Gi Down-regulation as a Mechanism for Heterologous Desensitization in Adipocytes”

Prolonged incubation of rat adipocytes with (-)N6-phenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) leads to down-regulation of each of the three subtypes of Gi (Green, A., Johnson, J. L., and Milligan, G. (1990) J. Biol. Chem. 265, 5206-5210). To determine whether other inhibitors of adenylylcyclase would have similar actions, we incubated adipocytes in primary culture with PIA, prostaglandin E1 (PGE1), or nicotinic acid. After various times cells were homogenized, and crude membrane fractions were analyzed on Western blots using antipeptide antisera to alpha- and beta-subunits of G-proteins (SG1 (which binds to alpha i1 and alpha i2), I3B (which binds to alpha i3), BN2 (binds to beta-subunits) and CS1 (recognizes forms of alpha s)). PIA and PGE1 caused approximately 90% down-regulation of alpha i1 and alpha i3, and about 50% loss of alpha i2 and beta-subunits. In contrast, nicotinic acid at concentrations up to 1 mM had no effect on levels of any of these Gi subtypes. None of the compounds altered levels of either a 43- or 47-kDa form of alpha s. PIA caused about a 50% decrease in binding of [3H]DPCPX (an A1 adenosine receptor antagonist), indicating adenosine receptor down-regulation; however, neither PGE1 nor nicotinic acid treatment altered [3H]DPCPX binding. None of the treatments affected the activity of adenylylcyclase when measured in the presence of 100 microM forskolin and 10 mM Mn2+, indicating that the catalytic subunit of adenylylcyclase is not altered. To determine whether Gi down-regulation results in heterologous desensitization, we incubated adipocytes with maximally effective concentrations of PIA (300 nM), PGE1 (3 microM), or nicotinic acid (1 mM) for 4 days. The cells were then washed and incubated for an additional 30 min with various concentrations of these compounds to determine their ability to inhibit lipolysis. PIA caused a (marked) decrease in the sensitivity of the cells to both PIA and PGE1, thus indicating heterologous desensitization. Similarly, PGE1 decreased the sensitivity of the cells to both PGE1 and PIA, again demonstrating heterologous desensitization. In contrast, prolonged incubation with nicotinic acid decreased the sensitivity of the cells to nicotinic acid but had no effect on the sensitivity of the cells to PIA. Adenylylcyclase in membranes from PGE1-treated cells showed decreased sensitivity to inhibition by PIA. In contrast, adenylylcyclase showed normal sensitivity to PIA in membranes from nicotinic acid-treated cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolonged incubation of rat adipocytes with (-)Nephenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) leads to down-regulation of each of the three subtypes of Gi ( T o determine whether other inhibitors of adenylylcyclase would have similar actions, we incubated adipocytes in primary culture with PIA, prostaglandin El (PGEl), or nicotinic acid. After various times cells were homogenized, and crude membrane fractions were analyzed on Western blots using antipeptide antisera to a-and &subunits of G-proteins (SG1 (which binds to ail and ai2), 13B (which binds to ai3), BN2 (binds to &subunits) and CS1 (recognizes forms of as)). PIA and PGE1 caused approximately 90% down-regulation of ail and ai3, and about 50% loss of ai2 and &subunits. In contrast, nicotinic acid at concentrations up to 1 m M had no effect on levels of any of these Gi subtypes. None of the compounds altered levels of either a 43-or 47-kDa form of a,. PIA caused about a 60% decrease in binding of ['HIDPCPX (an A1 adenosine receptor antagonist), indicating adenosine receptor down-regulation; however, neither PGEl nor nicotinic acid treatment altered ['HIDPCPX binding. None of the treatments affected the activity of adenylylcyclase when measured in the presence of 100 FM forskolin and 10 m M Mn2+, indicating that the catalytic subunit of adenylylcyclase is not altered.
To determine whether Gi down-regulation results in heterologous desensitization, we incubated adipocytes with maximally effective concentrations of PIA (300 nM), PGEl (3 FM), or nicotinic acid (1 mM) for 4 days.
The cells were then washed and incubated for an additional 30 min with various concentrations of these compounds to determine their ability to inhibit lipolysis. PIA caused a (marked) decrease in the sensitivity of the cells to both PIA and PGE1, thus indicating heterologous desensitization. Similarly, PGEl decreased the sensitivity of the cells to both PGEl and PIA, again demonstrating heterologous desensitization. In contrast, prolonged incubation with nicotinic acid decreased the sensitivity of the cells to nicotinic *This work was supported by Grant R-01 DK 40061 from the National Institutes of Health (to A.  acid but had no effect on the sensitivity of the cells to PIA. Adenylylcyclase in membranes from PGE1-treated cells showed decreased sensitivity to inhibition by PIA. In contrast, adenylylcyclase showed normal sensitivity to PIA in membranes from nicotinic acid-treated cells. Together with the finding that PGEl has no effect on either AI adenosine receptors nor on the catalytic subunit of adenylylcyclase, these findings suggest that heterologous desensitization of lipolysis is at least partly due to down-regulation of the G-protein(s) responsible for inhibition of adenylylcyclase.
It is well established that exposure of a cell to an agonist can cause desensitization, so that a second exposure to the agonist is less effective than the first. This phenomenon has been studied extensively, and various mechanisms have been described. First, relatively short term exposure to a ligand (minutes) can induce a change in the receptor. For example, 0-adrenergic receptors can become phosphorylated by a specific enzyme @-adrenergic receptor kinase) after exposure to an agonist (1,2). Second, after more prolonged exposure to an agonist, the number of receptors on the cell surface can decrease, by a process termed down-regulation. Down-regulation is generally considered to occur in two steps. First, fairly rapid sequestration of receptors from the cell surface through endocytosis. This is followed by a slower loss of total cellular receptors involving intracellular degradation, although at least a portion are often recycled back to the cell surface (3, 4). In addition to increased degradation of receptors, recent studies have suggested that alterations in receptor mRNA turnover may also play a role (5,6).
The phenomena described above can readily explain homologous desensitization, where exposure of a cell to a hormone results in subsequent insensitivity to that hormone. Another commonly reported phenomenon is known as heterologous desensitization, in which treatment of a cell with a hormone subsequently can make the cell less sensitive to another hormone that works through a different, distinct receptor. Heterologous desensitization has been described for many signaling systems, including receptors coupled to adenylylcyclase (7-9) and to phospholipase C (10, 11). Mechanisms of heterologous desensitization, however, are relatively poorly understood.
We reported that A, adenosine receptors can be downregulated in rat adipocytes following prolonged incubation 3223 Heterologous Desensitization in Adipocytes with an agonist, namely PIA' (12). The A, adenosine receptor is coupled to inhibition of adenylylcyclase through one or more of a group of GTP-dependent regulatory proteins (Gproteins), termed Gi, of which three subtypes have been identified, termed Gil, Gi2, and Gi3. By pertussis toxin labeling we found that, in addition to adenosine receptor down-regulation, chronic exposure of adipocytes to PIA causes selective loss of Gi from the cells (12). More recently, we have used a series of specific antisera to demonstrate that PIA causes a marked (approx 90%) loss of Gil and Gi3, with a more modest (50%) loss of Gi2 (13). When PIA is washed away, Gi down-regulation is reversible, suggesting that it is a real regulatory phenomenon rather than a simple toxic effect. Furthermore, Stiles and co-workers (14,15) have reported similar findings after chronic infusion of PIA into rats in uiuo.
More recent studies suggest that G-protein down-regulation is a common phenomenon resulting from chronic exposure to agonists. Thus, down-regulation of Gi2 was observed following prolonged treatment of hamster smooth muscle DDTl MF-2 cells with PIA (16). Gil is down-regulated by chronic treatment of rat spinal cord-dorsal root ganglion cocultures with opiates (17), and G. down-regulation has been observed following treatment of NG108-15 cells with PGE, (18). Since a number of agonists can presumably couple through a single G-protein, it is clear that this phenomenon of G-protein downregulation could account for heterologous desensitization.
In the current studies, we have evaluated effects of other inhibitors of adenylylcyclase: first, to determine whether Gprotein down-regulation is specific to the adenosine receptor system in adipocytes or may be a more general regulatory phenomenon, and second, to determine whether G-protein down-regulation is a mechanism for heterologous desensitization. We have used prostaglandin El and nicotinic acid, both of which are potent inhibitors of adenylylcyclase and lipolysis in fat cells (19).

MATERIALS AND METHODS AND RESULTS~
The effects of prolonged treatment of adipocytes with PIA, PGEl and nicotinic acid on G-proteins are summarized in Table I. PIA and PGE, caused about a 90-95% loss of ail and ai3 and about 50% loss of ai2 and &subunits. In contrast, nicotinic acid did not affect levels of any of the G-protein subunits. These findings demonstrate, first, that Gi downregulation is not unique to the A, adenosine receptor. Second, the findings demonstrate that down-regulation of ail, ai2, and ai3 is not secondary to chronic inhibition of lipolysis, because nicotinic acid was used at a concentration (1 mM) that was equally effective as an inhibitor of lipolysis as either of the other agents (Fig. 1). Furthermore, experiments using a wide range of concentrations of nicotinic acid (10 M M to 1 mM) failed to demonstrate an effect of chronic exposure to nicotinic acid on levels of any of the Gi subtypes (not shown). Finally, to ensure that the lack of effect of nicotinic acid is not due to breakdown of the compound, cells were incubated for up to 4   days with 1 mM nicotinic acid. Media was then assayed for nicotinic acid based on its ability to inhibit lipolysis in freshly isolated cells, which revealed that nicotinic acid levels did not alter appreciably over the 4-day incubation period (data not shown). The stability of nicotinic acid was also confirmed by analyzing the media for nicotinic acid on thin layer chromatography (see "Materials and Methods"), which demonstrated that the nicotinic acid was intact after the 4-day incubations. Insulin binding was determined as a marker for the plasma membrane, since previous studies have demonstrated that at least 95% of cellular insulin receptors are located on the cell surface in adipocytes (3,36). None of the treatments altered insulin binding to the membranes, indicating that the recovery of plasma membranes is similar in the various groups of cells (Table I). Similarly, the activity of adenylylcyclase, maximally stimulated with a combination of forskolin and Mn2+ ions, was not altered by any of the treatments. Furthermore, Coomassie Blue-stained gels run on membranes following the various treatments revealed an essentially identical pattern of visible bands (not shown). Together, these findings strongly suggest that the recovery of plasma membranes was very similar following the different treatments.
Time-course experiments, using a maximally effective concentration of PGE, (3 PM), demonstrated that down-regulation was detectable by 1 day and maximal by about 3 days (Fig. 6). This time course is similar to that observed in the presence of a maximally effective concentration of PIA (13). Fig. 8 demonstrates the effect of chronic treatment of adipocytes with PIA, PGEI, and nicotinic acid on the subsequent sensitivity of lipolysis to PIA and PGE1. Cells were incubated for 4 days with the compounds at concentrations determined to be maximally effective for Gi down-regulation (ie. 300 nM PIA or 3 PM PGE,), or with 1 mM nicotinic acid. The cells were then washed and incubated with various concentrations of PIA (Fig. &I) or PGEl (Fig. 8B), and the rate of glycerol release was determined over a 30-min incubation. As we reported previously (12), prolonged treatment of adipocytes with PIA decreased the subsequent sensitivity of the cells to PIA (Fig. &I). Prolonged treatment with PGEl also resulted in decreased sensitivity to PGEl (Fig. 8B). More interestingly, the PGE1-treated cells also showed decreased sensitivity to PIA (Fig. a), and similarly, the PIA-treated cells showed reduced sensitivity to PGEI (Fig. 8B). These findings demonstrate that PIA and PGE, induce both homol-

Heterologous Desensitization in Adipocytes 3225
Day: 0 1 2 3 4 7 ai1 . ogous and heterologous desensitization. In contrast, nicotinic acid did not alter the sensitivity of the cells to either PIA or PGEl. Since nicotinic acid did not induce Gi down-regulation, these findings suggest that down-regulation of Gi could be involved in the mechanism of heterologous desensitization.
There are differences in response to PIA and PGEl following the various treatments. Thus, both PIA and PGEl caused only a shift to the right in the dose-response curve to PIA (Fig. 8 A ) . In contrast, both compounds markedly reduced the maximal inhibition produced by PGEl. This apparent difference is difficult to interpret, but may be related to the biphasic response to PGEl that is seen in the control cells (Fig. 8B); while low concentrations of PGE, inhibit lipolysis, at higher concentrations the inhibition is partially reversed. Although this is a small effect, the rate of lipolysis was higher in the presence of 1000 nM than in the presence of 100 nM PGEl in each of the five experiments performed, and the difference was statistically significant ( p < 0.05) in both the controls and the nicotinic acid-treated cells. It is likely that in the PIA-and PGEl-treated cells the inhibitory effect of PGEl is diminished, such that the stimulatory part of the dose-response curve is superimposed on the inhibitory phase, and hence the net response is that maximal inhibition is decreased.
In Fig. 9, adipocytes were incubated with PIA or nicotinic acid for 4 days as before and then washed, and the effect of nicotinic acid on lipolysis was determined over a 30-min period. Nicotinic acid-treated cells showed a markedly decreased sensitivity to nicotinic acid. In addition, the PIAtreated cells showed a markedly decreased sensitivity to nicotinic acid. Therefore, nicotinic acid can induce homologous desensitization, even though it does not induce heterologous desensitization. The decreased sensitivity of PIA-treated cells again demonstrates heterologous desensitization. From these findings it is evident that the compounds that cause Gi downregulation, i.e. PIA and PGEl, will induce both homologous and heterologous desensitization. In contrast, however, nicotinic acid, which does not cause Gi down-regulation, causes only homologous desensitization.
The findings described above suggest that Gi down-regulation is central to the mechanism of heterologous desensitization in this system. However, it is also possible that changes at the level of the receptors occur. To investigate this question, adipocytes were incubated with the various compounds for 4 days as before, and then A, adenosine receptors were measured using the antagonist radioligand [3H]DPCPX (32). PIA caused about 58% loss of binding (Table I), consistent with adenosine receptor down-regulation as we have reported (12). In contrast, PGEl did not affect [3H]DPCPX binding. These experiments were performed with ['HJDPCPX at a concentration of 0.2 nM, which is close to the reported K d of A1 adenosine receptors for this antagonist (32). Therefore changes in the receptor, whether number or affinity, would have been detected by this approach. This demonstrates that PGEl-induced heterologous desensitization is not due to an alteration at the level of the adenosine receptor.
Since the antilipolytic activity of the compounds described is thought to be due primarily to inhibition of adenylylcyclase (19), we determined the ability of PIA to inhibit adenylylcyclase in membranes from cells treated with PIA, PGEl, or nicotinic acid (Fig. 10). The effect of PIA was determined in the presence of 10 p~ isoproterenol, because preliminary experiments revealed that in the absence of a stimulator, adenylylcyclase activity was very low. In membranes from control cells, PIA produced a maximal inhibition of about 50%, with a half-maximally effective concentration of about 1 nM (Fig. 1OA). Adenylylcyclase in membranes from both PIA-treated and PGEl-treated cells was much less sensitive to inhibition by PIA, as revealed by a rightward shift in the dose-response curve. This demonstrates heterologous desen-  sitization at the level of adenylylcyclase. In contrast, nicotinic acid had no effect on the sensitivity of cyclase to inhibition by PIA. Membranes from both PIA-treated and PGE1-treated cells also showed decreased sensitivity to PGEl-inhibition of adenylylcyclase (Fig. lOB), which is again indicative of both homologous and heterologous desensitization. Again, nicotinic acid treatment did not affect the sensitivity of adenylylcyclase to PGE,. However, all three treatments did decrease the sensitivity of adenylylcyclase to nicotinic acid (Fig. 1OC). This is again consistent with heterologous desensitization induced by PIA and PGEI, while nicotinic acid induces only homologous desensitization, presumably because it does not down-regulate Gi. Interestingly, PIA and PGE, caused similar changes in the sensitivity of cyclase to inhibition by PIA and PGEl, while PIA had the greater effect on intact cells, measuring lipolysis (compare Figs. 8 and 10). This observation suggests that PIA may have other effects distal to those identified here (see "Discussion"). DISCUSSION We reported previously that the three subtypes of Gi can be down-regulated to various degrees by PIA, an A, adenosine receptor agonist, in isolated adipocytes. The experiments presented in the current report were designed to answer two questions. First, is the phenomenon of Gi down-regulation unique to the adenosine receptor? The findings clearly demonstrate that, like PIA, PGE, can down-regulate each form of Gi in adipocytes, with a time course similar to that which we reported for PIA-induced down-regulation. Furthermore, the pattern was the same for the two agents, i.e. ail and ai3 were down-regulated by approximately 90%, whereas ai2 was decreased by only about 50%. Thus it is clear that Gi downregulation is not uniquely an effect of A, adenosine receptorcoupled agonists. Gi down-regulation is also not produced by all receptor agonists coupled to inhibition of adenylylcyclase, because nicotinic acid did not cause down-regulation. However, this latter observation is difficult to interpret, since the mechanism of action of nicotinic acid is poorly understood. This finding does demonstrate that the loss of Gi is not secondary to chronic inhibition of lipolysis, since nicotinic acid was just as effective at inhibiting lipolysis as was either PIA or PGEl at the concentrations used. Inhibition of lipolysis by nicotinic acid is pertussis toxin-sensitive (37, 38), and furthermore nicotinic acid inhibition of adenylylcyclase is GTP-dependent (39). Therefore nicotinic acid works through a G-protein-coupled receptor, although the identity of the receptor to which it binds is unknown, Consequently, it is not clear why nicotinic acid failed to down-regulate Gi, while both PIA and PGEl did. One possibility is that the cells have very few "nicotinic acid receptors." There may be enough nicotinic acid receptors to inhibit lipolysis, but an insufficient number to cause detectable down-regulation of G-proteins. However, this is little more than speculation.
Relatively few receptors are known to couple to inhibition of adenylylcyclase in adipocytes. In addition to AI adenosine receptors and prostaglandin receptors, a2-adrenergic receptors are thought to be important in inhibition of adenylylcyclase and lipolysis in man. Most investigators have been unable to demonstrate a2-receptors on rat adipocytes, although there is a report that they are indeed present (40). Unfortunately we could not demonstrate an effect of a highly selective a2-agonist (UK 14304) on either lipolysis or relative levels of Gi (data not shown). Therefore, at present we cannot determine whether a2-receptor activation would lead to down-regulation The second question these studies were designed to address is whether Gi down-regulation could form a basis for heterologous desensitization. This type of desensitization, in which exposure to one agonist results in resistance to another agonist that works through a distinct receptor, has been commonly observed, but the mechanism is not clear. G-proteins can couple several different classes of receptor to a single effector. For example, glucagon, ACTH, and /?-adrenergic receptors can all activate adenylylcyclase, and all couple through G,. Conversely as described above, several different receptor types can couple to inhibition of adenylylcyclase through one or more of the subtypes of Gi. Therefore, a change at the level of a G-protein could clearly decrease cellular sensitivity to a range of agonists, providing a mechanism for heterologous desensitization. A number of studies have indicated that a functional change in G. can account for heterologous desensitization of agonists coupled to activation of adenylylcyclase (8, 41). The findings reported here demonstrate that prolonged exposure of adipocytes to either PIA or PGEl, agonists that inhibit adenylylcyclase, causes actual loss of immunologically detectable Gi from the cells. Furthermore, PIA and PGEl induce heterologous desensitization of lipolysis. In contrast nicotinic acid, another potent inhibitor of adenylylcyclase and lipolysis, failed to induce Gi down-regulation and resulted in only homologous desensitization. Thus it appears that in this system, Gi down-regulation is required for heterologous but not homologous desensitization.
Clearly, heterologous desensitization could be caused by changes at the level of the receptor, the G-proteinb), the catalytic subunit of adenylylcyclase, or at some downstream location between adenylylcyclase and lipolysis, such as protein kinase A, triglyceride lipase, or cyclic AMP phosphodiesterase, Changes in phosphodiesterase activity have been suggested as a mechanism for desensitization (42), and indeed Conti and co-workers (43) have reported that follicle-stimulating hormone can induce more than a 100-fold increase in mRNA encoding a high affinity CAMP phosphodiesterase in Sertoli cells (43). However, Hoffman et al. (44) have demonstrated that infusion of PIA in vivo induced desensitization in subsequently isolated adipocytes but did not seem to alter the activity of phosphodiesterase. Together with the finding that heterologous desensitization can be seen at the level of adenylylcyclase in membranes isolated from cells after the of Gi.
various treatments, this suggests that the most likely mechanism involves either receptor changes, G-protein changes, or alterations in adenylylcyclase. We were unable to evaluate the effect of PIA on PGE, receptors. However, it was clear that while PIA caused a decrease in A, adenosine receptor binding (as would be expected), PGE, did not affect adenosine receptors. The catalytic subunit of adenylylcyclase was measured by incubating membranes with a combination of forskolin and Mn", which is thought to reflect the activity of the catalytic subunit of adenylylcyclase alone (45-47); none of the treatments affected the catalytic subunit, although they were able to alter the sensitivity of cyclase to inhibition. Since both PIA and PGE, did down-regulate the various forms of Gi, it seems very likely that Gi down-regulation is involved in heterologous desensitization in this situation. Furthermore this is strengthened by the finding that nicotinic acid, which did not down-regulate Gi, induced homologous but not heterologous desensitization. Since multiple mechanisms are clearly involved in desensitization the possibility that other changes occur, such as in phosphodiesterase or triglyceride lipase, cannot be ruled out. This may account for the differences in adenylylcyclase desensitization and desensitization of lipolysis. For example, treatment with PIA causes greater desensitization of lipolysis to PIA than to PGE,, while the shift is equal at the level of adenylylcyclase. This suggests that chronic PIA treatment causes additional effects distal to cyclase inhibition. Further studies will be required to evaluate this possibility.
An interesting observation is that prolonged treatment with PIA causes predominantly a shift to the right in dose-response curve, with little or no decrease in maximal effect. This is true both at the level of adenylylcyclase and at the level of lipolysis and has been reported previously both by us (12) and by Hoffman et al. (44, 48). Since PIA causes a 50-60% decrease in adenosine receptors, this shift in dose-response curve with little change in maximal inhibition suggests that there are "spare receptors" for adenosine on adipocytes.
Very little is known regarding the mechanism of G-protein down-regulation. Longabaugh et al. (15) have reported that steady state levels of mRNA for the various G-proteins are unaltered in adipose tissue from rats infused chronically with PIA, and we have made similar observations in primary cultured adipocytes? While the possibility of altered mRNA turnover cannot be ruled out, it seems likely that G-protein down-regulation is due to increased degradation. Indeed Hadcock et al. (16) have recently reported that PIA increases the rate of ai2 degradation in hamster smooth muscle cells. Therefore the stoichiometry of receptor and G-protein down-regulation is of interest. The concentration of A, adenosine receptors in adipocyte plasma membranes appears to be in the range of 0.5-1.5 pmol/mg protein (14,32,49). Our estimates suggest that each form of Gicu is present at about 7-10 pmol/ mg protein. Treatment of adipocytes with maximal concentrations of PIA results in about 60% down-regulation of A, adenosine receptors (12), 90% loss of Gil and Gi3, and 50% loss of Gi2. Thus it appears that the stoichiometry of downregulation is such that at least 10 mol of both Gil and Gi3 and 2 mol of Gi2 are lost for each mole of adenosine receptors. One possible explanation for this difference could be that the G-proteins and receptors are cointernalized, but the receptors are recycled back to the plasma membrane more efficiently than the G-proteins. This is supported to a certain extent by the finding that treatment of adipocytes with isoproterenol causes redistribution of G. from the plasma membrane to a less dense cellular fraction (50). Further work is clearly rea A. Green and G. Milligan, unpublished observations. quired on the mechanism of G-protein down-regulation.
Recent evidence suggests that Gi2 is the G-protein responsible for coupling receptors to inhibition of adenylylcyclase. Thus, both in neuroblastoma X glioma hybrid, NG108-15 cells (51) and in platelets (52), antibodies directed against ai2 blocked inhibition of adenylylcyclase, but antibodies directed against other forms of ai had no effect. Both of these cell types contain Gi2 and Gi3; hence, the findings suggest that Gi3 is not involved in inhibition of adenylylcyclase. However, neither NG108-15 cells nor platelets appear to express Gil, and so the findings do not rule out a role for Gil in cyclase inhibition.
Interestingly, both PIA and PGE, down-regulated Gi2 to a lesser degree than either Gil or Gi3. The significance of the effects of these compounds on Gil and Gi3 is difficult to interpret without more knowledge of the mechanism of Gprotein down-regulation. If G-proteins are down-regulated as a direct consequence of receptor activation, the findings might suggest that receptors for these compounds can couple to all three of these G-proteins. Further studies will be required to determine to which effector system(s) Gil and Gi3 might couple in adipocytes, and whether these effector systems show even more pronounced resistance after treatment with PIA and PGE, than does lipolysis, which is presumably regulated primarily by adenylylcyclase and hence, most likely, Gi2.

52.
blot analysis. are illustrated in Zg, 2. In this experiment. adipmyles were mcubated for 4 days with or 'Ihe e f f e~ of the antilipol ic mmpounds on relative lweb of Gp subypu. determined by Western without each of the antilipolyic a ents. then homogenized. and membranes were isolated. re arnted on SDS-PAGE tramferred to nttroce~ulme, and incubated with either antiserum SGI or 13B. as d%dbedjn the Methods seclim. Blols conlalnlng plasma mcmhrrnes from mnlrol and treated cells were probed unth antiserum SGI. which hmdr to the o.subunm of G I and G.2 equally. (Fig. 2A). As we have revmusl reponed (13). PIA caused a marked decrease in lsbehnf ?foil. and a somewhat bs pronounced Ls of a& As can be see% PGE had the ramc effect as PIA on re atwe levels of .-I and -2 In mntrast. nimtinic acid had no effect on the level of eirhcra-subunit. Similarly. PIAand PCB, holhbbwnregulated a>. whereas nicolmic acid was without effect (Fig ZB). prevcnl endogenous adenmine from accumulating and affecting G . m e i n levels (ree Methods). However The uprimentr illustrated were performed with adenmine deaminase in the incubation medium to ~n practice il was found that adenmine deaminav did not affect &r the levels of any of the Grotein d svbumtr nor the ability of PIA or PGE to induce Gprotcin downregulation data not shown?. In the f?ima~blturer. adlpocytes arc tncubated a1 a mncentration of only a b u t 25,006 cells/ml. and hence it is tkdy I at endogenous adenmine docs not reach ruffiaently high mncentratiom to cause G, domrcgulation.

A B
. . cells (13). Fig. 4 illustrates that PGEt causes a deocav in la&lingof the 8.ruhunit. but nicotinic acid does In our previous report we also found that PIA caused P proximately a 50% IOU of p-subunits from the not. which IS COnsiStent with mubunit damregularion bein linked to 10s of ..subunits. h r demilomety of the blots (see figurc legend) revealed that both PIA and &E, treatment caused approximately B 50% 10s of8-rubunit labeling from the cells. PGEl-treated: Innc4. nicotinic acid-treated. One of three similar blos in the IC end to Fig. 2. Western blots were probed with antiserum BN2 Io detect PsubuniU. h e 1.

B -
is illustrated.