Hiramatsu Y, Horn V J, Baum B J, Ambudkar I S
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Arch Biochem Biophys. 1992 Sep;297(2):368-76. doi: 10.1016/0003-9861(92)90686-q.
Hydrolysis of exogenously added, [3H]inositol-labeled, phosphatidylinositol 4,5-bisphosphate (PIP2) by rat parotid membranes was increased, dose-dependently, by the muscarinic cholinergic agonist carbamylcholine (carbachol) in the presence of guanosine 5'-O-thiotriphosphate (GTP gamma S). The stimulation was inhibited by atropine and guanosine 5'-O-thiodiphosphate (GDP beta S). GTP gamma S alone stimulated PIP2 hydrolysis, with half-maximal activation at 0.1 microM. This was inhibited by GDP beta S but not by atropine. Agonist stimulation of PIP2 hydrolysis was dependent on the presence of lipids (phosphatidylserine:phosphatidylethanolamine:PIP2 = 1:1:1). When PIP2 was added as micelles with detergent (sodium deoxycholate) only, basal hydrolysis was elevated, thus decreasing the relative stimulation by GTP gamma S and carbachol. The water-soluble hydrolysis products formed under either condition were 1,4,5-inositol trisphosphate, 1,4-inositol bisphosphate, and cyclic inositol trisphosphate. Hydrolysis of exogenous phosphatidylinositol (PI) was also stimulated by carbachol in the presence of GTP gamma S but the extent of PI hydrolysis was 44-fold lower than PIP2 hydrolysis. When [Ca2+] in the medium was increased from 100 nM to 1 microM, basal hydrolysis of both PI and PIP2 increased (9.3- and 19.2-fold, respectively). However, levels of basal and stimulated PIP2 hydrolysis were higher (37.9- and 29.6-fold, respectively) than those of PI hydrolysis. Antibodies (both polyclonal and monoclonal) raised against phospholipase C (PLC beta 1) from bovine brain did not react with any component in either rat parotid membranes or cytosol, although a reactivity was detected in rat brain membranes. A monoclonal antibody against bovine brain PLC gamma 1 detected a approximately 150-kDa protein only in the parotid cytosol, while antisera against bovine brain PLC delta 1 enzyme showed no reactivity with parotid membranes or cytosol. Together, these observations suggest that while there appears to be a protein similar to bovine brain PLC gamma 1 in parotid gland cytosol, the PLC which mediates PIP2 hydrolysis in rat parotid membranes and can be regulated by the muscarinic receptor via a G-protein is distinct from the well-characterized PLC enzymes gamma 1, delta 1, and beta 1.
在鸟苷 5'-O-硫代三磷酸（GTPγS）存在的情况下，毒蕈碱胆碱能激动剂氨甲酰胆碱（卡巴胆碱）可使大鼠腮腺膜对外源性添加的、[3H]肌醇标记的磷脂酰肌醇 4,5-二磷酸（PIP2）的水解呈剂量依赖性增加。阿托品和鸟苷 5'-O-硫代二磷酸（GDPβS）可抑制这种刺激作用。单独的 GTPγS 可刺激 PIP2 水解，半最大激活浓度为 0.1μM。这一作用被 GDPβS 抑制，但不受阿托品抑制。激动剂对 PIP2 水解的刺激作用依赖于脂质的存在（磷脂酰丝氨酸：磷脂酰乙醇胺：PIP2 = 1:1:1）。当仅将 PIP2 与去污剂（脱氧胆酸钠）以胶束形式添加时，基础水解增加，从而降低了 GTPγS 和卡巴胆碱的相对刺激作用。在任何一种条件下形成的水溶性水解产物均为 1,4,5-肌醇三磷酸、1,4-肌醇二磷酸和环肌醇三磷酸。在 GTPγS 存在的情况下，卡巴胆碱也可刺激外源性磷脂酰肌醇（PI）的水解，但 PI 水解的程度比 PIP2 水解低 44 倍。当培养基中的[Ca2+]从 100 nM 增加到 1μM 时，PI 和 PIP2 的基础水解均增加（分别增加 9.3 倍和 19.2 倍）。然而，PIP2 的基础水解和刺激后水解水平均高于 PI 水解水平（分别为 37.9 倍和 29.6 倍）。针对牛脑磷脂酶 C（PLCβ1）产生的抗体（多克隆和单克隆）在大鼠腮腺膜或胞质溶胶中均未与任何成分发生反应，尽管在大鼠脑膜中检测到了反应性。一种针对牛脑 PLCγ1 的单克隆抗体仅在腮腺胞质溶胶中检测到一种约 150 kDa 的蛋白质，而针对牛脑 PLCδ1 酶的抗血清在腮腺膜或胞质溶胶中未显示反应性。总之，这些观察结果表明，虽然腮腺腺泡胞质溶胶中似乎存在一种与牛脑 PLCγ1 相似的蛋白质，但介导大鼠腮腺膜中 PIP2 水解且可通过 G 蛋白由毒蕈碱受体调节的 PLC 与已明确的 PLC 酶γ1、δ1 和β1 不同。
