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result from desensitization and downregulation of the [~-cell glucagon/GLP-1 receptors that serve to maintain the glucose-competent state z7. The restoration of glucose competence would then require pharmacological concentrations of the hormones, as recently demonstrated in clinical studies examining the actions of GLP-1 in Type II diabetic patients 28,29.

seem to be relatively unrelated signalling systems.

Acknowledgement The authors acknowledge the valuable contribution of Dr Wiel M. Kuhtreiber to the acquisition and analysis of electrophysiological data presented in this review.

References

Concludingremarks The glucose competence concept provides a simplified, but useful model of a complex process, namely the role of crosstalk in the regulation of insulin secretion. From this perspective it is possible to appreciate fully that there is a synergistic, bidirectional and mutually interdependent interaction between the [3-cell signalling pathways that subserve intermediary metabolism and second messenger-mediated signal transduction. The failure of these two systems to interact properly is hypothesized to be an important determinant for the onset and/or progression of a common metabolic disorder, Type II diabetes. These observations reinforce our view that cellular signal transduction is achieved through the coordinate and fully integrated interaction of what at first might

HOW MANY PHOSPHOLIPASES are required for transduction of extracellular signals across the cellular plasma membrane? The answer seems to be: many more than were anticipated only ten years ago. The activated hydrolysis of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] by a phosphoinositidespecific phospholipase C (ptdIns-PLC) emerged as a new signal transduction pathway for 'Ca2+-mobilizing' agonists in the early 1980s. lntracellular targets for the two messengers produced by PtdIns-PLC - inositol-l,4,5-trisphosphate [lns(1,4,5)P3] and diacylglycerol (DAG) were identified as Ca2*-storage organelles and protein kinase C (PKC), respectively (see reviews by Nishiznka and Taylor and Marshall, this issue). In the following decade it became apparent that many additional products of

1 Wollheim, C. B. and Sharp, G. W. G. (1981) Physiol. Rev. 61, 914-973 2 Zawalich, W. S. and Rasmussen, H. (1990) Mol. Cell. Endocrinol. 70, 119-137 3 Matschinsky, F. M. (1990) Diabetes 39, 647-652 4 Bell, G. I. et al. (1990) Diabetes Care 13, 198-208 5 Thorens, B., Charron, M. J. and Lodish, H. F. (1990) Diabetes Care 13, 209-218 6 Magnuson, M. A. and Shelton, K. D. (1989) J. Biol. Chem. 264, 15936-15942 7 lynedjian, P. B. eta/. (1989) Proc. Natl Acad. Sci. USA 86, 7838-7842 8 MacDonald, M. J. (1990) Diabetes 39, 1461-1466 9 Rajah, A. S. et al. (1990) Diabetes Care 13, 340-363 10 Ashcroft, F. M. and Rorsman, P. (1991) Prog. Biophys. MoL Biol. 54, 87-143 11 Matschinsky, F. M. and Bedoya, F. J. (1989) in Endocrinology(DeGroot, L. J., ed.), pp. 1290-1303, W. B. Saunders 12 Meda, P. (1989) in Cell Interactions and Gap Junctions (Sperelakis, N. and Cole, W. C., eds), pp. 59-79, CRC Press 13 Grodsky, G. M., Curry, D., Landahl, H. and

14 15 16 17 18 19

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26 27 28

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Bennett, L. L. (1969) Acta Diabetol. Lat. 6 (suppl. 1), 554-579 Dupre, J. (1991) in The Endocrine Pancreas (Samols, E., ed.), pp. 253-281, Raven Press Pipeleers, D. G. et al. (1985) Endocrinology 117,824-833 Mojsov, S., Weir, G. C. and Habener, J. F. (1989) J. Clin. Invest. 79, 616-619 Fehman, H. C. and Habener, J. F. (1992) Trends Endocrinol. Metabol. 3, 158-163 Van Schravendijk, C. F. H. et al. (1985) Endocrinology117,841-848 Thorens, B. (1992) Diabetes 41 (suppl. 1), 12 Schuit, F. C. and Pipeleers, D. G. (1985) Endocrinology117,834-840 Drucker, D. J., Philipe, J., Mojsov, S., Chick, W. L. and Habener, J. F. (1987) Proc. Natl Acad. Sci. USA 84, 3434-3438 Pipeleers, D. G. (1987) Diabetologia 30, 277-291 Holz, G. G., Kuhtrieber, W. M. and Habener, J. F. (1992) The Endocrine Society 74th Annual Meeting, San Antonio, TX Anderson, M. P. et al. (1991) Cell 67,775-784 Unger, R. H. and Foster, D. W. (1992)in Williams Textbook of Endocrinology(Wilson, J. D. and Foster, D. W., eds), pp. 1255-1333, W. B. Saunders Bell, G. I. (1991) Diabetes 40, 413-422 Fehman, H. C. and Habener, J. F. (1991) Endocrinology128, 2880-2888 Nathan, D. M., Schreiber, E., Fogel, H., Mojsov, S. and Habener, J. F. (1992) Diabetes Care 15, 270-276 Gutniak, M., Orskov, C., Hoist, J. J., Ahren, B. and Efendic, S. (1992) N. Engl. J. Med. 326, 1316-1322 Horn, R. and Marty, A. (1988) J. Gen. Physiol. 92,145-159 Levitan, E. S. and Kramer, R. H. (1990) Nature 348, 546-547

Crosstalk among multiple signal-activated phospholipases Transduction of extracellular signals across the plasma membrane often involves activation of several phospholipases that generate multiple, sometimes interconvertible, lipid-derived messengers. Coordination and integration of these signal-activated phospholipases may require crosstalk between both the messengers and target protein constituents of these pathways.

phospholipid breakdown are biologically highly active. Furthermore, a number of phospholipases were found to be activated in a signal-dependent manner and thus may be defined as signalactivated phospholipases (SAPs). Among M. Liscovitch is at the Department of the phospholipids that are substrates Hormone Research, The Weizmann Institute for SAPs are not only minor membrane of Science, PO Box 26, Rehovot 76100, Israel. constituents such as PtdIns(4,5)P2 but © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00

also major structural building blocks of the bilayer such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) (Fig. 1). Thus, PtdIns-PLC turned out to be merely the tip of an iceberg and has been used as a model for an increasing number of SAPs, including phospholipases of the types A2, C and D (PLA2, PLC and PLD),

393

Second messenger generation and destruction which utilize PtdIns, PC, PE and SM as substrates (Fig. 1). Evidence for their critical role in cell regulation, coupled

with the fact that an agonist impinging on a single cell often activates several phospholipases in a defined cell type-

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- - c~- cu,,. COOH

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Figure 1 Major cellular phospholipids and their breakdown by signal-activated phospholipases. (a) Glycerophospholipids consist of a hydrophobic DAG moiety (tinted), linked via a phosphodiester bond to a polar headgroup which differ among phospholipid classes. The phospholipid depicted here is a 1-stearoyl-2-arachidonoyl-phospholipid; dozens of other forms (molecular species) exist with different fatty acid combinations. In addition, 1-O-alkyl or 1O-alk-l'-eny! phospholipids abound (especially in the nervous system) that also are substrates for SAPs. So far, only phospholipases of the A2, C and D types have been implicated in signal transduction processes (top panel). (b) Polar headgroups of phospholipids which are implicated in signaling include: o-myo-lns(4,5)P2, choline, ethanolamine and serine. (c) In sphingomyelin, a phosphorylcholine headgroup is esterified at C-1 to an Nacylated sphingoid base (a long chain amino alcohol possessing the general 1,3-diol-2amino structure). The ceramide produced by sphingomyelinase action on sphingomyelin is tinted.

394

TIBS 17 - OCTOBER1992 specific sequence, suggest that crosstalk among these multiple SAPs is involved in their coordinate activation and signal integration. Crosstalk is usually defined as a modulatory interaction between two distinct channels of information transfer. In the context of signal transduction, crosstalk may occur by three major modes, namely, if a constituent of one pathway is modulated by a constituent of another pathway, if two pathways converge upon a common target, or if a messenger of one pathway is converted to a messenger of another pathway. This article briefly reviews the current status of different signal-activated phospholipases, their coordinate activation by agonists, the possibilities for crosstalk among SAPs and the evidence for lipid messenger interconversion and its functional significance.

SAPs: an update A number of recent reviews have dealt with individual SAPs and their messenger products 1-7. By far the first and best characterized of the SAPs, the PtdIns-PLC superfamily, is now known to comprise at least 16 isoenzymes classified into three families. PtdIns-PLC isozymes exhibit distinct (albeit subtle) differences in their catalytic properties, their cell type-specific expression and their mode of activation (see Ref. 1 for recent review). The emergent picture is one where enzymes that belong in different Ptdlns-PLC families are regulated by different mechanisms. The mechanisms of activation of two isozymes, PLC-131 and PLC-?I, are particularly well understood and involve interactions with stimulatory guanine nucleotidebinding (G) proteins and tyrosine kinases, respectively. PLC-[31 is activated by members of the Gq class of pertussis toxin-insensitive G proteins 1. PLC-?I is activated upon the phosphorylation of specific tyrosine residues (Tyr783 and Tyr1254) by receptor tyrosine kinases (RTKs) as well as by cytoplasmic, receptor-activated tyrosine kinases 1,8. Soon after the second messenger function of DAG was established it became apparent that the bulk of DAG is most likely generated by hydrolysis of PC. The signal-activated formation of DAG is often biphasic: it consists of an early peak, which is rapid and transient [and parallels the increase in Ins(1,4,5)P3 and intracellular Ca 2- concentration], followed by a late phase, which is slow in onset but is sustained over many minutes (Fig. 2). While the

TIBS 17 - OCTOBER 1 9 9 2

source of 'early' DAG is most likely in phosphoinositides, 'late' DAG is probably derived by hydrolysis of other phospholipids, mainly PC (see extensive review in Ref. 2). DAG may be directly produced from PC by a phospholipase C; alternatively, DAG may be produced indirectly via PLD, yielding PA, which is then dephosphorylated by phosphatidic acid phosphohydrolase (PAP) (Fig. 3). Direct, PC-PLC-mediated generation of DAG has now been suggested to occur in a number of ceils, especially in response to certain cytokines. A recent report by Schfltze et al? is among the few studies that provide convincing evidence for activation of PC-PLC [by tumour necrosis factor cz (TNFc0] and in the absence of PtdInsPLC activation, Ca2÷ mobilization and PC-PLD activation. Several additional cytokines, e.g. interferon-a, interleukin 1, interleukin 3 and colony-stimulating factor 1, likewise do not affect Ptdlns breakdown, but stimulate production of DAG from PC, apparently via a PC-PLC pathway. Whereas cytokines appear to stimulate PC-PLC in the absence of any change in Ptdlns turnover, the same or a related enzyme is activated, along with PtdIns-PLC and/or PC-PLD, by certain Ca2+-mobilizing agonists such as bombesin, epinephrine, vasopressin and cholecystokinin (cited in Ref. 2). Van Blitterswijk et al. ~° uncovered a PCPLC in human fibroblasts that is stimulated within seconds by bradykinin in parallel to activation of PtdIns-PLC, and releases phosphorylcholine into the extracellular medium, suggesting that PC hydrolysis occurred at the outer leaflet of the plasma membrane. Similar observations were made in other types of ceils (cited in Refs 2,10). While the events discussed above are all shortterm affairs, Moscat and colleagues have reported a delayed activation of PC-PLC activity by platelet-derived growth factor and insulin, which seems to occur downstream of p21 ros activation 11. PLD activity increases rapidly upon stimulation by Ca2+-mobilizing agonists (most of which have previously also been shown to stimulate PtdIns breakdown) 3,4. By and large, the cellular PLD(s) that operate in stimulated cells are rather poorly defined in molecular terms. Studies with intact cells, labeled with different phospholipid polar headgroups, suggested that PLDs that hydrolyse PC, PE and Ptdlns are activated during signaling. Various "forms of

Second messengergeneration and destruction

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Time Figure 2 Dynamics of lipid-derived messengers produced by signal-activated phospholipases. A schematic representation of time-dependent changes in levels of Ins(1,4,5)P3, arachidonic acid (AA), PA and DAG that occur in an idealized cell in response to stimulation with a Ca2+mobilizing agonist. The sequence of phospholipase activation is highly cell type-specific. Ptdlns-PLC, PC-PLC, PC-PLD and PC-PLA2 are all reportedly activated within seconds in various cells. However, increased PC-PLD and PLA2 activity may also be secondary to initial activation of Ptdlns-PLC and PKC, while the DAG and AA thus produced may contribute to the sustained activation of PKC. A delayed activation/induction of PC-PLC provides elevated DAG levels several hours after mitogen stimulation11.

soluble and membrane-bound PLD activities have been characterized using in vitro assays (Refs 12-14 and citations therein). At present, it is difficult to determine which among these enzymes are activated in a signal-dependent manner. So far, none of the PLDs mentioned above have been purified, cloned or sequenced. The activation of PtdIns-PLC and PLD is often accompanied by agonistinduced release of arachidonic acid (AA) through PLA2-catalysed hydrolysis of phospholipids~.E A cytosolic 110 kDa PLA2 has recently been purified and cloned ~5. The PLA2 sequence contains a Ca2+/phospholipid-binding domain homologous to the C2 domain of translocatable, Ca2+-dependent PKC isozymes (a, [3 and 7). This suggests that elevated intracellular Ca2+ concentrations might be required for PLA2 translocation from cytosol to membrane, where the enzyme may be activated by PKC and/or a G protein and where its substrate(s) reside. However, a most intriguing recent report indicates that, in bombesinstimulated Swiss 3T3 cells, PLA 2 iS activated within 2 s, reaching a maximum

by 5-10 s and declines back to basal activity by 5 min 16. Furthermore, evidence that PLA2 activation is Ca2+- and PKC-independent was presented 16. (See below for more on mechanisms of PLA2 activation.) A recent addition to the field of lipidderived messengers is ceramide 7. A series of studies from Hannun's laboratory shows that agents that induce monocytic differentiation of HL-60 cells stimulate hydrolysis of SM by sphingomyelinase, with consequent elevation of cellular ceramide levels (Ref. 17 and citations therein). These agents include la,25-dihydroxyvitamin D3, TNFa and 7interferon. Recently, Dressier et al. TM reported the cell-free stimulation of SM breakdown in HL-60 homogenates by TNFa, implying that the coupling between TNFa receptor and sphingomyelinase is tight enough to withstand disruption of cellular organization. This contrasts with vitamin D3- (and perhaps 7-interferon)-induced SM breakdown, which requires protein synthesis. One immediate cellular target of ceramide might be a protein kinase that phosphorylates Thr669 of the epidermal

395

Second messenger generation and destruction

Ptdlns4P
PC
30% increases in blood pressure observed in many species when NO synthesis is inhibited with L-NMMA1.

Constitutive NO synthase in vascular endothelial cells The enzymes responsible for synthesis of NO from arginine, the NO synthases, each appear to consist of a single medium-sized subunit (125-160kDa), R. G. Knowles and S. Moncada are at The with two such subunits forming a homoWeltcome Research Laboratories, Langley Court, Beckenham, Kent, UK BR3 3BS. dimer in the native protein 4. However, © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00

this enzyme catalyses a reaction of considerable complexity in the synthesis of NO, as well as carrying out several 'side-reactions'. As depicted in Fig. 1, molecular oxygen is incorporated into both NO and citrulline during the (at least) two step reaction with arginine and NADPHs. It has been shown that N Ghydroxy arginine is an intermediate in this reaction 5,6. The NO synthases examined so far contain up to one FMN and FAD, one non-haem iron and o n e tetrahydrobiopterin (BH4) molecule per subunit4,7; these are presumed to participate in the five electron-oxidation of arginine necessary to produce NO. The other catalytic activities that have b e e n demonstrated with purified brain NO synthases are: NADPH-dependent, arginine-independent H 2 0 2 production, NADPH-dependent, arginine-independent dye reduction ('NADPH diaphorase') and cytochrome P-450 reductase activityT-l°; it remains to be seen whether

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Crosstalk among multiple signal-activated phospholipases.

Transduction of extracellular signals across the plasma membrane often involves activation of several phospholipases that generate multiple, sometimes...
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