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Recent hypotheses regarding the phosphatidylinositol effect
Life Sciences, Vol. 29, pp. Printed in the U.S.A.
1183-1194
Pergamon Press
MINIREVIEW RECENT HYPOTHESESREGARDING THE PHOSPHATIDYLINOSITOL EFFECT James W. Putney, Jr. Department of Pharmacology Medical College of V i r g i n i a V i r g i n i a Commonwealth University Richmond, VA. 23298 U.S.A. Summary In 1975, Michell f i r s t proposed that a c t i v a t i o n of phosp h a t i d y l i n o s i t o l turnover provided a d i r e c t l i n k between surface receptors and membrane Ca gates. Subsequently, a number of laboratories have begun to r e - i n v e s t i g a t e this phenomenon f i r s t described by Hokin and Hokin some twenty years e a r l i e r . As would be expected, some new hypothesis have emerged, most being extensions or revisions of M i c h e l l ' s o r i g i n a l concept. Despite d i f f i c u l t i e s in obtaining d i r e c t proof, i n d i r e c t evidence suggests that the plasma membrane is the primary locus of receptor-activated phosphatidylinositol turnover, at least for phosphatidylinositol breakdown and phosphatidic acid synthesis. The presence or absence of Na+ can markedly a f f e c t labelling of phosphatidylinositol by radioactive precursors but there is no compelling evidence that the i n i t i a l events are mediated by Na+. Prostaglandins are apparently formed in some tissues on receptor a c t i v a t i o n , but in most instances the evidence suggests that these compounds are not obligatory intermediates in tissues that show the phosphatidylinositol e f f e c t . Several laboratories have obtained evidence that phosphatidic acid newly synthesized following phosphatidylinositol breakdown, may function as an endogenous Ca ionophore under neurohumoral control. In 1953, Hokin and Hokin ( I ) reported that acetylcholine stimulates the incorporation of 32P04 into phospholipids of the pigeon pancreas in v i t r o . In a subsequent paper (2) these investigators demonstrated that the s p e c i f i c phospholipids responsible for the e f f e c t were phosphatidylinositol (PI) and phosphatidic acid (PA). This phenomenon by which stimuli enhance the radioactive l a b e l l i n g of phosphatidylinositol has been generally termed the "phospholipid e f f e c t " or more s p e c i f i c a l l y t h e " P l e f f e c t . " As discussed below, however, all instances of increased phosphatidylinositol l a b e l l i n g may not r e f l e c t the same mechanism, and additional constraints on d e f i n i t i o n s of the PI e f f e c t may be necessary.
0024-3205/81/121183-12502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
1184
On the PI Effect
Vol. 29, No.
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An i n i t i a l hypothesis as to the s i g n i f i c a n c e of the PI e f f e c t was that i t r e f l e c t e d a l t e r a t i o n s in the metabolism of membrane phospholipids r e s u l t i n g from the secretions of zymogens (3). A number of subsequent observations discouraged t h i s idea, however. For one, the concentration-effect r e l a t i o n s h i p f o r s t i m u l a t i o n of the PI e f f e c t lay somewhat to the r i g h t (higher concentrations) of that f o r s t i m u l a t i o n of protein secretion (4). Also, protein secretion could be prevented by removal of Ca, but the PI e f f e c t was l a r g e l y unaffected (5). F i n a l l y , the s u b c e l l u l a r locus of the m a j o r i t y of newly incorporated label was found to be the rough endoplasmic reticulum rather than the membranes of the plasmalemma and zymogen granules (6,7). When these studies were f i r s t carried to the s a l i v a r y glands (8), the secretory response and PI e f f e c t due to adrenergic s t i m u l a t i o n were c l e a r l y dissociable by the use of the m-adrenergic antagonist, ergotamine, which i n h i b i t e d the PI e f f e c t without a f f e c t i n g protein secretion; the l a t t e r is now known to occur in s a l i v a r y glands p r i m a r i l y through B-adrenoceptor a c t i v a t i o n (9). As is often the case, this apparent inconsistency ( i . e . d i s s o c i a t i o n of the PI e f f e c t from protein secretion) was r a t i o n a l i z e d (8) into obscurity and rediscovered sixteen years later (I0,II). Another hypothesis that received considerable a t t e n t i o n in the s i x t i e s was the idea that the reaction was in some way involved in the mechanism of the Na, K-pump (12,13). This concept was derived from the finding that in avian s a l t glands, a c t i v e Na transport and the PI e f f e c t were concomitantly activated by a c e t y l c h o l i n e . Again, however, f u r t h e r experimentation led to the clear d i s s o c i a t i o n of these two events (14,15). I t is now clear that no close association e x i s t s between the PI e f f e c t and a c t i v e Na transport. The concept, however, of a r e l a t i o n s h i p between membrane phospholipid composition and cation movements is quite s i m i l a r to some current ideas discussed below. (Compare, for example, Ref. 12 to Ref. 16 and Ref. 17). Enzymatic Pathways of the PI Effect There is considerably less data a v a i l a b l e on net changes of phospholipid l e v e l s as compared to data on l a b e l l i n g and turnover. In many cases, however, i t has been shown that associated with the increased l a b e l l i n g of the PI and PA, the tissue content of PI f a l l s and the content of PA increases (17-20). The simplest scheme consistent with these data is one proposed by Hokin and Hokin (15) for the avian s a l t gland, and generalized to other tissues by Michell (20). The pathways are i l l u s t r a t e d in Fig. I . For most tissues the evidence that agonists can a c t i v a t e t h i s cycle at the level of a phospholipase C is l a r g e l y c i r c u m s t a n t i a l . Unfortunately, a t tempts to demonstrate effects of agonists on phospholipase C a c t i v i t y in broken c e l l preparations have been l a r g e l y unsuccessful. A recent preliminary report suggests that 5-hydroxytryptamine accelerates phosphatidylinositol breakdown in homogenates of the blowfly s a l i v a r y gland (21). For the p l a t e l e t , there is strong evidence that thrombin acts by stimulation of a phospholipase C. Cyclic AMP is known to i n h i b i t the p l a t e l e t aggregation response to agents l i k e thrombin and the l a b e l l i n g of phosphatidic acid (22). B i l l a h and co-workers (23) have shown that p l a t e l e t s contain a phosphat i d y l i n o s i t o l s p e c i f i c phospholipase C. The enzyme a c t i v i t y when assayed in broken c e l l s , is also markedly depressed i f p l a t e l e t s are f i r s t treated with d i b u t y r y l c y c l i c AMP or phosphodiesterase i n h i b i t o r s . The i n h i b i t i o n by cycl i c AMP of both the phospholipase C and the thrombin stimulated PI e f f e c t provides the f i r s t demonstrated l i n k between a phospholipase C assayed in broken c e l l s and the effects of an agonist in the i n t a c t c e l l . Also of considerable s i g n i f i c a n c e is the demonstration by these same i n v e s t i g a t o r s that the enzyme is found in the soluble f r a c t i o n (24). This l a t t e r observation,
Vol. 29, No. 12, 1981
On the PI Effect
1185
i f i t turns out to be the general case, means that receptors may not d i r e c t ly activate the enzyme but rather could a l t e r the state of the substrate (phosphatidylinositol) increasing i t ' s s u s c e p t i b i l i t y to hydrolysis.
AGONIST + PI
RECEPTOR
INOSITOL/CDP-DIP D IACYLG LYCEROL
CDP-DIACYLGLYCEROL
DGKZ A T
P P i ~PACT CTP
P
PA FIG. 1
Enzymatic pathways of the PI effect. According to this scheme, receptor activation leads to the breakdown of PI to diacylglycerol by a phospholipase C (PLC). Diacylglycerol is phosphorylated by ATP and diacylglycerol kinase (DGK) to form PA. PA is subsequently conjugated with CTP by the action of phosphatidic acid: CTP cytidyltransferase (PACT) to form CDP-diacylglycerol. PI is f i n a l l y formed by exchange of the nucleotide with free inositol by the enzyme CDP-diacylglycerol inositol phosphatidyltransferase (CDP-DIP). One d i f f i c u l t y with the scheme in Fig. 1 is that while the enzyme that synthesizes PA (diglyceride kinase) apparently resides in the plasmalemma (20), i t appears that the enzymes involved in synthesizing PI from PA are localized largely to the endoplasmic reticulum (20,25). The relevance of this situation to patterns of phospholipid labelling is considered l a t e r in the section on "subcellul a r locus of the P l - e f f e c t . " Michel!'s Hypothesis From the late seventies to the present there has been a substantial increase in interest in the PI effect. This can be credited, in the reviewer's opinion, to an hypothesis f i r s t put forth in 1975 by Michell (20). Michell was apparently struck by the fact that the PI effect could be e l i c i t e d in salivary glands by muscarinic or~-adrenergic stimuli or in the pancreas by muscarinic agonists or certain peptides. The end response in each case, however, differed considerably. In the salivary glands, the receptors cont r o l l e d the K permeability response, while in the pancreas the receptors which caused PI turnover stimulated enzyme discharge. The common denominator was not the end response of the tissue but rather the second messenger, calcium. As the PI e f f e c t , in contrast to other c e l l u l a r responses, appeared independent of external Ca, Michell reasoned that PI turnover might pre-
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On the PI Effect
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cede Ca mobilization and might be involved in the mechanism by which receptors mediate Ca mobilization (20). Supportive of this idea was the finding that the a r t i f i c i a l introduction of Ca into pancreas and salivary gland c e l l s stimulates the appropriate end responses (26), but has l i t t l e e f f e c t on PI turnover (27-30). Similar experimental results have been obtained for a number of other tissues and receptor types that act through Ca (31), and thus the body of circumstantial evidence for the Ca-gating hypothesis continues to grow. There are also examples of stimulation of PI l a b e l l i n g that are not cons i s t e n t with the Michell hypothesis. One such example is the synaptosome (32) where stimulation of PI l a b e l l i n g by acetylcholine is Ca dependent and can be mimicked by the calcium ionophore A23187. In the neutrophil, both PI l a b e l l i n g and breakdown have been shown to be Ca mediated (33,34). In the case of the synaptosome, however, the Ca-dependence for only PI l a b e l l i n g has been investigated. Thus, i t is not certain that the i n i t i a l event is necessarily PI breakdown, at least in regard to the d i r e c t cause of the !abelling. For example, Ca-mediated breakdown of polyphosphoinositides to diglyceride is well documented in smooth muscle (35) and in syn~Rtosomes (36). The d i g l y c e r i d e is r a p i d l y phosphorylated in the presence of °LP-ATP to form radioactive PA, the precursor for PI synthesis (25). Other major phospholipids are p r i m a r i l y synthesized from diglyceride d i r e c t l y and would not show an increased l a b e l l i n g in p a r a l l e l to PA. There is no proof as to whether such a scheme can explain any s i t u a t i o n in which PI l a b e l l i n g appears to be Ca mediated. I t does serve to point out an important caveat, however: Stimul a t i o n of phospholipase C a c t i v i t y against any phospholipid (not necessarily PI) w i l l generate d i g l y c e r i d e and w i l l t h e o r e t i c a l l y r e s u l t in an increased l a b e l l i n g of PA and PI. Other t h e o r e t i c a l treatments of these anomalous results have been considered by Michell and Kirk (37). Regardless of the explanation, i t is important to point out that there is no basis f o r extrapolation of these results to other tissues where s t r i c t adherence to the appropriate c r i t e r i a of the Ca-gating hypothesis has been established. Table i l i s t s a few well studied tissues in which the PI e f f e c t has been c l e a r l y shown to be: (a) Ca-independent, (b) poorly or not at all stimulated by calcium ionophore, and (c) associated with net breakdown of PI. For such cases, M i c h e l l ' s hypothesis remains tenable, and i t is within the confines of these well-defined systems that some new postulates on the significance of the PI e f f e c t w i l l be considered. I n o s i t o l Dependency of the Fly Salivary Gland In the blowfly salivary gland, stimulation of membrane receptors by 5hydroxytryptamine (5-HT) activates both adenylate cyclase and Ca-gating (46). Probably, by analogy with the case for ~- and ~-adrenoceptors, these effects are mediated by separate receptors (47). The two second messengers, Ca and c y c l i c AMP, act in concert to increase e p i t h e l i a l CI~ permeability and K+ transport which results in salivary f l u i d secretion (46). Concomitant with the a c t i v a t i o n of f l u i d secretion and Ca-gating, 5-HT also stimulates breakdown of PI, apparently at the basal surface of the gland (42). The f l y gland is an especially useful preparation for these studies, since a c t i v a t i o n ~ Ca gating and PI breakdown can be monitored simultaneously as rel~ase of Ca and H - i n o s i t o l . Fain and Berridge (42) found that release of H-inositol was neither i n h i b i t e d by EGTA nor stimulated by A23187, and was independent of the synthesis of c y c l i c AMP (42). In addition, these authors (48,49) also found that prolonged stimulation of the f l y gland led
Kirk et al (40)
Lloyd et al (41)
Platelet
Fly Salivary Gland
Smooth Muscle
Salmon and Honeyman* (16)
*Salmon and Honeyman (16) found that elevation of [Ca2+] i with high [K+] contracted isolated smooth muscle cells but did not stimulate PA labelling. This experiment is equlvalent to the use of an ionophore for the sake of the arguments advanced here. I t should be mentioned, however, that J a f f e r j i and Michell (45), using smooth muscle fragments, obtained a stimulation of PI labelling by high [K+].
(43)
Jafferji
and Michell
Jafferji
(44)
Fain and Berridge (42)
Fain and Berridge (42)
Fain and Berridge (42)
and Michell
Lapetina and Cuatrecasas (22)
(Relatively) Lapetina and Cuatrecasas (22)
(39)
Billah and Michell
(Partially) Billah and Michell Kirk et al (40)
Liver (39)
Jones and Michell (19) Weiss and Putney (38)
Rossignol et al (27) Oron et a l - ~ 2 ~ Jones and Michell (28)
Oron et al (29) Jones and Michell (28)
Hokin (18)
Calderon et al (30)
Parotid Gland
PI-Breakdown Occurs
lonophore Insensitive
Hokin (5) Calderon et al (30)
Ca Independent
C r i t e r i a Met (Reference)
Exocrine Pancreas
Tissue
Tissues meeting the c r i t e r i a for an involvement of PI turnover in Ca-gating
TABLE 1
O0 "-.I
r-t
~-h
F~
0
OO
Z 0
< 0
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On the PI Effect
Vol. 29, No. 12, 1981
gradually to an i n a b i l i t y to secrete or to gate Ca. For recovery to occur, i n o s i t o l was required in the medium. These results suggest that when the relevant pool of PI is exhausted, the Ca channels are " f i x e d " in a "closed" rather than "open" state. This would indicate that simply removing the head group from PI is not s u f f i c i e n t alone to open Ca gates. Possibly, e i t h e r the breakdown reaction i t s e l f or some product of that reaction may be necessary. A l t e r n a t i v e l y , PI breakdown could mediate the closing of the gate (47,48). Perhaps more s i g n i f i c a n t l y , these observations provided the f i r s t evidence for a cause-effect r e l a t i o n s h i p between PI hydrolysis and Ca-gating. Subcellular Locus of the PI Effect When tissues are subjected to s u b c e l l u l a r f r a c t i o n a t i o n following stimul a t i o n of PI turnover, the majority of radioactive PI (or PI breakdown) is found in the microsomal f r a c t i o n , the largest portion of which is derived from endoplasmic reticulum (40,50). As Michell and his colleagues (20,40) point out, there are two possible explanations for this r e s u l t . F i r s t , receptor stimulation at the plasma membrane could lead to the generation of a heretofore undisclosed second messenger which activates phospholipid metabolism in the endoplasmic reticulum. On the other hand, the stimulus could act i v a t e PI breakdown at the plasma membrane, the resulting d i g l y c e r i d e could be phosphorylated to radioactive PA which would then be transported to the endoplasmic reticulum via phospholipid exchange proteins. Such proteins (which r e a d i l y catalyze PI exchange) have recently been characterized (51-54), but none s p e c i f i c for PA has yet been described. At the endoplasmic reticulum, synthesis of radioactive PI would occur. Because of the large pool of PI in the endoplasmic reticulum ( i . e . due to isotope d i l u t i o n ) , non-radioactive PI would be transported back to the plasma membrane, and the cycle continued. This is consistent with the generally held view (20) that PI synthesis is res t r i c t e d to the endoplasmic reticulum while d i g l y c e r i d e kinase (and incident a l l y , phosphatidate phosphohydrolase) are present in the plasma membrane. This l a t t e r scheme predicts that the primary hormone-sensitive pool of PI may be quite small. In the f l y salivary gland, resynthesis of PI is s u f f i c i e n t ly slow that a l l of the hormone-sensitive pool of PI can be hydrolyzed leading to complete i n a c t i v a t i o n of Ca-gating (48). Under these conditions net loss of PI is not s a t i s t i c a l l y detectable (49) and Fain and Berridge estimate that resynthesis of less than 10% of the PI in the gland w i l l completely reactivate hormone s e n s i t i v i t y . Again, these results could indicate a small pool of PI, perhaps in the plasma membrane, that is the primary s i t e of hormone action. Another point in favor of the proposed r e l a t i o n s h i p between endoplasmic reticulum and plasma membrane is the nature of the PI e f f e c t in the avian s a l t gland. Unfortunately, that Ca acts as a coupling factor in these c e l l s has not yet been c l e a r l y established. As the receptors are muscarinic (13), and since receptor a c t i v a t i o n does stimulate PA and PI l a b e l l i n g , the assumption that the phospholipid e f f e c t r e f l e c t s the same reactions as in the more extensively characterized mammalian system seems reasonable. Unlike the mammalian exocrine glands, the avian s a l t glands do not secrete protein; there does however appear to be an a c e t y l c h o l i n e - a c t i v a t e d (muscarinic) Na permeab i l i t y , probably at the basal membrane (55). A consequence of this is that avian s a l t glands, in contrast to mammalian exocrine glands, have very l i t t l e endoplasmic reticulum. Due to this paucity of endoplasmic reticulum and the extensive i n t e r d i g i t a t i o n s of the basal (plasma) membrane, the quantity of plasmalemma would be considerably 9reater than internal membranes (with the possible exception of mitochondria).
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The pattern of the PI e f f e c t in this tissue is consistent with the hypothesis that the plasma membrane is the primary s i t e of action of acetylcholine. F i r s t , the stimulation of 32P04 incorporation is not diminished in comparison to other tissues, despite the small quantity of endoplasmic r e t i c u lum. On the other hand, there is a considerably smaller increase in radioact i v i t y in PI (believed to be synthesized at the endoplasmic reticulum) with a larger and extremely rapid e f f e c t f o r PA (believed to be synthesized at the plasmalemma) (15,56). When the stimulus is removed (with a t r o p i n e ) , rapid resynthesis of PI occurs which is approximately stoichiometric for the loss in radioactive PA (15). Taken with the results obtained in mammalian systems, these findings are consistent with the suggestion that at the plasma membrane, activated receptors stimulate PI breakdown followed by PA synthesis. As resynthesis of PI presumably takes place in internal membranes (endoplasmic reticulum), this would occur therefore with r a p i d i t y varying from tissue to tissue. The extent of l a b e l l i n g of PI would also vary with the size of the endoplasmic reticulum pool. Role of Na+ in the PI Effect Recently, i t has been reported that media d e f i c i e n t in Na+ i n h i b i t the receptor-stimulated l a b e l l i n g of PI by 3H-inositol (57) or 32p04 (58). These observations have led to the suggestion that Na+ i n f l u x might precede the PI e f f e c t (57,58). Such a scheme is not consistent with previous data on control of Na+ movements in the parotid gland, however. Thus, in the absence of Ca, cholinergic and ~-adrenergic agonists stimulate PI l a b e l l i n g (28,29) but not Na÷ i n f l u x (59). Also, the d i v a l e n t cationophore A23187, has l i t t l e e f f e c t on PI turnover, (27,29) but does stimulate Ca-dependent Na uptake (59). In addition, Williams (60) has shown that Na+ removal does not a f f e c t Ca mobilization due to caerulein in pancreatic acini and concludes that Na+ i n f l u x has no d i r e c t role in coupling receptor occupancy to protein secretion. One explanation f o r the e f f e c t s of Nat may be that i t is the s p e c i f i c a c t i v i t y of precursors rather than the reaction rates themselves which is affected. In support of t h i s , A b d e l - L a t i f (61) found that in the absence of external Na+, the s p e c i f i c r a d i o a c t i v i t y of ATP was les§ than 5% of control. Also, Keryer, Herman and Ross~gnol (57) found that H-inositol uptake was s u b s t a n t i a l l y reduced by Na omission. These results suggest that i t would be more appropriate to examine the breakdown of PI d i r e c t l y rather than to deal with pools of precursors with unknown and changing s p e c i f i c radioactivity. In the only study in which net changes in PI content were measured, Jones and Michell (62) found that net breakdown of PI in parotid fragments due to carbachol was not prevented by decreasing external Na+ to less ~han 1 mM. Thus, while there appear to be r e a d i l y demonstrated e f f e c t s of Na- omission on the PI l a b e l l i n g cycle, the~e is no compelling evidence to support the idea that receptor-activated Na fluxes precede oY-mediate the primary e f f e c t s of agonists on PI breakdown. Prostagl andi ns In mammals, the number 2 position of PI is highly enriched in arachidonic acid (20). I t would not be unreasonable, therefore, to a n t i c i p a t e that a l t e r a t i o n s in PI metabolism might lead u l t i m a t e l y to the l i b e r a t i o n of this r a t e - l i m i t i n g precursor for synthesis of prostaglandins and other products of cyclo-oxygenase or lipoxygenase (63). These oxidative metabolites could subsequently function in Ca-gating or in other c e l l u l a r responses.
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There is good evidence that some tissues that show altered PI l a b e l l i n g can also make and respond to prostaglandins [mast c e l l s (64), neutrophils (65,66), p l a t e l e t s (67)]. In order to invoke prostaglandins as o b l i g a t o r y intermediates in the responses to receptor a c t i v a t i o n one should be able to (a) mimic the action of a receptor agonist by application of a prostaglandin or the r a t e - l i m i t i n g precursor, arachidonate, (b) demonstrate stimulation of prostaglandin formation, or at least arachidonate mobilization, and (c) demonstrate a p a r a l l e l r e l a t i o n s h i p between pharmacological i n h i b i t i o n of arachidonate metabolism and i n h i b i t i o n of the appropriate c e l l u l a r response. Failure to s a t i s f y c r i t e r i o n (c) has cast serious doubt on any o b l i g a t o ry role for prostaglandins in stimulus-response coupling in a v a r i e t y of cell types. In p l a t e l e t s for example, exogenous arachidonate or thromboxane can mimic receptor a c t i v a t i o n and stimulation of p l a t e l e t receptors leads to thromboxane synthesis (67). However, stimulation of p l a t e l e t secretion by thrombin is t o t a l l y unaffected by 20 ~M eicosatetraynoic acid (ETYA), an agent that (at 20 uM) completely blocks arachidonate metabolism by both cyclooxygenase and lipoxygenase (22). S i m i l a r l y , in neutrophils, ETYA is about ten times less e f f e c t i v e in antagonizing the chemotactic peptide, F-Met-Leu-Phe, than exogenous arachidonate, and is almost t o t a l l y i n e f f e c t i v e against the complement fragment, C5a (66). Recently, Marshall, Dixon and Hokin (68) reported data demonstrating that receptor-mediated secretion of amylase by the mouse exocrine pancreas might involve prostaglandin formation. Thus, arachidonic acid and certain prostaglandins stimulated amylase secretion from slices of mouse pancreas (68). The responses to receptor a c t i v a t i o n as well as to exogenous arachidonate were antagonized by low concentrations of the cyclo-oxygenase i n h i b i t o r , indomethacin. In addition, recent reports have documented stimulation of prostaglandin E synthesis by acetylcholine in mouse pancreatic fragments (69,78). These observations c o n s t i t u t e a strong case for prostaglandin involvement in secretory responses in the mouse pancreas. This does not appear to be a general phenomenon f o r the exocrine glands, however, since arachidonic acid and i n h i b i t o r s of prostaglandin systhesis do not appear to a f f e c t responses of the rat pancreas [(70 and unpublished observation] or the rat parotid or lacrimal glands (unpublished observation). Taken c o l l e c t i v e l y , these findings argue against an o b l i g a t o r y role of arachidonate metabolites in c e l l s that show a PI e f f e c t . I t is conceivable that prostaglandins may, to extents that vary considerably with tissue type or species, exert a modulatory action on receptor mediated responses. As i t is generally believed that arachidonate l i b e r a t i o n is a Ca-mediated event (71,72), such modulation would seem to occur as a l a t e event in the stimulusresponse coupling scheme rather than as a primary mechanism for Ca-gating. Role of Phosphatidic Acid As discussed above, the PI e f f e c t in most of the systems l i s t e d in Table 1 has been shown to involve a net increase in tissue levels of PA. One system in which the formation of PA is p a r t i c u l a r l y pronounced is the avian salt gland (56). The secretory c e l l s of t h i s organ contain l i t t l e endoplasmic reticulum and t h i s is consistent with the idea that the major s i t e of PA synthesis under these conditions is the plasma membrane. I t may be that in tissues in which increases in PA are more modest [in the parotid gland, for example ( 1 7 ) ] , the r e l a t i v e increase s p e c i f i c a l l y in the plasma membrane may be considerably larger. What then might be the r e l a t i o n s h i p between t h i s marked a l t e r a t i o n in phospholipid composition of the plasma membrane and Ca-gating mechanisms?
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One suggested hypothesis is that the altered phospholipid p r o f i l e of the membrane results in an a l t e r a t i o n in membrane physical properties ( f l u i d i t y ? ) which permit the opening of s p e c i f i c structures, the hypothetical Ca "gates" (57,73,74). This is an a t t r a c t i v e hypothesis but unfortunately i t is a d i f f i c u l t one to t e s t experimentally. I t r e l i e s considerably on the existence of a hypothetical Ca "gate" molecule, perhaps an integral protein, for which there is as yet no d i r e c t experimental evidence (at least for systems with a well documented PI e f f e c t ) . Another hypothesis which deals with the altered phospholipid content of the plasmalemma suggests that newly synthesized phosphatidate may d i r e c t l y mediate Ca-gating by acting as a Ca-ionophore. In e a r l i e r reports, the Hokins considered the potential role of phosphatidate in Na transport (12,13,15,56). Noting that phosphatidate had been shown to function as a Ca ionophore in a Pressman chamber (75), Michell and his colleagues (73) were the f i r s t to consider the p o s s i b i l i t y of PA acting as a Ca-ionophore within the context of the PI e f f e c t . The idea was l a r g e l y dismissed because of the purported lack of ionic s p e c i f i c i t y of PA and, because of evidence that phosphatidate did not appear to gate Ca in red c e l l s (73). Salmon and Honeyman, however, found that not only did the tissue level of PA r i s e in response to acetylcholine in smooth muscle, but also that the e f f e c t of acetylcholine on smooth muscle c e l l s (contraction) could be mimicked by exogenously applied PA (16). Accordi n g l y , these investigators proposed that the mechanism by which PI metabolism activates Ca-influx is through formation of PA which functions as a Ca-ionophore Recent studies on Ca-gating in the rat parotid gland strongly support this contention. In parotid s l i c e s , exogenous phosphatidate mimicked the e f f e c t s of receptor stimulation (17), although the magnitude of this e f f e c t is somewhat variable. More important was the r e s u l t of i n v e s t i g a t i n g the r e l a t i o n s h i p between Ca-antagonists and PA. The binding of Ca to PA was assayed by measuring the PA-dependent p a r t i t i o n i n g of 45Ca from an aqueous to an organic solvent phase. Graded amounts of several Ca antagonists added to this system produced a concentration-dependent i n h i b i t i o n of this p a r t i tioning a c t i v i t y . These data provided r e l a t i v e estimates of potencies of the antagonists in blocking binding of Ca to the negatively charged head group of PA. When these values were compared to the dissociation constants of these antagonists for c h o l i n e r g i c a l l y activated Ca channels (estimated by pharmacoZogical techniques), a s t r i k i n g q u a n t i t a t i v e c o r r e l a t i o n was obtained (r ~ ~ 0.993). T r i v a l e n t cations (La +3, Tm+3) were most potent, neomycin and d i v a l e n t cations (Co2+, Ni 2+) less potent and Mg2+, considerably less potent. These data provide (apparently) the f i r s t q u a n t i t a t i v e c o r r e l a t i o n between an ion-phospholipid i n t e r a c t i o n measured in a nonbiological system with a hormone-regulated event in l i v i n g c e l l s , S t a t i s t i c a l l y significant increases in PA formation are seen within 10 sec in smooth muscle (16) andparotid cells (unpublished observation). In platelets, the formation of PA following receptor activation is extremely rapid. Lapetina and Cuatrecasas (22) were able to observe elevations in PA labelling as early as 2-5 sec after a thrombin stimulus, and extrapolation of their data suggests a latency of less than l sec. I t should be pointed out that unlike the more commonly employed Ca ionophores, the effects of exogenous PA are not always predictable. Thus, PA activates Ca-dependent K+ release from parotid slices, but not from lacrimal gland slices (unpublished observation). Also, whereas PA w i l l release Ca from membrane vesicles made from platelets, added PA w i l l not cause whole platelets to aggregate (76). This is not p a r t i c u l a r l y surprising, since PA is an amphiphile and only very small amounts are l i k e l y to be present in
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aqueous solution, e x p e c i a l l y in the presence of Ca. Thus the a b i l i t y to del i v e r s i g n i f i c a n t q u a n t i t i e s of PA from micellar suspensions may depend on a wide v a r i e t y of unknown factors r e l a t i n g to the a b i l i t y of PA micelles to be incorporated in the plasma membrane and to accumulate p r i o r to any f u r t h e r metabolic d i s p o s i t i o n . One possible c r i t i c i s m of the PA-ionophore hypothesis relates to the significance of data obtained with organic solvents ( i . e . , Pressman chamber) to phenomena as they may occur in biological bilayers. Recently, however, Serhan and co-workers (77) have demonstrated that PA at reasonably low concentrations (1.5 mol %) acts as a Ca-ionophore in m u l t i l a m e l l a r liposomes. The procedure involves the use of the Ca-sensitive dye, arsenazo I I I , entrapped in the liposomes. Thus the method measures Ca translocated into the aqueous compartment of the liposome, rather than simply bound or adsorbed to the surface. In addition, there was no stimulation of Mg uptake, and there was no (or very l i t t l e ) leakage of dye from the liposomes. These observations demonstrated that Ca did not gain entry through rupture of the vesicles or other such non-specific processes (77). There are, therefore, three points of evidence supportive of the PAionophore hypothesis: (a) On receptor a c t i v a t i o n , PA synthesis is stimulated and PA levels increase both probably occurring in the plasma membrane. (b) In a simple b i l a y e r , PA is an e f f i c i e n t , selective Ca ionophore. (c) Agents that competively block Ca entry through receptor activated "gates" also block Ca binding to phosphatidate and in the same order of potency. These points, i t would seem, c o n s t i t u t e a strong argument for p a r t i c i p a t i o n of PA, acting as a Ca ionophore, in receptor regulation of membrane Ca-gating mechanims. Acknowledgements Some of the research described in this review was supported by a grant from the NIH # DE-04067. References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
M. R. HOKIN and L.E. HOKIN, J. Biol.
Chem. 203:967-977 (1953). L. E. HOKIN and M.R. HOKIN, Biochem. Biophys.--Acta 18:102-110 (1955). L. E. HOKIN and M.R. HOKIN, J. Physiol. (Lond.) 132--442-453 (1956). M. R. HOKIN and L.E. HOKIN, J. Biol. Chem. 209:549-558 (1954). L. E. HOKIN, Biochim. Biophys. Acta 115:219-221 (1966). C. M. REDMANand L.E. HOKIN, J. Biophys. Biochem. Cytol. 6:207-214 (1959). 33:421-530 (1967). L. E. HOKIN and D. HUEBNER, J. Cell Biol. L. E. HOKIN and A.L. SHERWIN, J. Physiol. (Lond.) 135:18-29 (1957). M. SCHRAMM and Z. SELINGER, J. Cyclic Nucleotide Res. 1:181-192 (1975). Y. ORON, M. LOWE and Z. SELINGER, F.E.B.S. Letters 3 4 : i 9 8 - 2 0 0 (1973). R. H. MICHELL and L.M. JONES, Biochem. J. 138:47-52 (1974). L. E. HOKIN and M.R. HOKIN, Nature 184:1068-1069 (1959). L. E. HOKIN and M.R. HOKIN, J. Gen. Physiol. 4 4 : 6 1 - 8 5 (1960). M. R. HOKIN and L.E. HOKIN, J. Biol. Chem. 239--2116-2122 (1964). M.R. HOKIN and L.E. HOKIN, in Metabolism and Physiological Significance of Lipids, ( e d i t . by Dawson, R.M.C. and Rhodes, D.N.), pp. 423-435, John Wiley and Sons, London (1964). D.M. SALMON and T.W. HONEYMAN, Nature 284:344-345 (1980). J.W. PUTNEY, J r . , S.J. WEISS, C.M. VAN DE WALLE and R.A. HADDAS, Nature 284:345-347 (1980). M.~. HOKIN, in Secretor~ Mechanisms of Exocrine Glands, ( e d i t . by Thorn, N.A. and Petersen, O.H.), pp. 110-112, Munksgaard, Copenhagen (1974). L.M. JONES and R.H. MICHELL, Biochem. J. 142:583-590 (1974). R.H. MICHELL, Biochem. Biophys. Acta 415:81-147 (1975).
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I. LITOSCH and J.N. FAIN, Fed. Proc. in the press (1981). E.G. LAPETINA and P. CUATRECASAS, Biochem. Biophys. Acta 573:394-402 (1979). M.M. BILLAH, E.G. LAPETINA and P. CUATRECASAS, Biochem. Biophys. Res. Comm. 9 0 : 9 2 - 9 8 (1979). M.M. BILLAH, E.G. LAPETINA and P. CUATRECASAS, J. Biol. Chem. 255: 10227-10231 (1980). R.M. BELL and R.A. COLEMAN, Ann. Rev. Biochem. 49:459-487 (1980). Z. SELINGER, S. EIMERL, N. SAVlON and M. SCHRAMM, in Secretory Mechanisms of Exocrine Glands, ( e d i t . by Thorn, N.A. and Petersen, O.H.), pp. 68-78, Munksgaard, Copenhagen (1974). B. ROSSIGNOL, G. HERMAN, A.M. CHAMBAUTand G. KERYER, F.E.B.S. Letters 43:241-246 (1974). L.M. JONES and R.H. MICHELL, Biochem. J. 148:479-485 (1975). Y. ORON, M. LOWE and Z. SELINGER, Mol. Pharmacol. 11:79-86 (1975). P. CALDERON, J. FURNELLE and J. CHRISTOPHE, Am. J. Physiol. 238: G247-G254 (1980). R.H. MICHELL, T.I.B.S. 4:128-131 (1979). S.K. FISHER and B.W. AGRANOFF, J. Neurochem. 34:1231-1240 (1980). S. COCKROFT, J.P. BENNET and B.D. GOMPERTS, F.E.B.S. Letters i i 0 : 115118 (1980). S. COCKROFT, J.P. BENNET and B.D. GOMPERTS, Nature 288:275-277 (1980). R.A. AKHTAR and A.A. ABDEL-LATIF, J. Pharmacol. Exp. Ther. 204: 655668 (1978). H.D. GRIFFIN and J.N. HAWTHORNE, Biochem. J. 176:541-552 (1978). R.H. MICHELL and C.J. KIRK, T.I.P.S. in the press (1981). S.J. WEISS and J.W. PUTNEY, JR., Biochem. J. in the press (1981). M.M. BILLAH and R.H. MICHELL, Biochem. J. 182:661-668 (1979). C.J. KIRK, R.H. MICHELL and D.A. HEMS, Biochem. J. in the press (1981). J.V. LLOYD, E.E. NISHIZAWA and J.F. MUSTARD, B r i t . J. Haematol. 25: 77-79 (1973). J.N. FAIN and M.J. BERRIDGE, Biochem. J. 178:45-58 (1979). S.S. JAFFERJI and R.H. MICHELL, Biochem. J. 160:163-169 (1976). S.S. JAFFERJI and R.H. MICHELL, Biochem. J. 154:653-657 (1976). S.S. JAFFERJI and R.H. MICHELL, Biochem. J. 160:397-399 (1976). M.J. BERRIDGE, in Transport of Ions and Water in Animals, ( e d i t . by Gupta, B.L., Moreton, R.B., Oschman, J.L. and Wall, B . J . ) , pp. 225-238, Academic Press, New York (1977). M.J. BERRIDGE, T.I.P.S. 1:419-424 (1980). M.J. BERRIDGE and J.N. FAIN, Biochem. J. 178:59-69 (1979). J.N. FAIN and M.J. BERRIDGE, Biochem. J. 180:655-661 (1979). D. GERBER, M. DAVIES and L.E. HOKIN, J. Cell. Biol. 56:736-745 (1973). K.W.A. WIRTZ and D.B. ZILVERSMIT, Biochim. Biophys. Acta. 193:105-116 (1969). R.A. DEMEL, R. KALSBEEK, K.W.A. WIRTZ and L.L.M. VAN DEENEN, Biochim. Biophys. Acta 466:10-12 (1977). P.E. DI CORLETO, J.B. WARACHand D.B. ZILVERSMIT, J. Biol. Chem. 254: 7795-7802 (1979). P.J. BROPHY, P. BURBACH, S.A. NELEMANS, J. WESTERMAN, K.W.A. WIRTZ and L.L.M. VAN DEENEN, Biochem. J. 174:413-420 (1978). M.J. BERRIDGE and J.L. OSCHMAN, Transportin 9 E p i t h e l i a , Academic Press, New York (1972). M.R. HOKIN and L.E. HOKIN, J. Gen. Physiol. 50:793-811 (1967). G. KERYER, G. HERMANand B. ROSSIGNOL, F.E.B.S. Letters 1 0 2 : 4 - 8 (1979). A.A. ABDEL-LATIF and B. LUKE, Biochim. Biophys. Acta in the press (1981). C.A. LANDIS and J.W. PUTNEY, JR., J. Physiol. (Lond.) 297:369-377 (1979). J.A. WILLIAMS, Cell Tiss. Res. 210:295-303 (1980). A.A. ABDEL-LATIF, Biochem. Pharmacol. in the press (1981). L.M. JONES and R.H. MICHELL, Biochem. J. 158:505-507 (1976).
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S. MONCADAand J.R. VANE, Pharm. Rev. 30:293-331 (1978). W.F. STENSON, C.W. PARKER and T.J. SULLI--VAN, Biochem. Biophys. Res. Comm. 96:1045-1052 (1980). W.F. STETSON and C.W. PARKER, J. Clin. Invest. 64:1457-1465 (1979). P.H. NACCACHE, H.J. SHOWELL, E.L. BECKER and R.I. SHA'AFI, Biochem. Biophys. Res. Comm. 87:292-299 (1979). M. HAMBERG, J. SVENSSONand B. SAMUELSSON, Proc. Nat. Acad. Sci. U.S.A. 72:2994-2998 (1975). P.J. MARSHALL, J.F. DIXON and L.E. HOKIN, Proc. Nat. Acad. Sci. U.S.A. 77: 3292-3296 (1980). M.W. BANSCHBACHand M. HOKIN-NEAVERSON, F.E.B.S. Letters 117:131-133 (1980). L. CHAUVELOT, S. HEISLER, J. HUOT and D. GAGNON, Life Sciences 25: 913920 (1979). R.P. RUBIN and S.G. LAYCHOCK, in Calcium in Dru 9 Action, ( e d i t . by Weiss, G.B.) pp. 135-155, Plenum, New York (1978). R.P. RUBIN and S.G. LAYCHOCK, Ann. N.Y. Acad. Sci. 307:377-389 (1978). R.H. MICHELL, S.S. JAFFERJI and L.M. JONES, Adv. Exp. Biol. Med. 83: 447-464 (1977). G. KERYERand B. ROSSIGNOL, Eur. J. Biochem. 8 5 : 7 7 - 8 3 (1978). C.A. TYSON, H.V. ZANDE and D.E. GREEN, J. Biol:--Chem. 251:1326-1332 (1976). S.M. GERRARD, A.M. BUTLER, D.A. PETERSONand J.G. WHITE, Prostaglandins and Medicine 1:387-396 (1978). C. SERHAN, P. ANDERSON, E. GOODMAN, P. DUNHAMand G. WEISSMANN, J. Biol. Chem. in the press (1981). P.J. MARSHALL, D.E. BOATMANand L.E. HOKIN, J. Biol. Chem. 256:844-847 (1981).
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MINIREVIEW RECENT HYPOTHESESREGARDING THE PHOSPHATIDYLINOSITOL EFFECT James W. Putney, Jr. Department of Pharmacology Medical College of V i r g i n i a V i r g i n i a Commonwealth University Richmond, VA. 23298 U.S.A. Summary In 1975, Michell f i r s t proposed that a c t i v a t i o n of phosp h a t i d y l i n o s i t o l turnover provided a d i r e c t l i n k between surface receptors and membrane Ca gates. Subsequently, a number of laboratories have begun to r e - i n v e s t i g a t e this phenomenon f i r s t described by Hokin and Hokin some twenty years e a r l i e r . As would be expected, some new hypothesis have emerged, most being extensions or revisions of M i c h e l l ' s o r i g i n a l concept. Despite d i f f i c u l t i e s in obtaining d i r e c t proof, i n d i r e c t evidence suggests that the plasma membrane is the primary locus of receptor-activated phosphatidylinositol turnover, at least for phosphatidylinositol breakdown and phosphatidic acid synthesis. The presence or absence of Na+ can markedly a f f e c t labelling of phosphatidylinositol by radioactive precursors but there is no compelling evidence that the i n i t i a l events are mediated by Na+. Prostaglandins are apparently formed in some tissues on receptor a c t i v a t i o n , but in most instances the evidence suggests that these compounds are not obligatory intermediates in tissues that show the phosphatidylinositol e f f e c t . Several laboratories have obtained evidence that phosphatidic acid newly synthesized following phosphatidylinositol breakdown, may function as an endogenous Ca ionophore under neurohumoral control. In 1953, Hokin and Hokin ( I ) reported that acetylcholine stimulates the incorporation of 32P04 into phospholipids of the pigeon pancreas in v i t r o . In a subsequent paper (2) these investigators demonstrated that the s p e c i f i c phospholipids responsible for the e f f e c t were phosphatidylinositol (PI) and phosphatidic acid (PA). This phenomenon by which stimuli enhance the radioactive l a b e l l i n g of phosphatidylinositol has been generally termed the "phospholipid e f f e c t " or more s p e c i f i c a l l y t h e " P l e f f e c t . " As discussed below, however, all instances of increased phosphatidylinositol l a b e l l i n g may not r e f l e c t the same mechanism, and additional constraints on d e f i n i t i o n s of the PI e f f e c t may be necessary.
0024-3205/81/121183-12502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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An i n i t i a l hypothesis as to the s i g n i f i c a n c e of the PI e f f e c t was that i t r e f l e c t e d a l t e r a t i o n s in the metabolism of membrane phospholipids r e s u l t i n g from the secretions of zymogens (3). A number of subsequent observations discouraged t h i s idea, however. For one, the concentration-effect r e l a t i o n s h i p f o r s t i m u l a t i o n of the PI e f f e c t lay somewhat to the r i g h t (higher concentrations) of that f o r s t i m u l a t i o n of protein secretion (4). Also, protein secretion could be prevented by removal of Ca, but the PI e f f e c t was l a r g e l y unaffected (5). F i n a l l y , the s u b c e l l u l a r locus of the m a j o r i t y of newly incorporated label was found to be the rough endoplasmic reticulum rather than the membranes of the plasmalemma and zymogen granules (6,7). When these studies were f i r s t carried to the s a l i v a r y glands (8), the secretory response and PI e f f e c t due to adrenergic s t i m u l a t i o n were c l e a r l y dissociable by the use of the m-adrenergic antagonist, ergotamine, which i n h i b i t e d the PI e f f e c t without a f f e c t i n g protein secretion; the l a t t e r is now known to occur in s a l i v a r y glands p r i m a r i l y through B-adrenoceptor a c t i v a t i o n (9). As is often the case, this apparent inconsistency ( i . e . d i s s o c i a t i o n of the PI e f f e c t from protein secretion) was r a t i o n a l i z e d (8) into obscurity and rediscovered sixteen years later (I0,II). Another hypothesis that received considerable a t t e n t i o n in the s i x t i e s was the idea that the reaction was in some way involved in the mechanism of the Na, K-pump (12,13). This concept was derived from the finding that in avian s a l t glands, a c t i v e Na transport and the PI e f f e c t were concomitantly activated by a c e t y l c h o l i n e . Again, however, f u r t h e r experimentation led to the clear d i s s o c i a t i o n of these two events (14,15). I t is now clear that no close association e x i s t s between the PI e f f e c t and a c t i v e Na transport. The concept, however, of a r e l a t i o n s h i p between membrane phospholipid composition and cation movements is quite s i m i l a r to some current ideas discussed below. (Compare, for example, Ref. 12 to Ref. 16 and Ref. 17). Enzymatic Pathways of the PI Effect There is considerably less data a v a i l a b l e on net changes of phospholipid l e v e l s as compared to data on l a b e l l i n g and turnover. In many cases, however, i t has been shown that associated with the increased l a b e l l i n g of the PI and PA, the tissue content of PI f a l l s and the content of PA increases (17-20). The simplest scheme consistent with these data is one proposed by Hokin and Hokin (15) for the avian s a l t gland, and generalized to other tissues by Michell (20). The pathways are i l l u s t r a t e d in Fig. I . For most tissues the evidence that agonists can a c t i v a t e t h i s cycle at the level of a phospholipase C is l a r g e l y c i r c u m s t a n t i a l . Unfortunately, a t tempts to demonstrate effects of agonists on phospholipase C a c t i v i t y in broken c e l l preparations have been l a r g e l y unsuccessful. A recent preliminary report suggests that 5-hydroxytryptamine accelerates phosphatidylinositol breakdown in homogenates of the blowfly s a l i v a r y gland (21). For the p l a t e l e t , there is strong evidence that thrombin acts by stimulation of a phospholipase C. Cyclic AMP is known to i n h i b i t the p l a t e l e t aggregation response to agents l i k e thrombin and the l a b e l l i n g of phosphatidic acid (22). B i l l a h and co-workers (23) have shown that p l a t e l e t s contain a phosphat i d y l i n o s i t o l s p e c i f i c phospholipase C. The enzyme a c t i v i t y when assayed in broken c e l l s , is also markedly depressed i f p l a t e l e t s are f i r s t treated with d i b u t y r y l c y c l i c AMP or phosphodiesterase i n h i b i t o r s . The i n h i b i t i o n by cycl i c AMP of both the phospholipase C and the thrombin stimulated PI e f f e c t provides the f i r s t demonstrated l i n k between a phospholipase C assayed in broken c e l l s and the effects of an agonist in the i n t a c t c e l l . Also of considerable s i g n i f i c a n c e is the demonstration by these same i n v e s t i g a t o r s that the enzyme is found in the soluble f r a c t i o n (24). This l a t t e r observation,
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i f i t turns out to be the general case, means that receptors may not d i r e c t ly activate the enzyme but rather could a l t e r the state of the substrate (phosphatidylinositol) increasing i t ' s s u s c e p t i b i l i t y to hydrolysis.
AGONIST + PI
RECEPTOR
INOSITOL/CDP-DIP D IACYLG LYCEROL
CDP-DIACYLGLYCEROL
DGKZ A T
P P i ~PACT CTP
P
PA FIG. 1
Enzymatic pathways of the PI effect. According to this scheme, receptor activation leads to the breakdown of PI to diacylglycerol by a phospholipase C (PLC). Diacylglycerol is phosphorylated by ATP and diacylglycerol kinase (DGK) to form PA. PA is subsequently conjugated with CTP by the action of phosphatidic acid: CTP cytidyltransferase (PACT) to form CDP-diacylglycerol. PI is f i n a l l y formed by exchange of the nucleotide with free inositol by the enzyme CDP-diacylglycerol inositol phosphatidyltransferase (CDP-DIP). One d i f f i c u l t y with the scheme in Fig. 1 is that while the enzyme that synthesizes PA (diglyceride kinase) apparently resides in the plasmalemma (20), i t appears that the enzymes involved in synthesizing PI from PA are localized largely to the endoplasmic reticulum (20,25). The relevance of this situation to patterns of phospholipid labelling is considered l a t e r in the section on "subcellul a r locus of the P l - e f f e c t . " Michel!'s Hypothesis From the late seventies to the present there has been a substantial increase in interest in the PI effect. This can be credited, in the reviewer's opinion, to an hypothesis f i r s t put forth in 1975 by Michell (20). Michell was apparently struck by the fact that the PI effect could be e l i c i t e d in salivary glands by muscarinic or~-adrenergic stimuli or in the pancreas by muscarinic agonists or certain peptides. The end response in each case, however, differed considerably. In the salivary glands, the receptors cont r o l l e d the K permeability response, while in the pancreas the receptors which caused PI turnover stimulated enzyme discharge. The common denominator was not the end response of the tissue but rather the second messenger, calcium. As the PI e f f e c t , in contrast to other c e l l u l a r responses, appeared independent of external Ca, Michell reasoned that PI turnover might pre-
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cede Ca mobilization and might be involved in the mechanism by which receptors mediate Ca mobilization (20). Supportive of this idea was the finding that the a r t i f i c i a l introduction of Ca into pancreas and salivary gland c e l l s stimulates the appropriate end responses (26), but has l i t t l e e f f e c t on PI turnover (27-30). Similar experimental results have been obtained for a number of other tissues and receptor types that act through Ca (31), and thus the body of circumstantial evidence for the Ca-gating hypothesis continues to grow. There are also examples of stimulation of PI l a b e l l i n g that are not cons i s t e n t with the Michell hypothesis. One such example is the synaptosome (32) where stimulation of PI l a b e l l i n g by acetylcholine is Ca dependent and can be mimicked by the calcium ionophore A23187. In the neutrophil, both PI l a b e l l i n g and breakdown have been shown to be Ca mediated (33,34). In the case of the synaptosome, however, the Ca-dependence for only PI l a b e l l i n g has been investigated. Thus, i t is not certain that the i n i t i a l event is necessarily PI breakdown, at least in regard to the d i r e c t cause of the !abelling. For example, Ca-mediated breakdown of polyphosphoinositides to diglyceride is well documented in smooth muscle (35) and in syn~Rtosomes (36). The d i g l y c e r i d e is r a p i d l y phosphorylated in the presence of °LP-ATP to form radioactive PA, the precursor for PI synthesis (25). Other major phospholipids are p r i m a r i l y synthesized from diglyceride d i r e c t l y and would not show an increased l a b e l l i n g in p a r a l l e l to PA. There is no proof as to whether such a scheme can explain any s i t u a t i o n in which PI l a b e l l i n g appears to be Ca mediated. I t does serve to point out an important caveat, however: Stimul a t i o n of phospholipase C a c t i v i t y against any phospholipid (not necessarily PI) w i l l generate d i g l y c e r i d e and w i l l t h e o r e t i c a l l y r e s u l t in an increased l a b e l l i n g of PA and PI. Other t h e o r e t i c a l treatments of these anomalous results have been considered by Michell and Kirk (37). Regardless of the explanation, i t is important to point out that there is no basis f o r extrapolation of these results to other tissues where s t r i c t adherence to the appropriate c r i t e r i a of the Ca-gating hypothesis has been established. Table i l i s t s a few well studied tissues in which the PI e f f e c t has been c l e a r l y shown to be: (a) Ca-independent, (b) poorly or not at all stimulated by calcium ionophore, and (c) associated with net breakdown of PI. For such cases, M i c h e l l ' s hypothesis remains tenable, and i t is within the confines of these well-defined systems that some new postulates on the significance of the PI e f f e c t w i l l be considered. I n o s i t o l Dependency of the Fly Salivary Gland In the blowfly salivary gland, stimulation of membrane receptors by 5hydroxytryptamine (5-HT) activates both adenylate cyclase and Ca-gating (46). Probably, by analogy with the case for ~- and ~-adrenoceptors, these effects are mediated by separate receptors (47). The two second messengers, Ca and c y c l i c AMP, act in concert to increase e p i t h e l i a l CI~ permeability and K+ transport which results in salivary f l u i d secretion (46). Concomitant with the a c t i v a t i o n of f l u i d secretion and Ca-gating, 5-HT also stimulates breakdown of PI, apparently at the basal surface of the gland (42). The f l y gland is an especially useful preparation for these studies, since a c t i v a t i o n ~ Ca gating and PI breakdown can be monitored simultaneously as rel~ase of Ca and H - i n o s i t o l . Fain and Berridge (42) found that release of H-inositol was neither i n h i b i t e d by EGTA nor stimulated by A23187, and was independent of the synthesis of c y c l i c AMP (42). In addition, these authors (48,49) also found that prolonged stimulation of the f l y gland led
Kirk et al (40)
Lloyd et al (41)
Platelet
Fly Salivary Gland
Smooth Muscle
Salmon and Honeyman* (16)
*Salmon and Honeyman (16) found that elevation of [Ca2+] i with high [K+] contracted isolated smooth muscle cells but did not stimulate PA labelling. This experiment is equlvalent to the use of an ionophore for the sake of the arguments advanced here. I t should be mentioned, however, that J a f f e r j i and Michell (45), using smooth muscle fragments, obtained a stimulation of PI labelling by high [K+].
(43)
Jafferji
and Michell
Jafferji
(44)
Fain and Berridge (42)
Fain and Berridge (42)
Fain and Berridge (42)
and Michell
Lapetina and Cuatrecasas (22)
(Relatively) Lapetina and Cuatrecasas (22)
(39)
Billah and Michell
(Partially) Billah and Michell Kirk et al (40)
Liver (39)
Jones and Michell (19) Weiss and Putney (38)
Rossignol et al (27) Oron et a l - ~ 2 ~ Jones and Michell (28)
Oron et al (29) Jones and Michell (28)
Hokin (18)
Calderon et al (30)
Parotid Gland
PI-Breakdown Occurs
lonophore Insensitive
Hokin (5) Calderon et al (30)
Ca Independent
C r i t e r i a Met (Reference)
Exocrine Pancreas
Tissue
Tissues meeting the c r i t e r i a for an involvement of PI turnover in Ca-gating
TABLE 1
O0 "-.I
r-t
~-h
F~
0
OO
Z 0
< 0
1188
On the PI Effect
Vol. 29, No. 12, 1981
gradually to an i n a b i l i t y to secrete or to gate Ca. For recovery to occur, i n o s i t o l was required in the medium. These results suggest that when the relevant pool of PI is exhausted, the Ca channels are " f i x e d " in a "closed" rather than "open" state. This would indicate that simply removing the head group from PI is not s u f f i c i e n t alone to open Ca gates. Possibly, e i t h e r the breakdown reaction i t s e l f or some product of that reaction may be necessary. A l t e r n a t i v e l y , PI breakdown could mediate the closing of the gate (47,48). Perhaps more s i g n i f i c a n t l y , these observations provided the f i r s t evidence for a cause-effect r e l a t i o n s h i p between PI hydrolysis and Ca-gating. Subcellular Locus of the PI Effect When tissues are subjected to s u b c e l l u l a r f r a c t i o n a t i o n following stimul a t i o n of PI turnover, the majority of radioactive PI (or PI breakdown) is found in the microsomal f r a c t i o n , the largest portion of which is derived from endoplasmic reticulum (40,50). As Michell and his colleagues (20,40) point out, there are two possible explanations for this r e s u l t . F i r s t , receptor stimulation at the plasma membrane could lead to the generation of a heretofore undisclosed second messenger which activates phospholipid metabolism in the endoplasmic reticulum. On the other hand, the stimulus could act i v a t e PI breakdown at the plasma membrane, the resulting d i g l y c e r i d e could be phosphorylated to radioactive PA which would then be transported to the endoplasmic reticulum via phospholipid exchange proteins. Such proteins (which r e a d i l y catalyze PI exchange) have recently been characterized (51-54), but none s p e c i f i c for PA has yet been described. At the endoplasmic reticulum, synthesis of radioactive PI would occur. Because of the large pool of PI in the endoplasmic reticulum ( i . e . due to isotope d i l u t i o n ) , non-radioactive PI would be transported back to the plasma membrane, and the cycle continued. This is consistent with the generally held view (20) that PI synthesis is res t r i c t e d to the endoplasmic reticulum while d i g l y c e r i d e kinase (and incident a l l y , phosphatidate phosphohydrolase) are present in the plasma membrane. This l a t t e r scheme predicts that the primary hormone-sensitive pool of PI may be quite small. In the f l y salivary gland, resynthesis of PI is s u f f i c i e n t ly slow that a l l of the hormone-sensitive pool of PI can be hydrolyzed leading to complete i n a c t i v a t i o n of Ca-gating (48). Under these conditions net loss of PI is not s a t i s t i c a l l y detectable (49) and Fain and Berridge estimate that resynthesis of less than 10% of the PI in the gland w i l l completely reactivate hormone s e n s i t i v i t y . Again, these results could indicate a small pool of PI, perhaps in the plasma membrane, that is the primary s i t e of hormone action. Another point in favor of the proposed r e l a t i o n s h i p between endoplasmic reticulum and plasma membrane is the nature of the PI e f f e c t in the avian s a l t gland. Unfortunately, that Ca acts as a coupling factor in these c e l l s has not yet been c l e a r l y established. As the receptors are muscarinic (13), and since receptor a c t i v a t i o n does stimulate PA and PI l a b e l l i n g , the assumption that the phospholipid e f f e c t r e f l e c t s the same reactions as in the more extensively characterized mammalian system seems reasonable. Unlike the mammalian exocrine glands, the avian s a l t glands do not secrete protein; there does however appear to be an a c e t y l c h o l i n e - a c t i v a t e d (muscarinic) Na permeab i l i t y , probably at the basal membrane (55). A consequence of this is that avian s a l t glands, in contrast to mammalian exocrine glands, have very l i t t l e endoplasmic reticulum. Due to this paucity of endoplasmic reticulum and the extensive i n t e r d i g i t a t i o n s of the basal (plasma) membrane, the quantity of plasmalemma would be considerably 9reater than internal membranes (with the possible exception of mitochondria).
Vol. 29, No.
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On the PI Effect
1189
The pattern of the PI e f f e c t in this tissue is consistent with the hypothesis that the plasma membrane is the primary s i t e of action of acetylcholine. F i r s t , the stimulation of 32P04 incorporation is not diminished in comparison to other tissues, despite the small quantity of endoplasmic r e t i c u lum. On the other hand, there is a considerably smaller increase in radioact i v i t y in PI (believed to be synthesized at the endoplasmic reticulum) with a larger and extremely rapid e f f e c t f o r PA (believed to be synthesized at the plasmalemma) (15,56). When the stimulus is removed (with a t r o p i n e ) , rapid resynthesis of PI occurs which is approximately stoichiometric for the loss in radioactive PA (15). Taken with the results obtained in mammalian systems, these findings are consistent with the suggestion that at the plasma membrane, activated receptors stimulate PI breakdown followed by PA synthesis. As resynthesis of PI presumably takes place in internal membranes (endoplasmic reticulum), this would occur therefore with r a p i d i t y varying from tissue to tissue. The extent of l a b e l l i n g of PI would also vary with the size of the endoplasmic reticulum pool. Role of Na+ in the PI Effect Recently, i t has been reported that media d e f i c i e n t in Na+ i n h i b i t the receptor-stimulated l a b e l l i n g of PI by 3H-inositol (57) or 32p04 (58). These observations have led to the suggestion that Na+ i n f l u x might precede the PI e f f e c t (57,58). Such a scheme is not consistent with previous data on control of Na+ movements in the parotid gland, however. Thus, in the absence of Ca, cholinergic and ~-adrenergic agonists stimulate PI l a b e l l i n g (28,29) but not Na÷ i n f l u x (59). Also, the d i v a l e n t cationophore A23187, has l i t t l e e f f e c t on PI turnover, (27,29) but does stimulate Ca-dependent Na uptake (59). In addition, Williams (60) has shown that Na+ removal does not a f f e c t Ca mobilization due to caerulein in pancreatic acini and concludes that Na+ i n f l u x has no d i r e c t role in coupling receptor occupancy to protein secretion. One explanation f o r the e f f e c t s of Nat may be that i t is the s p e c i f i c a c t i v i t y of precursors rather than the reaction rates themselves which is affected. In support of t h i s , A b d e l - L a t i f (61) found that in the absence of external Na+, the s p e c i f i c r a d i o a c t i v i t y of ATP was les§ than 5% of control. Also, Keryer, Herman and Ross~gnol (57) found that H-inositol uptake was s u b s t a n t i a l l y reduced by Na omission. These results suggest that i t would be more appropriate to examine the breakdown of PI d i r e c t l y rather than to deal with pools of precursors with unknown and changing s p e c i f i c radioactivity. In the only study in which net changes in PI content were measured, Jones and Michell (62) found that net breakdown of PI in parotid fragments due to carbachol was not prevented by decreasing external Na+ to less ~han 1 mM. Thus, while there appear to be r e a d i l y demonstrated e f f e c t s of Na- omission on the PI l a b e l l i n g cycle, the~e is no compelling evidence to support the idea that receptor-activated Na fluxes precede oY-mediate the primary e f f e c t s of agonists on PI breakdown. Prostagl andi ns In mammals, the number 2 position of PI is highly enriched in arachidonic acid (20). I t would not be unreasonable, therefore, to a n t i c i p a t e that a l t e r a t i o n s in PI metabolism might lead u l t i m a t e l y to the l i b e r a t i o n of this r a t e - l i m i t i n g precursor for synthesis of prostaglandins and other products of cyclo-oxygenase or lipoxygenase (63). These oxidative metabolites could subsequently function in Ca-gating or in other c e l l u l a r responses.
1190
On the PI Effect
Vol. 29, No.
12, 1981
There is good evidence that some tissues that show altered PI l a b e l l i n g can also make and respond to prostaglandins [mast c e l l s (64), neutrophils (65,66), p l a t e l e t s (67)]. In order to invoke prostaglandins as o b l i g a t o r y intermediates in the responses to receptor a c t i v a t i o n one should be able to (a) mimic the action of a receptor agonist by application of a prostaglandin or the r a t e - l i m i t i n g precursor, arachidonate, (b) demonstrate stimulation of prostaglandin formation, or at least arachidonate mobilization, and (c) demonstrate a p a r a l l e l r e l a t i o n s h i p between pharmacological i n h i b i t i o n of arachidonate metabolism and i n h i b i t i o n of the appropriate c e l l u l a r response. Failure to s a t i s f y c r i t e r i o n (c) has cast serious doubt on any o b l i g a t o ry role for prostaglandins in stimulus-response coupling in a v a r i e t y of cell types. In p l a t e l e t s for example, exogenous arachidonate or thromboxane can mimic receptor a c t i v a t i o n and stimulation of p l a t e l e t receptors leads to thromboxane synthesis (67). However, stimulation of p l a t e l e t secretion by thrombin is t o t a l l y unaffected by 20 ~M eicosatetraynoic acid (ETYA), an agent that (at 20 uM) completely blocks arachidonate metabolism by both cyclooxygenase and lipoxygenase (22). S i m i l a r l y , in neutrophils, ETYA is about ten times less e f f e c t i v e in antagonizing the chemotactic peptide, F-Met-Leu-Phe, than exogenous arachidonate, and is almost t o t a l l y i n e f f e c t i v e against the complement fragment, C5a (66). Recently, Marshall, Dixon and Hokin (68) reported data demonstrating that receptor-mediated secretion of amylase by the mouse exocrine pancreas might involve prostaglandin formation. Thus, arachidonic acid and certain prostaglandins stimulated amylase secretion from slices of mouse pancreas (68). The responses to receptor a c t i v a t i o n as well as to exogenous arachidonate were antagonized by low concentrations of the cyclo-oxygenase i n h i b i t o r , indomethacin. In addition, recent reports have documented stimulation of prostaglandin E synthesis by acetylcholine in mouse pancreatic fragments (69,78). These observations c o n s t i t u t e a strong case for prostaglandin involvement in secretory responses in the mouse pancreas. This does not appear to be a general phenomenon f o r the exocrine glands, however, since arachidonic acid and i n h i b i t o r s of prostaglandin systhesis do not appear to a f f e c t responses of the rat pancreas [(70 and unpublished observation] or the rat parotid or lacrimal glands (unpublished observation). Taken c o l l e c t i v e l y , these findings argue against an o b l i g a t o r y role of arachidonate metabolites in c e l l s that show a PI e f f e c t . I t is conceivable that prostaglandins may, to extents that vary considerably with tissue type or species, exert a modulatory action on receptor mediated responses. As i t is generally believed that arachidonate l i b e r a t i o n is a Ca-mediated event (71,72), such modulation would seem to occur as a l a t e event in the stimulusresponse coupling scheme rather than as a primary mechanism for Ca-gating. Role of Phosphatidic Acid As discussed above, the PI e f f e c t in most of the systems l i s t e d in Table 1 has been shown to involve a net increase in tissue levels of PA. One system in which the formation of PA is p a r t i c u l a r l y pronounced is the avian salt gland (56). The secretory c e l l s of t h i s organ contain l i t t l e endoplasmic reticulum and t h i s is consistent with the idea that the major s i t e of PA synthesis under these conditions is the plasma membrane. I t may be that in tissues in which increases in PA are more modest [in the parotid gland, for example ( 1 7 ) ] , the r e l a t i v e increase s p e c i f i c a l l y in the plasma membrane may be considerably larger. What then might be the r e l a t i o n s h i p between t h i s marked a l t e r a t i o n in phospholipid composition of the plasma membrane and Ca-gating mechanisms?
Vol. 29, No.
12, 1981
On the PI Effect
1191
One suggested hypothesis is that the altered phospholipid p r o f i l e of the membrane results in an a l t e r a t i o n in membrane physical properties ( f l u i d i t y ? ) which permit the opening of s p e c i f i c structures, the hypothetical Ca "gates" (57,73,74). This is an a t t r a c t i v e hypothesis but unfortunately i t is a d i f f i c u l t one to t e s t experimentally. I t r e l i e s considerably on the existence of a hypothetical Ca "gate" molecule, perhaps an integral protein, for which there is as yet no d i r e c t experimental evidence (at least for systems with a well documented PI e f f e c t ) . Another hypothesis which deals with the altered phospholipid content of the plasmalemma suggests that newly synthesized phosphatidate may d i r e c t l y mediate Ca-gating by acting as a Ca-ionophore. In e a r l i e r reports, the Hokins considered the potential role of phosphatidate in Na transport (12,13,15,56). Noting that phosphatidate had been shown to function as a Ca ionophore in a Pressman chamber (75), Michell and his colleagues (73) were the f i r s t to consider the p o s s i b i l i t y of PA acting as a Ca-ionophore within the context of the PI e f f e c t . The idea was l a r g e l y dismissed because of the purported lack of ionic s p e c i f i c i t y of PA and, because of evidence that phosphatidate did not appear to gate Ca in red c e l l s (73). Salmon and Honeyman, however, found that not only did the tissue level of PA r i s e in response to acetylcholine in smooth muscle, but also that the e f f e c t of acetylcholine on smooth muscle c e l l s (contraction) could be mimicked by exogenously applied PA (16). Accordi n g l y , these investigators proposed that the mechanism by which PI metabolism activates Ca-influx is through formation of PA which functions as a Ca-ionophore Recent studies on Ca-gating in the rat parotid gland strongly support this contention. In parotid s l i c e s , exogenous phosphatidate mimicked the e f f e c t s of receptor stimulation (17), although the magnitude of this e f f e c t is somewhat variable. More important was the r e s u l t of i n v e s t i g a t i n g the r e l a t i o n s h i p between Ca-antagonists and PA. The binding of Ca to PA was assayed by measuring the PA-dependent p a r t i t i o n i n g of 45Ca from an aqueous to an organic solvent phase. Graded amounts of several Ca antagonists added to this system produced a concentration-dependent i n h i b i t i o n of this p a r t i tioning a c t i v i t y . These data provided r e l a t i v e estimates of potencies of the antagonists in blocking binding of Ca to the negatively charged head group of PA. When these values were compared to the dissociation constants of these antagonists for c h o l i n e r g i c a l l y activated Ca channels (estimated by pharmacoZogical techniques), a s t r i k i n g q u a n t i t a t i v e c o r r e l a t i o n was obtained (r ~ ~ 0.993). T r i v a l e n t cations (La +3, Tm+3) were most potent, neomycin and d i v a l e n t cations (Co2+, Ni 2+) less potent and Mg2+, considerably less potent. These data provide (apparently) the f i r s t q u a n t i t a t i v e c o r r e l a t i o n between an ion-phospholipid i n t e r a c t i o n measured in a nonbiological system with a hormone-regulated event in l i v i n g c e l l s , S t a t i s t i c a l l y significant increases in PA formation are seen within 10 sec in smooth muscle (16) andparotid cells (unpublished observation). In platelets, the formation of PA following receptor activation is extremely rapid. Lapetina and Cuatrecasas (22) were able to observe elevations in PA labelling as early as 2-5 sec after a thrombin stimulus, and extrapolation of their data suggests a latency of less than l sec. I t should be pointed out that unlike the more commonly employed Ca ionophores, the effects of exogenous PA are not always predictable. Thus, PA activates Ca-dependent K+ release from parotid slices, but not from lacrimal gland slices (unpublished observation). Also, whereas PA w i l l release Ca from membrane vesicles made from platelets, added PA w i l l not cause whole platelets to aggregate (76). This is not p a r t i c u l a r l y surprising, since PA is an amphiphile and only very small amounts are l i k e l y to be present in
1192
On the PI Effect
Vol. 29, No.
12, 1981
aqueous solution, e x p e c i a l l y in the presence of Ca. Thus the a b i l i t y to del i v e r s i g n i f i c a n t q u a n t i t i e s of PA from micellar suspensions may depend on a wide v a r i e t y of unknown factors r e l a t i n g to the a b i l i t y of PA micelles to be incorporated in the plasma membrane and to accumulate p r i o r to any f u r t h e r metabolic d i s p o s i t i o n . One possible c r i t i c i s m of the PA-ionophore hypothesis relates to the significance of data obtained with organic solvents ( i . e . , Pressman chamber) to phenomena as they may occur in biological bilayers. Recently, however, Serhan and co-workers (77) have demonstrated that PA at reasonably low concentrations (1.5 mol %) acts as a Ca-ionophore in m u l t i l a m e l l a r liposomes. The procedure involves the use of the Ca-sensitive dye, arsenazo I I I , entrapped in the liposomes. Thus the method measures Ca translocated into the aqueous compartment of the liposome, rather than simply bound or adsorbed to the surface. In addition, there was no stimulation of Mg uptake, and there was no (or very l i t t l e ) leakage of dye from the liposomes. These observations demonstrated that Ca did not gain entry through rupture of the vesicles or other such non-specific processes (77). There are, therefore, three points of evidence supportive of the PAionophore hypothesis: (a) On receptor a c t i v a t i o n , PA synthesis is stimulated and PA levels increase both probably occurring in the plasma membrane. (b) In a simple b i l a y e r , PA is an e f f i c i e n t , selective Ca ionophore. (c) Agents that competively block Ca entry through receptor activated "gates" also block Ca binding to phosphatidate and in the same order of potency. These points, i t would seem, c o n s t i t u t e a strong argument for p a r t i c i p a t i o n of PA, acting as a Ca ionophore, in receptor regulation of membrane Ca-gating mechanims. Acknowledgements Some of the research described in this review was supported by a grant from the NIH # DE-04067. References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
M. R. HOKIN and L.E. HOKIN, J. Biol.
Chem. 203:967-977 (1953). L. E. HOKIN and M.R. HOKIN, Biochem. Biophys.--Acta 18:102-110 (1955). L. E. HOKIN and M.R. HOKIN, J. Physiol. (Lond.) 132--442-453 (1956). M. R. HOKIN and L.E. HOKIN, J. Biol. Chem. 209:549-558 (1954). L. E. HOKIN, Biochim. Biophys. Acta 115:219-221 (1966). C. M. REDMANand L.E. HOKIN, J. Biophys. Biochem. Cytol. 6:207-214 (1959). 33:421-530 (1967). L. E. HOKIN and D. HUEBNER, J. Cell Biol. L. E. HOKIN and A.L. SHERWIN, J. Physiol. (Lond.) 135:18-29 (1957). M. SCHRAMM and Z. SELINGER, J. Cyclic Nucleotide Res. 1:181-192 (1975). Y. ORON, M. LOWE and Z. SELINGER, F.E.B.S. Letters 3 4 : i 9 8 - 2 0 0 (1973). R. H. MICHELL and L.M. JONES, Biochem. J. 138:47-52 (1974). L. E. HOKIN and M.R. HOKIN, Nature 184:1068-1069 (1959). L. E. HOKIN and M.R. HOKIN, J. Gen. Physiol. 4 4 : 6 1 - 8 5 (1960). M. R. HOKIN and L.E. HOKIN, J. Biol. Chem. 239--2116-2122 (1964). M.R. HOKIN and L.E. HOKIN, in Metabolism and Physiological Significance of Lipids, ( e d i t . by Dawson, R.M.C. and Rhodes, D.N.), pp. 423-435, John Wiley and Sons, London (1964). D.M. SALMON and T.W. HONEYMAN, Nature 284:344-345 (1980). J.W. PUTNEY, J r . , S.J. WEISS, C.M. VAN DE WALLE and R.A. HADDAS, Nature 284:345-347 (1980). M.~. HOKIN, in Secretor~ Mechanisms of Exocrine Glands, ( e d i t . by Thorn, N.A. and Petersen, O.H.), pp. 110-112, Munksgaard, Copenhagen (1974). L.M. JONES and R.H. MICHELL, Biochem. J. 142:583-590 (1974). R.H. MICHELL, Biochem. Biophys. Acta 415:81-147 (1975).
Vol. 29, No. 12, 1981
21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.
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