum responses for carbachol. Paired Student’s t-test was used for statistical comparisons. Carbachol produced concentration–dependant contractions of the detrusor smooth muscle with a mean pEC50 of 5.51±0.11 and a maximum contraction of 9.86g±2.09g (n=13). The presence of ryanodine (30 µM), which inhibits intracellular calcium release, had no significant effect on carbachol-induced contractions (control max = 4.5 ± 0.9g; ryanodine max = 4.9 ± 1.3g) or the pEC50 (control = 5.5 ± 0.1; ryanodine pEC50 5.1 ± 0.1). The IP3 receptor antagonist 2-aminoethoxydiphenylborane (2APB, 30µM) also had no significant effect on maximal contractions (control max = 14.9 ± 1.7g, 2ABPmax = 13.7 ± 1.7g) or pEC50 (control = 5.3 ± 0.04, with 2ABP pEC50 = 5.3 ± 0.04). In contrast, the L- type calcium channel blocker nifedipine (1µM), significantly reduced responses to carbachol by 85.9 ± 4.5% (P=0.003, n=11). Incubation with low calcium Krebs(0.95mM n=8) or calcium-free Krebs (nominal n=8) caused significant falls in maximum contraction (39.7 ± 9.6 %, P=0.006 and 98.1 ± 0.8 %, P=0.0001 respectively). The phospholipase C inhibitor U71322 (30uM), had no significant effect on carbachol induced contractions, but the phospholipase D inhibitor butan-1-ol (n= 8) significantly reduced maximal responses to carbachol by 73.7 ± 4.6% (p=0.001). When using Ach (in the presence of physostigmine, 100nM), nifedipine and 2ABP had similar effects as with carbachol, nifedipine significantly inhibiting responses by 83.0% ± 2.8 (control pEC50 = 5.0 ± 0.1; nifedipine pEC50=4.3 ± 0.2) and 2ABP having no effect (control pEC50 = 4.81 ± 0.1; 2ABP = 4.84 ± 0.1). The results suggest that pig detrusor contraction to carbachol is mediated with little contribution from intracelluar calcium stores. A pathway may be involved whereby M3 stimulation activates PLD resulting in DAG formation and contraction via extracellular calcium influx. No differences were observed between carbachol and Ach in these experiments.
Schneider, T. Hein , P. Michel, MC.(2004), Signal transduction underlying carbachol-induced contraction of rat urinary bladder. Phospholipases and Ca2+ sources. J Pharmacol Exp Ther, 308, 47-53. |
