Volume 25, number 3

MOLECULAR & CELLULAR BIOCHEMISTRY

June 15, 1979

REGULATION OF ADENYLATE CYCLASE BY ADENOSINE* John N. FAIN and Craig C. MALBONi"

Section of Physiological Chemistry, Division of Biology and Medicine, Brown University, Box (3, Providence, Rhode Island 02912 (Received January 22, 1979)

Summary Adenosine may well be as important in the regulation of adenylate cyclase as hormones. SATrIN and RALL first demonstrated in 1970 that adenosine was a potent stimulator of adenylate cyclase in the brain. However, adenosine is an equally potent inhibitor of adenylate cyclase in other cells such as adipocytes. The concentration of adenosine required for this regulation of adenylate cyclase is in the nanomolar range (10 to 100 riM). Both the inhibitory and stimulatory effects of low concentrations of adenosine on adenylate cyclase are antagonized by methylxanthines. This antagonism of adenosine action may account for all or part of the effects of methyl xanthines on cyclic A M P levels in many tissues. Adenosine appears to be a particularly important endogenous regulator of adenylate cyclase in brain, smooth muscle and fat cells. Under conditions in which intracellular AMP rises, adenosine formation and release is accelerated. In addition to its direct effects on adenylate cyclase, adenosine (at higher concentrations approaching millimolar) exerts multiple effects on cellular metabolism as a result of its intracellular metabolism and especially conversion to nucleotides. The effects of nanomolar concentrations of adenosine on adenylate cyclase are mediated * This work was supported by United States Public Health Service Research Grant AM-10149 from the National Institute of Arthritis, Metabolism and Digestive Diseases. t Present address: Department of PharmacologicalSciences, School of Basic Health Sciences, State University of New York at Stony Brook, New York, 11794.

through an adenosine site possessing strict structural specificity for the ribose moiety of the molecule (the " R " adenosine site) which is presumably located on the external surface of the plasma membrane. In brain, lung, platelets, bone, lymphocytes, skin, adrenals, Leydig tumors, and coronary arteries adenosine stimulates adenylate cyclase via this site. However, in rat adipocytes, brain astroblasts and ventricular myocardium adenosine inhibits adenylate cyclase through the " R " or adenosine site. Although the " R " site requires an intact ribose moiety, adenosine analogs modified in the purine ring such as N 6phenylisopropyladenosine appear to be potent agonists for this site. All effects of adenosine mediated via the " R " site are competitively antagonized by methyl xanthines. The effects of micromolar concentrations of adenosine appear to be mediated via a site with strict structural specificity with respect to the purine moiety of the molecule (the " P " or adenine adenosine site). This " P " site is postulated to be located on the intracellular face of the plasma membrane and mediates the effects of adenosine due to conversion of adenosine to 5'-AMP or perhaps other nucleotides. The effects of high concentrations of adenosine are always inhibitory to adenylate cyclase activity, are readily demonstrated in broken cell preparations, and are unaffected by methylxanthines. An intact purine ring is required for these adenosine effects but modifications of the ribose moiety of the molecule generally increases the potency of the analog. A prime example is

Dr. W. Junk b.v. Publishers- The Hague, The Netherlands

143

2',5'-dideoxyadenosine, which is the most potent known "R"-site specific adenosine analog. We propose a unitary model which explains both the stimulatory and inhibitory effects of low concentrations of adenosine on adenylate cyclase. In brief, adenylate cyclase is postulated to exist in three interconvertible activity states: (i) an inactive state (E0); (ii) a GTP-liganded state with high activity (EGTp); and (iii) a GDP-liganded s t a t e (EGDP) which is inactive in cells where adenosine stimulates adenylate cyclase, but active in cells where adenosine inhibits adenylate cyclase. We postulate that the enzyme cycles through these states in the following manner: the Eo state binds GTP and forms the EGTP state; hydrolysis of bound GTP converts the EGT P to the EGD P state; and release of bound GDP converts EGDP to the Eo state. The Eo state is the only form of the enzyme which can be stimulated by either hormones or GTP and its formation from the EGDp state is rate-limiting in this cycle. The conversion of EGO P t o E 0 regulates the ability of hormones and GTP to activate adenylate cyclase and is postulated to be adenosine sensitive. In cells where both EGD v and E0 states are inactive, adenosine stimulates adenylate cyclase activity. In cells where E0 is inactive, but EGDa is active, adenosine inhibits adenylate cyclase activity. In addition we suggest that in cells where adenosine inhibits adenylate cyclase activity (cells postulated to have a n EGD a state which is active) high concentrations of GTP favor accumulation of the enzyme in EGOp and thus are inhibitory to activity. Prostaglandins may also regulate adenylate cyclase in a manner similar to that described above for adenosine. We conclude that adenosine is an important regulator of adenylate cyclase whose role has only been appreciated recently. Further studies are warranted on both its binding to cells and mechanisms by which it regulates adenylate cyclase.

Introduction

SATrIN and RALL 1 first reported in 1970 that adenosine was a potent activator of cyclic AMP accumulation in brain slices. The importance of this observation was no more recognized at the time than was the original report on the 144

discovery of cyclic AMP from the same laboratory by SUTHERLANDand RALL 2. The discovery of a role for adenosine in regulating cyclic AMP resulted from the finding that cyclic AMP was markedly elevated by electrical pulses applied to slices of guinea pig cerebral cortex 3. In an effort to determine if electrical pulses result in the formation of a messenger which elevated cyclic AMP, SATrIN and RALL 1 tested acid extracts of brain and found that they elevated cyclic AMP. Subsequently it was established that the release of adenosine can account for most of the effects of electrical depolarization on brain slices. SATTIN and RALL 1 also reported that the elevations in cyclic AMP due to adenosine were antagonized by methyl xanthines. Previously the only known effects of methyl xanthines had been to potentiate the increases in cyclic AMP due to hormones. The interest of this laboratory in adenosine regulation of adenylate cyclase stemmed from some unrelated studies initiated in 1971 on the effects of the RNA synthesis inhibitor 3'deoxyadenosine on rat fat cell ghosts. We noted that this compound inhibited the rise in adenylate cyclase due to both fluoride and norepinephrine 4. Interestingly, 2'deoxyadenosine and adenosine had similar effects4. A variety of adenosine analogs were tested in the search for the significance of this observation with surprising results. Westermann et al. 5 had reported that N 6phenylisopropyladenosine* inhibited hormonestimulated lipolysis in vivo and in vitro in isolated rat fat cells. However, this compound was totally inactive as an inhibitor of adenylate cyclase activity of rat fat cell ghosts. In contrast, 2',5'-dideoxyadenosinel" markedly inhibited adenylate cyclase4. Although inhibiting cyclic AMP accumulation by rat fat cells adenosine stimulates cyclic AMP accumulation by brain slices and nearly all other cells tested. Furthermore, SATrIN and RALL 1 had * N6(Phenylisopropyl)adenosine was originally developed in the search of adenosine analogs which might be clinically useful as vasodilators and is now available from Boehringer Mannheim. All studies have apparently been done with the levoisomer ( - ) which is some 50 to-1000-fold more active on lipid mobilization than is the dextro (+) isomer. 2',5'-Dideoxyadenosine was originally tested in our laboratory because of the desire to have an adenosine analog which could not be converted to nucleotides and is now available from P-L Biochemicals, Milwaukee.

reported that 2'-deoxyadenosine did not stimulate cyclic A M P accumulation by rat brain slices. In fact, the deoxy analogs of adenosine such as 2'-deoxy, 3'-deoxy and 2'5'dideoxyadenosine were subsequently shown to inhibit cyclic A M P formation by brain slices 6. These so-called "P"-site adenosine analogs 7 possessing an intact purine moiety but with modified ribose moiety such as 2'-deoxy-, 3'deoxy, 5'-deoxy, 2'5'-dideoxy-. and xylofuranosyladenosine are primarily inhibitors of cyclic AMP accumulation in brain slices. Adenosine analogs with an intact ribose group but with modifications on the purine ring such as 2-fluoro, 2-chloro or 6-phenylisopropyl adenosine (so-called "R"-site compounds because of the need for an unaltered ribose group), in contrast, elevate cyclic A M P accumulation by brain slices. These data are best explained by the hypothesis that low concentrations of adenosine and other R-site analogs (with intact ribose but altered purine rings such as N6-phenylisopropyladenosine) activate brain adenylate cyclase while higher concentrations of adenosine and the P-site compounds such as 2'5'-dideoxyadenosine interact at a different site to inhibit adenylate cyclase. This hypothesis can explain the original finding that N 6phenylisopropyladenosine and adenosine at low concentrations (below 10 -6 M) inhibited cyclic A M P accumulation due to hormones in fat cells but had little effect on adenylate cyclase activity of broken cell preparations 4. In contrast 2'5'-

dideoxyadenosine at concentrations between 10 -s and 10-4M had similar effects on cyclic A M P accumulation by intact fat cells and adenylate cyclase activity of fat cell ghosts. FAIN et al. 8 suggested that there are two separate types of adenosine effects on fat cells as on brain slices. The R-site is sensitive to low concentrations of adenosine or N 6phenylisopropyladenosine and mediates the physiological effects of adenosine but is difficult to demonstrate on adenylate cyclase activity of broken cell preparations. The P-site, whose physiological significance is obscure, is responsible for inhibition of adenylate cyclase by 2'5'dideoxyadenosine or relatively high concentrations of adenosine and is easily demonstrated in broken cell preparations. The physiological regulator of the postulated intracellular site may be 5'-AMP which could be considered as a P-site compound since it is an analog of adenosine with an intact purine ring but a modified ribose moiety. Alternatively the regulator of the intracellular or P-site could be a compound such as 2'-deoxy 3-AMP which is a potent inhibitor of adenylate cyclase activity derived from a variety of cells 9'1°. LoNoos and WOLFF7 first divided adenosine effects on adenylate cyclase into " P " - and "R"-site mediated effects. This concept was an important advance in our understanding of adenosine effects on cellular metabolism and will be more thoroughly discussed in subsequent sections. In Table 1 we suggest that the " R " -

Table 1 Differences between R and P effects of adenosine. Comparison of adenosine effects on adenylate cyclase mediated via "R" versus "P" sites. Effects mediated via:

Require intact ribose moiety but accept compounds substituted on the purine ring such as N6-phenylisopropyladenosine Require intact purine ring but accept ribose modified compounds such as 2'5'-dideoxyadenosine Are antagonized by compounds which block adenosine uptake into cells Are antagonized by theophylline Are readily demonstrated on adenylate cyclase activity of solubilized membrane preparations Always inhibit adenylate cyclase Require high concentrations of adenosine

Adenosine or "R" site

AMP or "P" site

Yes

No

No

Yes

No Yes

Yes No

No No No

Yes Yes Yes 145

site is the site which mediates the playsiological effects of adenosine. In contrast, the " P " - s i t e appears to be an adenine nucleotide or deoxyadenosine site which requires rather high concentrations of adenosine or the deoxy analogs. Perhaps the "P"-site is an intracellular nucleotide regulatory site whose physiological regulator is an adenine nucleotide rather than adenosine itself. In any case, it is important to realize that effects of adenosine on the "P"-site represent primarily a complication in our understanding of adenosine action. The other features of the "R"-site versus "P"-site effects of adenosine listed in Table 1 will be detailed below. The literature on adenosine is extensive. A Medline* search indicated that 617 articles were published during 1976, 1977 and 1978 in which adenosine was the main. focus of the article. Another indication of the growing interest in adenosine action was the convening of the First International Conference on the regulatory functions of adenosine and adenine nucleotides in June, 1978. This conference emphasized the effects of adenosine on smooth muscle, the cardiovascular system and cyclic AMP metabolism. The published proceedings of this symposium contain over 30 articles reviewing research related to adenosine aS. Some of the effects of millimolar concentrations of adenosine on cellular metabolism are secondary to elevations in total intracellular adenine nucleotides. This review is restricted to the effects of adenosine seen at micromolar concentrations which are not due to elevations in ATP.

General Effects of Adenosine on Metabolism and Cyclic A M P Accumulation in Intact Cells Since the original demonstration by Sattin and Rail 1 that addition of adenosine to brain slices increases the accumulation of cyclic AMP, similar results have been seen in a variety of cells (Table 2). The rise in cyclic A M P due to low concentrations of adenosine may result from an activation of adenylate cyclase by mechanisms similar to those by which hormones activate this enzyme. Adenosine probably acts as a local A Medline search is a computer-based retrieval system based on journals listed in Index Medicus.

146

hormone or messenger released by cells under certain conditions which regulates adenylate cyclase activity. There are some cells (Table 3) in which adenosine inhibits the ability of hormones to activate adenylate cyclase and cellular metabolism. It should be realized that in cells where low concentrations of adenosine either inhibit or activate adenylate cyclase through " R " or "adenosine" site there is a second effect of high concentrations of adenosine which is always inhibitory to adenylate cyclase. This effect is mediated via the so-called " P " or " A M P " site. In intact cells this is rather difficult to demonstrate since the high levels of added adenosine required for demonstration of this effect also elevate intracellular A T P which directly alters cellular metabolism. The pathways for adenosine release and metabolism are shown in Figure 1. Adenosine accumulates extracellularly via direct release by cells or can be formed via extracellular metabolism of A T P or AMP derived from cell lysis. Adenosine is removed from the extracellular space either through re-uptake by cells and conversion to A T P via adenosine kinase or by metabolism to inosine via adenosine deaminase. Inosine is unable to mimic the actions of adenosine on cyclic A M P metabolism, highlighting the strict requirement for an amino or N'-substituted group at the 6-position of the purine moiety for either action of adenosine. Inosine reacts with purine nucleoside phosphorylase to give hypoxanthine which is either oxidized to uric acid or salvaged by cells to serve as a precursor of adenine nucleotides. The important feature is that the level of adenosine depends on the sum of removal by phosphorylation and deamination. Figure 1 also shows that the deamination of adenosine can be blocked by coformycin, deoxycoformycin or erythro-9-(2hydroxy-3-nonyl)adenine (EHNA), potent inhibitors of adenosine deaminase 26'27.

Effects of Nucleotides due to Adenosine Many of the reported effects of cYclic AMP, A T P or A M P addition to isolated cells or tissues may have been due to adenosine formed during extracellular catabolism of these nucleotides. By adding adenosine deaminase along with nucleotide the effects due to adenosine can

Table 2 Tissues in which adenosine increases cyclicAMP accumulation. Tissues (Listed in order of reports)

Effect antagonized by methylxanthines

Brain slices (guinea pig)

yes

Rat lung slices Platelets (human)

-yes

Cultured mouse neuroblastoma cells Cultured human astrocytoma cells Bone cells (fetal rat) Lymphocytes Ventricular myocardium (guinea pig) Pig skin slices Cultured adrenal and Leydig tumor cells Pig and cow coronary artery strips

ye s yes yes -yes yes yes yes

be eliminated. WOLFF and COOK 21 found that the addition of cyclic A M P to cultured adrenal cells stimulated steroidogenesis but this affect was abolished in the presence of adenosine deaminase. Similar increases in steroidogenesis were seen following the addition of adenosine, N A D or A T P which, with the exception of a small part of the stimulatory effect of ATP, were abolished by the presence of added adenosine deaminase 21. SNYDER and SEEGMILLER 28 found that culture medium supplemented with fetal calf serum or horse serum converted cyclic A M P to adenosine. Perhaps many of the reported effects of cyclic A M P on cultured cells were, in fact, due to adenosine formation. In H e L a cells the inhibition of growth seen in the presence of cyclic A M P was shown to result from adenosine formation rather than any direct effect of cyclic A M P 29'3°. Adenosine is toxic to a wide variety of cells. This toxicity may result from an

References SATTIN and RALL, 19701 SHIMIZU and DALY, 197011 PALMER, 197112 MILLS and SMITH, 197113 HASLAM and RossoN, 197514 BLUME et al., 197315 CLARK et al., 197416 PECK et al., 197417 WOLBERG et al., 1975 TM HUANG and DRUMMOND, 197619 IIZUKA et al., 197620 WOLFF and COOK, 19772~ KUKOVETZ, 197822

elevation in A T P which inhibits the formation of U T P 31. It should be noted that these effects of adenosine appear to be unrelated to regulation of cyclic AMP metabolism but are rather the result of direct conversion to ATP. Adenosine formation from added cyclic A M P may account for the unusual findings reported some years ago by DOLE 32'33 and VAUGHAN34'35 that cyclic A M P had effects on fat cells exactly the opposite of hormones which elevate cyclic AMP. This is to be expected since adenosine antagonizes the increases in cyclic AMP in response to hormones 4'23. SOLOMON et al. 36 also found that cyclic A M P stimulated glucose oxidation by rat fat cells while dibutyryl cyclic A M P inhibited glucose oxidation. Presumably cyclic AMP served as a source of adenosine which lowers intracellular cyclic AMP while the dibutyryl derivative enters cells and increases the cyclic A M P pool. BURNSTOCK 37 suggested that the autonomic

Table 3. Tissues showing a decrease in hormone-stimulated cyclic AMP accumulation in response to low concentrations of adenosine.* Rat fat cells Slices of rat ventricular myocardium Cultured epitheloid astroblasts from mouse brain

FAIN et al., 19724 SCHWABE et al., 197323 DOBSON, 197824

VAN CALKERet al., 1978132

* In liver cells adenosine fails to stimulate or inhibit cyclic AMP accumulation at low concentration, but does decrease cyclic AMP accumulation at fairly high concentrations. In nearly all cells in which low concentrations of adenosine stimulate cyclic AMP accumulation the addition of higher concentrations of adenosine are also inhibitory. 2

147

ATP

l

•

ADENYLATE(AMP)

~ 5'-Nucleotidase ,~l".................. ADENOSINE

~l~i:iilFiiiiilliJiJJJiii~J--~lTq~ll~!!!!!! i!i:ii;i[;it;PLASMAM~M~RANE ATP.-~-~. AMP---~ADENOSINE EXTRACELLULAR SPACE

l

Deaminase Blocked by c o | o r m y c i n and erythro-9-(2-hydroxy-3-nonyl)adenlne

--Adenosine

INOSINE

Fig. 1. Pathwaysfor adenosinerelease and uptake by cells. nervous system has, in addition to adrenergic and cholinergic nerves, purinergic nerves which release a purine nucleotide as the transmitter. Whether ATP or adenosine is the transmitter is notclear since ATP is a good source for adenosine. More recently BURNSTOCK38 suggested that adenosine is also a purinergic transmitter and divided the purinergic receptors into two types. One receptor (P1) is for adenosine while the P2 receptor binds ATP. Hopefully, the studies with added ATP will now be repeated in the presence of adenosine deaminase to differentiate those receptors for ATP from those for adenosine. The P1 purinergic receptors respond to adenosine, are antagonized by methyl xanthines, and apparently modulate cyclic AMP levels. In contrast, the P2 or ATP receptors do not mediate changes in cyclic AMP, are insensitive to antagonism by methyl xanthines, but are antagonized by compounds such as quinidine, 2-substituted imidazolines and 2'2-pyridilisatogenas. BURNSTOCK suggested that adenosine is the primary purinergic transmitter in the trachea while ATP is the transmitter in the bladder and taenia coli. In the taenia coli, bladder and trachea the addition of adenosine results in muscular relaxation. However, ATP causes relaxation of the taeni coli but contractions of the bladder and trachea.

Adenosine Action on Hepatic Metabolism

The liver is a good example of a tissue in which there is little evidence that adenosine is a physiological regulator of cyclic AMP accumulation. Adenosine is so rapidly metabolized by rat liver cells that in order to see any appreciable effects of adenosine on cellular metabolism rather high concentrations of adenosine are 148

required. FAIN and SHEPHERD 39 found that in the presence of an inhibitor of adenosine deaminase 0.2 mM adenosine blocked glucagon-stimulated glycogenolysis in liver cells from fed rats. This effect was not secondary to the small inhibitory effect of adenosine on glucagon-stimulated cyclic AMP accumulation since 2'5'dideoxyadenosine was a more potent inhibitor of cyclic AMP accumulation than was adenosine but had little effect on glycogenolysis as measured by glucose release. These results are not consistent with the current view that cyclic AMP is the only regulator of glycogenolysis and indeed there is some evidence that cyclic AMP may not be the sole mediator of glucagonstimulated hepatic glycogenolysis4°. Similar results have also been noted in isolated rat fat cells where inhibition of cyclic AMP accumulation by 2'5'-dideoxyadenosine was not associated with any inhibition of catecholamineactivated lipolysis8. The inhibition of glucagonstimulated glycogenolysis in liver cells by adenosine may, in fact, be secondary to an elevation in ATP. LUND et al. 41 found that 0.5 mM adenosine increased the total intracellular concentration of ATP by almost three-fold. In addition, LUND et a l / 1 noted an inhibition of hepatic gluconeogenesis by adenosine. Adenosine has also been shown to stimulate vasoconstriction in the perfused liver42. Thus the action of adenosine on hepatic metabolism requires very high concentrations of adenosine and appears to reflect changes in nucleotide metabolism.

Adenosine Action on Fat Cells

DOLE reported in 1961 that adenosine and adenosine-containing nucleotides inhibited hormone-stimulated lipolysis by incubated rat adipose tissue ~2. Subsequently this report was confirmed43-46 and extended to include inhibition of hormone-stimulated cyclic AMP accumulation4,23. Not all of the effects of nucleotides are due to adenosine formation since there are inhibitory effects of 1-10/zM NAD on cyclic AMP accumulation by isolated fat cells even in the presence of adenosine deaminase which are unrelated to adenosine47. NAD acts like a weak analog of nicotinic acid which at concentrations

in the range of 0.033 to 0.33 p,M markedly reduces cyclic A M P accumulation by fat cells in the presence of hormones 47. The inhibitory effect of nicotinic acid is also unaffected by adenosine deaminase. CARLSON 48 originally reported that nicotinic acid is a potent inhibitor of lipolysis and this may account for its hypolipidemic action 49. Whether adenosine and nicotinic acid interact at the same site to inhibit cyclic A M P accumulation by cells is not known. There is no evidence that nicotinic acid binds to the same site as adenosine in isolated fat cell membranes 5°. The administration of 2 mg/kg of nicotinic acid or 80 mg/kg of adenosine to rats produced a transient drop in free fatty acids 5. However, the administration of N 6phenylisopropyladenosine at 0.02 mg/kg resulted in an equal but more sustained drop in the plasma free fatty acids. WESTE~MAN et al. 5 also reported that 0.1 t~M N 6phenylisopropyladenosine gave a maximal inhibition of the lipolytic action of A C T H , theophylline or norepinephrine but failed to inhibit the lipolytic action of dibutyryl cyclic AMP on rat adipose tissue. We were initially perplexed by the inability of N6-phenylisopropyladenosine to inhibit adenylate cyclase activation in fat cell membranes 4. In contrast, 2'5'-dideoxyadenosine which had no inhibitory effect on fat cell lipolysis was the most potent inhibitor of adenylate cyclase. These results are summarized in Table 4 and can now be explained on the assumption that there are two sites at which adenosine inhibits cyclic A M P accumulation.

The true adenosine or R-site is difficult to demonstrate in cell-free preparations while the AMP or P-site is easily seen in these systems. Inhibition of adenylate cyclase at the adenosine or R-site can only be seen under special conditions. LONDOS e t al. 53 found that in the presence of 0.1 to 10tzM G T P and the presence of exogenous adenosine deaminase to remove bound adenosine there was a partial inhibition of adenylate cyclase activity by 0.01 to 0.1 ~M concentrations of R-type compounds such as N6-methyladenosine, N 6phenylisopropyladenosine and 2chloroadenosine. The effects of the R-type adenosine analogs were reversed by methyl xanthines 53. Figure 2 shows similar data obtained in our laboratory by a slightly different procedure. The addition of N 6phenylisopropyladenosine reversed the stimulatory effect of theophylline on adenylate cyclase activity of fat cell ghosts previously incubated with the non-hydrolyzable G T P analog guanyl-5'-yl imidodiphosphate, Gpp(NH)p. These effects are discussed in the section on adenosine regulation of adenylate cyclase. Adenosine interaction at the physiological or R-site is antagonized with respect to inhibition of hormone-stimulated cyclic A M P accumulation in intact cells by methyl xanthines. In contrast, the ability of 2'5'-dideoxyadenosine to inhibit cyclic A M P accumulation in intact cells via the P-site is not antagonized by methyl xanthines 8. The marked drop in cyclic A M P accumulation produced by 2',5'dideoxyadenosine is not accompanied by any

Table 4 Comparison of the deoxyadenosineor AMP-site (P-site) and the adenosine site (R-site) effects in fat cells.* Adenylate Cyclase Agent

2',5'-dideoxyadenosine adenosine N 6-phenylisOprOpyl adenosine

Cyclic AMP Parameter: accumulation

Lipolysis

AMP or "P"-site

Adenosine or "R"-site

(concentration of adenosine or analogs required for 50% inhibition of catecholamine stimulation of indicated parameter) 10/xM > 50 /.tM 0.5 p,M -0.01 /XM 0.10 /XM 10 ~M -0.003 /.£M

0.01 /./,M

> 5 0 /.LM

2',5'dideoxyadenosine > ATP = ADP > cyclic AMP = AMP > inosine > N6-phenylisopropyladenosine. The competition studies suggested the highaffinity binding was to multiple population of sites with differing specificity and was not to a single, homogeneous class of binding sites. The high-affinity binding was sensitive both to trypsin-treatment and to heat denaturation and was inhibited by low concentrations of theophylline but not by 100/~M dipyridamole or p-nitrobenzylthioguanosine. 2',5'-Dideoxyadenosine was a potent inhibitor of adenosine binding to the fat cell membrane, whereas N6-phenylisopropyladenosine at even 100/~M failed to display any capacity to

Table 10 Systems in which specific plasma membrane receptors for adenosine have been postulated as the site of adenosine action. System Brain slices Platelets Mouse neuroblastoma cells Cultured human astrocytomacells Cultured fetal rat bone cells Transformed human lung fibroblasts Adrenal cells Leydigtumor cells Coronary arterial smooth muscle Isolated fat cells 164

Action of adenosine

Reference

Stimulates cAMP accumulation Stimulates cAMP accumulation Stimulates cAMP accumulation StimulatescAMP accumulation Stimulates cAMP accumulation

(1,129) (14,93) (103) (16,99) (17)

Stimulates cAMP accumulation Stimulates cAMP accumulation Stimulates cAMP accumulation

(13) (21) (21)

Stimulates cAMP accumulation Inhibits cAMP accumulation

(95) (45,51,54)

compete for these sites. These data indicate that under the conditions employed adenosine binding is predominantly to a "P" type site on the fat cell membrane s°. The inability of N 6phenylisopropyladenosine to inhibit adenosine binding to fat cell membrane may reflect the modification of this "R" site or the loss (perhaps via conversion to a "P" site) during membrane preparation. The ability of adenosine to stimulate adenylate cyclase in platelets 93 and mouse neuroblastoma cells ~°23°3 has been shown to be sensitive to the fractionation process. LoNoos et al. 53 reported that detection of "R' site mediated inhibition of hormonally activated fat cell adenylate cyclase could only be demonstrated in the presence of GTP and absence of endogenous adenosine. Detection of adenosine binding to "R" type sites on fat cell membranes may similarly require incubation conditions similar to those described by LONDOS et al. 53.

Curiously, theophylline, but not methylisobutyl xanthine, antagonized adenosine binding to the fat cell membrane 5°. One micromolar theophylline inhibited adenosine binding to fat cell membranes by 14%. Why theophylline antagonizes the binding of adenosine to fat cell membranes while N6-phenylisopropyladenosine has no effect remains obscure. ROSENBLITand LEVY 121 investigated adenosine binding components on plasma membranes of intact fat cells employing a tritiated, ph0toreactive derivative of adenosine, (2-[3H])8-azidoadenosine. Photocatalyzed labeling of fat cells with 8-azidoadenosine resulted in a general incorporation of radioactivity into membrane proteins ~2i. The specifiity of this technique, however, was not addressed and the suitability of this technique for identification of "adenosine receptors" on intact cells or membranes remains to be established. Identification of the receptors mediating the action of adenosine on adenylate cyclase may be possible utilizing the direct radioligand binding technique or flash photolysis labeling described above. Development of high specific activity, radiolabeled "R" site-specific and "P" sitespecific adenosine analogs will facilitate direct binding studies. In the turkey erythrocyte the number of adenosine receptors reported (--500700/cell) 1°~, approximates the reported number

of beta-adrenergic receptors 122. If the density of adenosine receptors is generally as high as that for beta-adrenergic receptors, direct binding studies with radiolabeled adenosine should be possible in most systems. However, studies employing radiolabeled adenosine rather than " R " or "P" site-specific analogs may reflect binding to both of these sites thus complicating interpretation of the data. Adenosine-binding activity has been detected in high-speed supernatant fractions obtained from lysed rabbit erythrocytes 123 and from both rat and mouse 125 liver homogenates. The adenosine binding proteins of the mouse liver 125 and rabbit erythrocyte x23 also bind cyclic AMP. Although adenosine completely inhibited cyclic AMP binding to these components, cyclic AMP had no effect on the binding of adenosine 123'125. The apparent dissociation constant (Kd) for the binding of adenosine to these components ranged between 0.05-0.25/x~ 123-a25. Ytm and TAO123 purifed the adenosine binding protein from rabbit erythrocytes and on the basis of sodium dodecyl-polyacrylamide gel electrophoresis reported that the receptor was composed of a single protein component of -48,000 daltons. UELANDand DOSKELAND125 reported that the adenosine binding component of mouse liver was composed of polypeptides with an approximate molecular weight of 45,000 daltons as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although no enzyme activity was attributed to the soluble adenosine-binding proteins described above, HERSHFIELDand KREDICH 126 r e c e n t l y suggested that these adenosine-binding proteins may, in fact, be S-adenosylhomocysteine hydrolase, the activity of which was not measured in the studies by YUH and T A O la3 o r UELAND and DOSKELAND125. HERSHFIELD and KREDICH 126 identified a cytoplasmic protein of human lymphoblasts and placenta capable of binding adenosine with high affinity (Kd 0.5 ~M) as S-adenosylhomocysteine hydrolase. Furthermore, these authors reported that adenosine binding activity in extracts of mouse liver copurified with this enzyme 126. The role of adenosine in regulating the activity of Sadenosylhomocysteine hydrolase is obscure, but may be an important facet of the immune dysfunction in adenosine deaminase-deficient humans 3~. 165

Conclusion Our understanding of the regulation of adenylate cyclase by adenosine has expanded in the eight years since the original observation by SA~tN and RALL1. A fundamental question is why does adenosine inhibit rather than stimulate fat cell adenylate cyclase activity. We propose that adenylate cyclase exists in three activity states: (i) An inactive state (Eo), (ii) a highly active state (EoTp), and (iii) a third state (EGDp), which is inactive in cells where adenosine stimulates adenylate cyclase but active in cells where adenosine inhibits adenylate cyclase. We postulate that the conversion of the EGDp state to the Eo is ordinarily rate-limiting for activation of adenylate cyclase by hormones and GTP (Figure 5). Adenosine and prostaglandins are postulated to. increase the conversion of ECDp to Eo as originally suggested by BLUME and FOSTER114. This might be a unitary mechanism for regulation of adenylate cyclase by adenosine and prostaglandins. In cells where

adenylate cyclase is activated by these agents the EODp state of the cyclase is postulated to be inactive and cannot be stimulated by either hormones or GTP. Before the inactive adenylate cyclase can be activated it must be converted to the Eo state by an adenosine sensitive process which is postulated to limit the ability of GTP and hormones to activate adenylate cyclase. Both adenosine and prostaglandins of the E series inhibit cyclic AMP accumulation by fat cells78'79 but stimulate cyclic AMP accumulation by cells such as platelets 9. Inhibition of adenylate cyclase by adenosine and prostaglandins occurs in cells where the EGDp state of the cyclase is active. Acceleration by adenosine of the EGDp to Eo transformation results in a loss of activity. This hypothesis can also explain the paradoxical ability of high levels of GTP to reduce fat cell adenylate cyclase activity and the difficulty in demonstrating adenosine inhibition of adenylate cyclase at the R or adenosine site in broken cell preparations. High levels of GTP

CHOLERA TOXIN:

x

HORMONES~E)

(V E RYACTIVE) FAT C E LL ADENYLATE CYCLASE

GTP O (INACTIVE) IN C Y C L A S E S

STIMULATED

TO RE INACTIVE

AND

BY ADENOSINE

CONVERSION

THE EGDP

OF EGDp

GDP (ACTIVE)"

~ADENOSI NE PGE INTERMEDIATE

T O E ° IS R A T E - L I M I T I N G

IS POSTULATED

FOR HORMONAL

ACTIVATION.

Fig. 5. Regulation of fat cell adenylate cyclaseactivityby adenosine and prostaglandins. 166

result in an acceleration of the EGav state (which is highly active) t o EGD P (which is less active in fat cells) state. D e m o n s t r a t i o n of an inhibitory effect of adenosine on adenylate cyclase will only occur w h e n there is an appreciable accumulation of the EGD P state as in the presence of G T P . In the studies of LONDOS et al. 53 this was just the condition in which inhibition of adenylate cyclase by adenosine was noted. W e suggest in conclusion that adenosine facilitates the rate-limiting conversion of EGD P (which is s o m e w h a t active) to the Eo e n z y m e state which is totally inactive. U n d e r the influence of adenosine the EGDP----~Eo conversion is faster than the E o + GTP----~EGTP process, Eo accumulates and e n z y m e activity is thus inhibited rather than stimulated. This model, however, is based solely on speculation and m u c h further study will be required to explore the validity of this and alternative models. T h e w o r k of LEVITZKI and coworkers 1°5'1°6 provides further insight into the m e c h a n i s m of adenosine regulation of adenylate cyclase and similar studies in other systems m a y provide additional support for their proposal that the adenosine receptor is precoupled to the cyclase. Radioligand binding studies, too, m a y provide m o r e detailed description of the adenosine receptor itself, its localization, and m a y permit purification of the receptor. Clearly m a n y new horizons await those investigators studying the action of adenosine on adenylate cyclase.

Acknowledgements This review is dedicated in m e m o r y of DRS. E. WESTERMANN and K. STOCK whose discovery that very low concentrations of N6-phenyl isopropyladenosine m a r k e d l y inhibited lipolysis in adipose tissue was responsible for m u c h of the recent interest in adenosine regulation of fat cell metabolism. T h e authors would also like to express their appreciation to the investigators w h o provided preprints of their w o r k especially DRS. C. LONDOS, W. KUKOVETZ, J. G. DOBSON, G. BURNSTOCK, J. W. DALY, B. B. FREDHOLM, D. M. PATON and J. PR~MONT. W e also thank DR. F. J. MORENO for permission to include the data shown in Figure 2, MRS. S. H. L~ and MR. RICHARD HERT for technical assistance and MRS.

GERL~NDE CELONA for her excellent secretarial assistance.

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