Phospholipase C-independent effects of 3M3FBS in murine colon

The muscarinic receptor subtype M3 is coupled to Gq/11 proteins. Muscarinic receptor agonists such as carbachol stimulate these receptors that result in activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and Ins(1,4,5)P3. This pathway leads to excitation and smooth muscle contraction. In this study the PLC agonist, 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benezenesulfonamide (m-3M3FBS), was used to investigate whether direct PLC activation mimics carbachol-induced excitation.We examined the effects of m-3M3FBS and 2, 4, 6-trimethyl-N-(ortho-3-trifluoromethyl-phenyl)-benzenesulfonamide (o-3M3FBS), on murine colonic smooth muscle tissue and cells by performing conventional microelectrode recordings, isometric force measurements and patch clamp experiments.Application of m-3M3FBS decreased spontaneous contractility in murine colonic smooth muscle without affecting the resting membrane potential. Patch clamp studies revealed that delayed rectifier K+ channels were reversibly inhibited by m-3M3FBS and o-3M3FBS. The PLC inhibitor, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), did not prevent this inhibition by m-3M3FBS. Both m-3M3FBS and o-3M3FBS decreased two components of delayed rectifier K+ currents in the presence of tetraethylammonium chloride or 4-aminopyridine. Ca2+ currents were significantly suppressed by m-3M3FBS and o-3M3FBS with a simultaneous increase in intracellular Ca2+. Pretreatment with U73122 did not prevent the decrease in Ca2+ currents upon m-3M3FBS application.In conclusion, both m-3M3FBS and o-3M3FBS inhibit inward and outward currents via mechanisms independent of PLC acting in an antagonistic manner. In contrast, both compounds also caused an increase in [Ca2+]i in an agonistic manner. Therefore caution must be employed when interpreting their effects at the tissue and cellular level.