Contraction of smooth muscle is initiated by an increase in cytosolic Ca2⁢ leading to activation of Ca2⁢/ calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of Ca2⁢ and the kinetic of Ca2⁢ mobilization. Ca2⁢ mobilizing agonists stimulate different phospholipases (PLC-β, PLD and PLA2) to generate one or more Ca2⁢ mobilizing messengers (IP3 and AA), and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of PLC-β, PLA2 and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of PIP2 and generation of IP3 and IP3-dependent Ca2⁢ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated Ca2⁢ influx, cADP ribose formation and Ca2⁢-induced Ca2⁢ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by Ca2⁢-independent mechanism involving activation of PKC-ε by DAG derived form PLD. A functional linkage between G13, RhoA, ROCK, PKC-ε, CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to M2 muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase A2 (cPLA2) to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic M3 receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the Gq/11 type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP2), producing inositol 1, 4, 5-trisphosphate (IP3) and DAG. IP3 causes release of intracellular Ca2⁢ and formation of a Ca2⁢-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.