Inositol Phosphate Metabolism and Signal 'Iransductlon' ARLENE R. HUGHES, DEBRA A. HORSTMAN, HARUO TAKEMURA, and JAMES W. PUTNEY, JR.
Introduction The examination ofthe relationship of phosphoinositide turnover to Ca2+ signaling began in the early 1950s with the observation by Mabel and Lowell Hokin that the muscarinic cholinergic receptor agonist, acetylcholine, selectively increased the incorporation of HPj into two minor plasma membrane phospholipids, phosphatidylinositol (PI) and phosphatidic acid (I). However, it was not until some 20 years later that Robert Michell, noting that the receptors that stimulated phosphoinositide turnover also activated Ca2+-dependent processesin the cell, proposed that receptor-stimulated phosphoinositideturnover results in a cellular Ca2+ response (2). PI is sequentially phosphorylated by kinases in the cell to phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositoI4,5-bisphosphate (PIP,). Indeed, wenow know that stimulation by any of a number of Ca2+-mobilizing receptor agonists initially results in the phospholipase C-catalyzed hydrolysisof PIP, (3). Furthermore, the Hokins' initial observation of the incorporation of HPj into PI results from receptor-stimulated PIP, hydrolysis, breakdown of the inositol phosphates to inositol, and resynthesis of PI. Phospholipase C-catalyzed PIP, hydrolysisresults in the formation ofthe water-soluble (I,4,5)inositol trisphosphate [(I,4,5)IP 3 ) and the lipid-soluble diacylglycerol.Berridge proposed that (1,4,5)IP3 was the likely intracellular messenger candidate that stimulated Ca2+ release from intracellular stores (4). Soon thereafter, the effects of (I,4,5)IP 3 on Ca2+ mobilization were demonstrated; micromolar concentrations of (I,4,5)IP 3 rapidly released Ca 2 ' from a nonmitochondrial store in permeabilized pancreatic acinar cells (5). These results wereconfirmed quickly in other tissues and laboratories (4, 6). Thus, the evidence is convincing that (I,4,5)IP3 , generated upon the activation of Ca2+-mobilizing receptors, releases Ca2+ from intracellular stores. Meanwhile, a parallel story evolvedin Nishizuka's laboratory (7-9), which demonstrated that the other product of PIP 2 hydrolysis, namely diacylglycerol, also was a potent intracellular messenger. Diacylglycerolremains in the plasma membrane to activate a ubiquitous protein kinase, designated as C-kinase by Nishizuka. This reviewbriefly summarizes some of the current issues regarding the regulation of cellular Ca 2 ' metabolism by inositol phosphates. The metabolic pathways by which the inositol phosphates are interconverted will be discussed. Finally, a recently proposed mechanism by which inositol phosphates elicit Ca2+ entry from the extracellularspace is described.
SUMMARY Activation of a variety of cell surface receptors results in a biphasic Increase in the cytoplasmic Ca2+ concentration, due to the release or mobilization of intracellular Ca" stores and to the entry of Ca2+ from the extracellular space. Stimulation of these same receptors also results in the hydrolysis of the minor plasma membrane phospholipid, phosphatldylinositoI4,5-bisphosphate, with the concomitant formation of (1,4,5)inositol trisphosphate [(1,4,5)IP,1 and diacylglycerol. It is well established that phosphatldylinositoI4,5-bisphosphate hydrolysis is responsible for the changes in Ca" homeostasis. There is strong evidence that (1,4,5)IP, stimulates Ca2+ release from intracellular stores. The Ca"-releasing actions of (1,4,5)IP, are terminated by Its metabolism through two distinct pathways: (1,4,5)IP,ls dephosphorylated by a S-phosphataseto (1,4)IP,;alternatively, (l,4,5)IP, is phosphorylated to (1,3,4,5)IP. by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known. A recent proposal that (1,4,5)IP" by emptying an Intracellular Ca2+ pool, secondarily elicits Ca" entry will be considered. This review summarizes recent studies of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations. AM REV RESPIR DIS 1990; 141:S115-S118
Metabolism of Inositol Phosphates Initially, the metabolism of inositol tris- and bisphosphates generated in response to the activation of surface membrane receptors was thought to be a rather simple process. It was believedthat (I,4,5)IP 3 wasdephosphorylated to (I,4)IP" to (I)IP, and finally to free inositol by a lithium-sensitive inositol l-phosphatase. More recently, the pathways of inositol phosphate metabolism have been revealed to be more complex (figure 1)(10, ll). This is attributable largelyto the development of HPLC analytical procedures that can resolve inositol phosphates with only subtle differences in structure. (1,4,5)IP 3 is dephosphorylated by a 5-phosphatase to (1,4)IP,(12), and (1,4)IP2 is believed to be dephosphorylated almost exclusively to (4)IP by (1,3,4)IP3/(I,4)IP,-Iphosphatase (13). In addition to the dephosphorylation of (I,4,5)IP 3 by the 5-phosphatase, there exists in most tissues a 3-kinase that transfers a phosphate from ATP to the 3 position of (I,4,5)IP 3 to form (1,3,4,5)IP. (14).This molecule is then dephosphorylated by the same 5-phosphatase that degrades (I,4,5)IP3 to form an isomeric inositol trisphosphate, (1,3,4)IP 3 • (I,3,4)IP 3 is then dephosphorylated by the (I,3,4)IP 3/(I,4)IP,-Iphosphatase to (3,4)IP, (13), and to (I,3)IP 2 (15) by an enzyme that is not well characterized. These bisphosphates are then dephosphorylated primarily to a mixture of (I)IP and (3)IP, which are stereoisomers and not resolved by conventional HPLC. The above discussion describes the routes of metabolism of (I,4,5)IP 3 derived from the phospholipase C-mediated hydrolysis of PIP 2 • In vitro, it is possible to demonstrate phospholipase C-mediated hydrolysis of PIP and PI as well (10, 16). If these reactions were to occur in vivo, then it would be possible to generate diacylglycerol and to cause activation of protein kinase C without an accompanying mobilization of intracellular Ca",
Some authors have speculated that PIP and PI might be degraded directly in stimulated cells, based primarily on the large quantities of(l)IP and (I,4)IP, formed (13,17,18). However, in one recent study, the kinetics of formation and breakdown of inositol mono- and bisphosphates in stimulated parotid acinar cells were examined (19). In that study, it was found that despite the accumulation of large quantities of (I)IP and (I,4)IP, after prolonged stimulation, the turnover of all of the mono- and bisphosphates formed could be accounted for from the calculated rates of metabolism of (1,4,5)IP3 through the 3-kinase and 5-phosphatase pathways. Similar kinetic studies have not as yet been carried out on other cellular systems. Thus, for the present, the extent of hydrolysis of inositol-containing lipids other than PIP 2 in agonist-activated cells must remain an open question. The complexity of the metabolic pathway for metabolism of (1,4,5)IP 3 suggests that in addition to the Ca'
Introduction The examination ofthe relationship of phosphoinositide turnover to Ca2+ signaling began in the early 1950s with the observation by Mabel and Lowell Hokin that the muscarinic cholinergic receptor agonist, acetylcholine, selectively increased the incorporation of HPj into two minor plasma membrane phospholipids, phosphatidylinositol (PI) and phosphatidic acid (I). However, it was not until some 20 years later that Robert Michell, noting that the receptors that stimulated phosphoinositide turnover also activated Ca2+-dependent processesin the cell, proposed that receptor-stimulated phosphoinositideturnover results in a cellular Ca2+ response (2). PI is sequentially phosphorylated by kinases in the cell to phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositoI4,5-bisphosphate (PIP,). Indeed, wenow know that stimulation by any of a number of Ca2+-mobilizing receptor agonists initially results in the phospholipase C-catalyzed hydrolysisof PIP, (3). Furthermore, the Hokins' initial observation of the incorporation of HPj into PI results from receptor-stimulated PIP, hydrolysis, breakdown of the inositol phosphates to inositol, and resynthesis of PI. Phospholipase C-catalyzed PIP, hydrolysisresults in the formation ofthe water-soluble (I,4,5)inositol trisphosphate [(I,4,5)IP 3 ) and the lipid-soluble diacylglycerol.Berridge proposed that (1,4,5)IP3 was the likely intracellular messenger candidate that stimulated Ca2+ release from intracellular stores (4). Soon thereafter, the effects of (I,4,5)IP 3 on Ca2+ mobilization were demonstrated; micromolar concentrations of (I,4,5)IP 3 rapidly released Ca 2 ' from a nonmitochondrial store in permeabilized pancreatic acinar cells (5). These results wereconfirmed quickly in other tissues and laboratories (4, 6). Thus, the evidence is convincing that (I,4,5)IP3 , generated upon the activation of Ca2+-mobilizing receptors, releases Ca2+ from intracellular stores. Meanwhile, a parallel story evolvedin Nishizuka's laboratory (7-9), which demonstrated that the other product of PIP 2 hydrolysis, namely diacylglycerol, also was a potent intracellular messenger. Diacylglycerolremains in the plasma membrane to activate a ubiquitous protein kinase, designated as C-kinase by Nishizuka. This reviewbriefly summarizes some of the current issues regarding the regulation of cellular Ca 2 ' metabolism by inositol phosphates. The metabolic pathways by which the inositol phosphates are interconverted will be discussed. Finally, a recently proposed mechanism by which inositol phosphates elicit Ca2+ entry from the extracellularspace is described.
SUMMARY Activation of a variety of cell surface receptors results in a biphasic Increase in the cytoplasmic Ca2+ concentration, due to the release or mobilization of intracellular Ca" stores and to the entry of Ca2+ from the extracellular space. Stimulation of these same receptors also results in the hydrolysis of the minor plasma membrane phospholipid, phosphatldylinositoI4,5-bisphosphate, with the concomitant formation of (1,4,5)inositol trisphosphate [(1,4,5)IP,1 and diacylglycerol. It is well established that phosphatldylinositoI4,5-bisphosphate hydrolysis is responsible for the changes in Ca" homeostasis. There is strong evidence that (1,4,5)IP, stimulates Ca2+ release from intracellular stores. The Ca"-releasing actions of (1,4,5)IP, are terminated by Its metabolism through two distinct pathways: (1,4,5)IP,ls dephosphorylated by a S-phosphataseto (1,4)IP,;alternatively, (l,4,5)IP, is phosphorylated to (1,3,4,5)IP. by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known. A recent proposal that (1,4,5)IP" by emptying an Intracellular Ca2+ pool, secondarily elicits Ca" entry will be considered. This review summarizes recent studies of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations. AM REV RESPIR DIS 1990; 141:S115-S118
Metabolism of Inositol Phosphates Initially, the metabolism of inositol tris- and bisphosphates generated in response to the activation of surface membrane receptors was thought to be a rather simple process. It was believedthat (I,4,5)IP 3 wasdephosphorylated to (I,4)IP" to (I)IP, and finally to free inositol by a lithium-sensitive inositol l-phosphatase. More recently, the pathways of inositol phosphate metabolism have been revealed to be more complex (figure 1)(10, ll). This is attributable largelyto the development of HPLC analytical procedures that can resolve inositol phosphates with only subtle differences in structure. (1,4,5)IP 3 is dephosphorylated by a 5-phosphatase to (1,4)IP,(12), and (1,4)IP2 is believed to be dephosphorylated almost exclusively to (4)IP by (1,3,4)IP3/(I,4)IP,-Iphosphatase (13). In addition to the dephosphorylation of (I,4,5)IP 3 by the 5-phosphatase, there exists in most tissues a 3-kinase that transfers a phosphate from ATP to the 3 position of (I,4,5)IP 3 to form (1,3,4,5)IP. (14).This molecule is then dephosphorylated by the same 5-phosphatase that degrades (I,4,5)IP3 to form an isomeric inositol trisphosphate, (1,3,4)IP 3 • (I,3,4)IP 3 is then dephosphorylated by the (I,3,4)IP 3/(I,4)IP,-Iphosphatase to (3,4)IP, (13), and to (I,3)IP 2 (15) by an enzyme that is not well characterized. These bisphosphates are then dephosphorylated primarily to a mixture of (I)IP and (3)IP, which are stereoisomers and not resolved by conventional HPLC. The above discussion describes the routes of metabolism of (I,4,5)IP 3 derived from the phospholipase C-mediated hydrolysis of PIP 2 • In vitro, it is possible to demonstrate phospholipase C-mediated hydrolysis of PIP and PI as well (10, 16). If these reactions were to occur in vivo, then it would be possible to generate diacylglycerol and to cause activation of protein kinase C without an accompanying mobilization of intracellular Ca",
Some authors have speculated that PIP and PI might be degraded directly in stimulated cells, based primarily on the large quantities of(l)IP and (I,4)IP, formed (13,17,18). However, in one recent study, the kinetics of formation and breakdown of inositol mono- and bisphosphates in stimulated parotid acinar cells were examined (19). In that study, it was found that despite the accumulation of large quantities of (I)IP and (I,4)IP, after prolonged stimulation, the turnover of all of the mono- and bisphosphates formed could be accounted for from the calculated rates of metabolism of (1,4,5)IP3 through the 3-kinase and 5-phosphatase pathways. Similar kinetic studies have not as yet been carried out on other cellular systems. Thus, for the present, the extent of hydrolysis of inositol-containing lipids other than PIP 2 in agonist-activated cells must remain an open question. The complexity of the metabolic pathway for metabolism of (1,4,5)IP 3 suggests that in addition to the Ca'
