Ethanol and guanine nucleotide binding proteins: a selective interaction. PAULA. L. HOFFMAN'. AND BORIS TABAKOFF. National Institute on Alcohol Abuse ...
Ethanol
and guanine
a selective PAULA
AND
BORIS
nucleotide
key roles in signal
on Alcohol Abuse and Alcoholism, USA
Division
of hormone and neurotransmitter ion channels,
metabolism.
One
member
receptors to adeny-
and polyphosphoinositide of this family of proteins,
G,, appears to represent a specific site of action of ethanol in the central nervous system. Ethanol is often perceived as a nonspecific drug, and its anesthetic effects may in fact arise from relatively nonspecific interactions with cell membrane lipids. However, recent investigations point to a selective effect of low concentrations of ethanol to promote the activation of G,, and thus to enhance adenylate cyclase activity. Ethanol seems to have little or no effect on the function of other identified G proteins. After chronic ingestion of ethanol by animals, or chronic exposure of cells in culture to ethanol, the sensitivity of adenylate cyclase to stimulation by guanine nucleotides and agonists that act via G, is decreased. The mechanism of this change may involve qualitative and/or quantitative alterations in G,, and seems to vary in different cell types. Studies of human platelets and lymphocytes also reveal differences in adenylate cyclase activity between alcoholics and control subjectst The differences are consistent with involvement of G,, and do not appear to reverse upon cessation of alcohol exposure. The results suggest that the plateletand/or lymphocyte
cyclase
system
may provide
a biochemical
genetic predisposition to alcoholism-HoFFMAN, L.; TABAKOFF, B. Ethanol and guanine nucleotide ing proteins: a selective interaction. FASEB
2612-2622,
since ethanol Because plained 2612
IS OFTEN
there
NUCLEOTIDE
of
P. bind-
j
4:
REGARDED
are apparently
.
poly-
as a nonspecific
no specific
receptors
Clinical
and Biological
Research,
Bethesda,
drug,
AND FUNCTIONS BINDING PROTEINS
OF GUANINE (G PROTEINS)
The properties and functions of regulatory G proteins involved in signal transduction have been reviewed in detail elsewhere (e.g., see ref 3), and will be described here only briefly. The G proteins are heterotrimers, consisting of a, f3, and -y subunits. The a subunits, which contain the guanine nucleotide binding site, as well as GTPase activity, vary among proteins and confer on them their specificity of interaction with receptors and effectors. Many of the genes encoding G protein a subunits have been identified, and the a subunits have been cloned (e.g., see ref 4). These experiments have provided the primary structure of various a subunits, and characterization of sites involved in guanine nucleotide binding and in interactions with receptor, effector, and f3/’y subunits (see ref 3). Two G proteins, G5 and G, medi-
ate hormone
1990.
Key Words: G proteins ethanol . adenylate cyclase phosphoinositide metabolism alcoholism . genetics
ETHANOL
PROPERTIES
adenylate
marker
of Intramural
potency of various alcohols had been related to their lipid-water partition coefficients (see ref 1), the membrane hypothesis of ethanol’s actions popularized the notion that ethanol-induced perturbation of the structure of cell membrane lipids can account for the myriad behavioral and biochemical effects of nonanesthetic doses of ethanol (1). A growing number of investigations, however, suggests that specific sites of action for ethanol exist within the cell membrane that might be proteinaceous. We have previously designated such sites as receptive elements (2). The selective interaction of ethanol with certain members of the family of guanine nucleotide binding proteins (G proteins) exemplifies such a receptive element.
binding proteins (0 proteins) play transduction, including the coupling
late cyclase,
proteins:
TABAKOFF
ABSTRACT Guanine
binding
interaction
L. HOFFMAN’
National Institute Ma?yland 20892,
nucleotide
and/or neurotransmitter
stimulation
or in-
hibition, respectively, of adenylate cyclase activity. There are at least two and possibly four forms of the a subunit of G (a5). Two forms of a5 that differ in apparent molecular weight (46- and 52-kDa forms) have been reported to derive from alternative splicing of mRNA for a (see ref 3). Three different G1 proteins (arbitrarily designated G1, G12, and G13) have been identified and found to be independent gene products (see ref 3).
for
as there are for many other psychoactive drugs. the anesthetic effects of ethanol could be exby the law of mass action, and the anesthetic
‘Address correspondence DICBR, 12501 Washington
to: Paula L. Hoffman, PhD, NIAAAI Ave., Rockvile, MD 20852, USA. 0892-6638/90/0004-261
2/$01 .50. © FASEB
It has recently been shown (5) that inhibition of adenylate cyclase in platelets by a2-adrenergic agonists can be blocked by pretreating platelet membranes with an antibody to a2, thus implicating G12 in the inhibition of adenylate cyclase. It has also been postulated that G13 may be a modulator of potassium channel activity (6). Similarly, in addition to coupling stimulatory receptors to adenylate cyclase, G5 has been suggested to regulate the activity of voltage-gated calcium channels in the heart (3). In addition to G5 and G, the brain contains a large quantity of another G protein, designated G0, whose function is still unknown (3), although on the basis of reconstitution studies it has been postulated to play a role in the control of voltage-sensitive calcium channels (7). G proteins have also been implicated in other signal transduction pathways, such as receptor-coupled polyphosphoinositide metabolism, but the identity of these G proteins is still being debated (3). One method for implicating G proteins in an interaction with an effector takes advantage of the fact that the a subunit of some of the proteins can be ADP-ribosylated by bacterial toxins. In the presence of NAD, cholera toxin catalyzes ADP-ribosylation of G, causing persistent activation of adenylate cyclase, whereas pertussis toxin catalyzes ADPribosylat ion of G0 and G1, inhibiting the function of the latter. The ADP-ribosylation sites have been identified as a cysteine residue close to the COOH terminus for G and G0, and an arginine residue in the portion of the protein coded for by exon 7 of the gene for G5 (3). In addition, toxin-insensitive G proteins have been reported (3).
In general, significant structural similarity exists among the a subunits of the 0 proteins (as well as with
other guanine
nucleotide
binding
proteins,
including
rod
and cone transducins, bacterial elongation factor Tu (EFTu), and ros gene products (3)). a5 shows the greatest differences in structure compared with the other characterized G protein a subunits (4). The /3/-y subunits of the various 0 proteins are very similar or identical (3). There are two closely related forms of the f3 subunit, encoded by two genes, and one function of the /3/-y subunit may be to anchor the 0 proteins in the cell membrane (3). Some evidence, however, shows that the f3/y subunit can activate ion channels (8). The -y subunit is the smallest of the three 0 protein subunits, and there is also some evidence for different forms of this subunit (3). In addition, the interaction of a5 with the f3/y subunit of transducin is different from its interaction with jf3/y subunits of other G proteins, and this difference has been attributed to the -y subunit (see ref 3).
The
mechanism
by which
G proteins
modulate
adenylate cyclase activity has been studied in detail (3). This mechanism is based on studies of stimulation of adenylate cyclase by agonists acting at 3-adrenergic receptors. In these systems, agonist interaction with the receptor results in an association of the agonistreceptor complex with the trimeric form of G, which has GDP bound to the a subunit. Interaction with the receptor enhances the clearance of GDP, perhaps by increasing the efficacy of Mg2 to promote this clearance,
ETHANOL
AND
GUANINE
NUCLEOTIDE
BINDING
PROTEINS
and allows binding of GTP to a5. The ternary complex of agonist-receptor G5 displays high affinity for agonist; when GTP binds to a5, the affinity of receptor for agonist is lowered, and the 0 protein dissociates into its component subunits. a5 with GTP bound to it (as*) activates adenylate cyclase. The activation of adenylate cyclase by a55 is terminated by hydrolysis of GTP and reassociation of a5 with f3/y to form the trimeric G protein. The protein-protein interactions within the cell membrane that are involved in agonist-induced activation of adenylate cyclase suggested that this process might be susceptible to the actions of ethanol. This has proved to be the case, but the evidence suggests that the effects of ethanol are not simply a result of nonspecific perturbations of cell membrane lipids and instead may entail specific interactions of ethanol either with particular lipid-protein microdomains or with hydrophobic regions of particular proteins. ETHANOL
AND
ON ADENYLATE Peripheral The
earliest
G PROTEINS:
ACUTE
EFFECTS
CYCLASE
tissue
of the effects of ethanol and other cyclase activity in peripheral tissues demonstrated that ethanol, when used at high concentrations, increased adenylate cyclase activity (see ref 9). However, these assays were performed in homogenates or membrane preparations without the addition of guanine nucleotides. Ethanol at much lower concentrations (approximately 20 mM) was reported to increase cyclic AMP levels in intact cells (lymphocytes, platelets, granulocytes, alveolar macrophages) in which endogenous GTP is expected to be present, and ethanol was also a more potent activator of adenylate cyclase in cell-free preparations of various tissues (adipocytes, pancreas, parotid gland) when guanine nucleotides
alcohols
studies
on adenylate
were present
(see ref 9). In most instances,
ethanol
had
little effect on basal adenylate cyclase activity, but enhanced the response to hormones or guanine nucleotides. These results suggested a role for G proteins in the actions of ethanol on adenylate cyclase, and in recent years a rather detailed description has emerged of
the interaction of ethanol the brain and in cultured neural
with 0 proteins, primarily in cells of both neural and non-
origin.
Brain In initial investigations of the interaction of ethanol with adenylate cyclase in the brain, levels of cyclic AMP in whole brain or large brain regions were measured, and in many but not all cases, ethanol, administered in vivo, was found to lower cyclic AMP levels (e.g., refs 10, 11). This approach, however, is confounded by the varying influences of different neurotransmitters and neuromodulators on cyclic AMP levels (and thus the lack of distinction between direct and indirect effects of
2613
ethanol), and because cyclic AMP levels in brain increase after most methods of (11). Levels may increase differentially in brains of ethanol-treated and control animals. Progress in determining the mechanism of the inter-
action
of ethanol
with adenylate
cyclase
and with G
proteins has come mainly from in vitro studies of intact cells and/or cell-free preparations of brain. The results of these studies have demonstrated a selective and potent effect of ethanol on G5, the stimulatory guanine nucleotide binding protein. Thus, in mouse striatal membranes, ethanol in vitro had little effect on basal (no guanine nucleotides added) adenylate cyclase activity, but a much greater effect in the presence of guanine nucleotides (12). Ethanol also increased dopaminestimulated adenylate cyclase activity (12, 13) without affecting dopamine binding to striatal receptors (13). Substantial effects of ethanol were observed in these studies at a concentration of 75 mM (approximately 300 mg/dI). Ethanol did not alter the efficacy (i.e, EC50) of the nonhydrolyzable guanine nucleotide analog 5-guanylylimidodiphosphate (Gpp(NH)p) to stimulate adenylate cyclase, which suggested that ethanol had no effect on Gpp(NH)p binding to a5 (12). However, in one study, a high concentration of ethanol was reported to enhance the rate of activation (dissociation) of G5 by Gpp(NH)p (14) (although this result was not found in another study) (15). Since ethanol could further increase adenylate cyclase activity in striatal membrane preparations in which the enzyme had been preactivated by treatment with cholera toxin or by preincubation with Gpp(NH)p, it was postulated that ethanol not only affected G5 activation, but also promoted the in-
Figure 1. Sites of action of ethanol within rotransmitter receptor-mediated stimulation activity. In this diagram, neurotransmitter
the scheme of neuof adenylate cyclase (N) interaction with receptor(R) promotes the exchange of GTP for GDP on the a
subunit of 0,, leadingto activation of G,. The activated a subunit of 0,, with GTP bound, interacts with the catalytic unit of adenylate cyclase (AC) to activate the enzyme. Activated forms of receptor, G protein, and AC are depictedas circles; inactiveforms are squares. The evidencesuggeststhat in some brain areas and cultured cells, a primary action of low concentrations of ethanol is to promote the activation of G,, leading to increased AC activity and formation of the low-affinity agonist binding form of the receptor (in red).Ethanol alsoappears to promote the interaction of activated a, with AC (in blue); in certain brain areas, this action may be predominant.
2614
Vol. 4
June 1990
teraction of striatal a55 with the catalytic unit of adenylate cyclase (12, 14) (Fig. 1). This latter action, which was postulated to be the major effect of ethanol in the striatum (12), was not simply the result of ethanol-induced membrane lipid fluidization, as another lipid-fluidizing agent, chloroform, did not share all of ethanol’s actions (12). Similarly, in a study of adenylate cyclase in L6 muscle cells, Rabin et a!. (16) found that other lipid-fluidizing agents (i.e., anesthetics such as halothane) did not act like ethanol, and that ethanol-induced adenylate cyclase stimulation was not well correlated with changes produced by ethanol in membrane fluidity, as measured by fluorescence
depolarization. Studies of ethanol’s effects on cerebral cortical f3adrenergic receptor-coupled adenylate cyclase have provided further support for a specific interaction of ethanol and G5. As in striatal tissue, ethanol increased
guanine
nucleotide
and
isoproterenol-stimulated
adenylate cyclase activity to a greater extent than basal activity in membranes of the cerebral cortex (17). The effect of ethanol on guanine nucleotide and agoniststimulated adenylate cyclase was apparent at a concentration of 50 mM (approximately 200 mg/dl). However, in cortical tissue, ethanol also increased the efficacy
(EC50) of Gpp(NH)p
and Mg2
to stimulate
adenyl#{225}te
cyclase activity. Furthermore, ethanol increased the rate of activation of adenylate cyclase by Gpp(NH)p. Thus, in a sense ethanol acted like an agonist (e.g., norepinephrine) in this system. These data, suggesting a key role for G5 in the actions of ethanol on cortical adenylate cyclase activity, were supported by ligand binding studies. Concentrations of ethanol as low as 20 mM (less than 100 mg/dl) decreased the affinity for isoproterenol of the high-affinity agonist binding form of the receptor (reflecting an effect on the ternary agonist-receptor-G5 complex), but ethanol did not alter antagonist binding (reflecting ligand binding to the receptor per se) (18). Ethanol also promoted the Gpp(NH)p-induced interconversion of high- and lowaffinity agonist binding forms of the receptor. All of these data are compatible with the hypothesis that, in cerebral cortical tissue, a primary site of action of ethanol within the receptor-coupled adenylate cyclase system is G5; ethanol appears to alter the rate of activation (dissociation) of G5 and to enhance the interaction of a with guanine nucleotides (Fig. 1). In this system, as in others (12-14), ethanol also had a relatively minor stimulatory effect on the catalytic unit of adenylate cyclase and on the interaction of as5 and adenylate cyclase (15, 17). Similar to results in the striatum, studies with other lipid-perturbing agents, such as halothane, produced results different from those with ethanol, indicating that changes in the properties of bulk membrane lipids cannot completely account for ethanol’s actions (17). The qualitative and quantitative differences in the effects of ethanol on G5 in striatal and cerebral cortical membranes (e.g., effects on agonist binding; relative effects on a5”-catalytic unit interaction) might be related to the content of the 52- and 46-kDa forms of a5 in neuronal membranes from these
The FASEB Journal
HOFFMAN
AND
TABAKOFF
two brain areas. We have recently observed, by Western blotting techniques, a difference in the proportion of the two (52- and 46-kDa) forms of a5 in mouse striatum and cerebral cortex (ratio of 46:52 kDa forms of a5 in striatum, 2.5; in cortex, 0.8) P. Whelan, P. L. Hoffman, B. Tabakoff, manuscript submitted). Although there is evidence that both of these forms of a3 can interact with adenylate cyclase, the 52-kDa form has been reported to be more rapidly activated by gua-
(J.
nine nucleotides Neural
cells
(see ref 3).
Mechanism
in culture
Studies of ethanol-adenylate tured cells for the most part
from experiments
cyclase interactions in culhave supported the findings
using brain membrane
preparations.
For example, ethanol at 75-100 mM increased basal, and to a greater extent, guanine nucleotideand fluoride-stimulated adenylate cyclase activity, in membrane preparations of wild-type (WT)2 S49 lymphoma cells as well as in preparations from the mutant Unc (in
which G5 and adenylate
cyclase are not coupled
to the
f3-adrenergic receptor). However, ethanol had little effect on adenylate cyclase activity in membrane preparations from the cyC mutant, which has no a8 (14). In
the WT S49 cells, as in cerebra! cortex, ethanol had a greater effect on agonist than antagonist binding to the /3-adrenergic receptor (19). In several other cultured cell systems, including N1E-115, NG 108-15, and rat pineal cells (20-22), ethanol has been reported to increase agonist-stimulated cyclic AMP formation, although the mechanism of the effect was not investigated. In NG 108-15 cells grown under conditions of reduced cell division, ethanol increased adenosinestimulated cyclic AMP production at concentrations as
low as 25 mM (20). We have also found ethanol production
culture On
creases
enhances agonist-stimulated and melatonin release from
that 25 mM
cyclic AMP pineal glands in
(23). the
other
adenylate
hand,
although
cyclase activity
ethanol
generally
in cell-free
in-
prepara-
tions and increases cellular levels of cyclic AMP in whole cell assay systems, other regulatory mechanisms that control cyclic AMP levels can influence the response of a cell to ethanol. This phenomenon was demonstrated in our studies of PCI2 cells (24). In
membrane preparations from two different subclones of PC12 cells, ethanol (50 mM) increased adenosine and other agonist-stimulated adenylate cyclase activity. However, although ethanol enhanced the cyclic AMP response to agonist in intact cells of one subclone, ethanol reduced this response in cells of the other subclone. Similarly, in a study of pineal cells in culture, it was found that ethanol enhanced the cyclic AMP response to isoproterenol, vasoactive intestinal peptide (VIP), and, to a lesser extent, phenylephrine. However, ethanol reduced the large increases in cyclic AMP production produced by concurrent addition of VIP and phenylephrine to the medium (22). It has also been reported that the ethanol-induced increase in cyclic AMP levels in intact N1E-l15 cells is not commensurate
ETHANOL
AND
GUANINE
with the effects of ethanol on adenylate cyclase activity in membrane preparations (21). A likely explanation for these discrepancies is that in intact cells, feedback mechanisms (including other second messenger systems) tend to stabilize cyclic AMP levels, and may influence the observed effects of ethanol. Nevertheless, transient increases in adenylate cyclase activity and cyclic AMP production by ethanol can have significant physiological effects, as observed with intact pineal glands in culture (23).
NUCLEOTIDE
BINDING
PROTEINS
of action
of ethanol
Differential responses of various cell types to ethanol, however, cannot be easily explained by invoking effects on other, related G proteins. A striking example of the selectivity of ethanol’s effects on signal transduction mechanisms in the central nervous system (CNS) is the observation that ethanol has little effect on agonistinduced inhibition of adenylate cyclase activity, which involves G1. The mechanism of G1-mediated inhibition of adenylate cyclase activity is still not completely resolved. In several instances, addition of Gia (ai) to reconstituted systems has produced little inhibition of adenylate cyclase, and in pertussis toxin-treated membranes, the addition of $/y subunits of G proteins
produced
more
inhibition
than did addition
of a (see
ref 25). These data supported the hypothesis that inhibition of adenylate cyclase occurs because activation and dissociation of G1 result in release of 13/’y subunits that can interact with a8. However, in cyc cells, which lack G5, agonist-induced inhibition of adenylate cyclase activity can be demonstrated (26, 27). In addition, a study (28) has shown that in a pituitary tumor cell line, quantitative cholera toxin-catalyzed ADPribosylation of a, which should reduce the affinity of a8 for f3/y, did not alter the ability of somatostatin to inhibit adenylate cyclase (28). Thus, a direct role for a, in adenylate cyclase inhibition remains a possibility. If ethanol increased G activation, similar to its effect on G5, and if either a1 or f3/y mediates adenylate cyclase
inhibition,
then one would expect
to observe
increased
agonist-induced inhibition of adenylate cyclase in the presence of ethanol. In fact, in striatal tissue, ethanol in vitro had no discernible effect on opiate-induced inhibition of adenylate cyclase activity, even though high concentrations of ethanol can alter ligand binding to opiate receptors (29, 30). Similarly, ethanol did not alter inhibition of striatal adenylate cyclase by acetylcholine (31). One possible explanation for an inability of ethanol to alter G-mediated inhibition of adenylate cyclase could be the high affinity of a1 for Mg25 (micromolar range) (32). For example, if ethanol acts by increasing the efficacy of Mg25 to promote the dissociation G, no effect of ethanol on this dissociation
2Abbreviations:
VIP,
vasoactive
intestinal
peptide;
CNS,
central
nervous system; P1, polyphosphoinositide; NE, norepinephrine; PGE, prostaglandinE,; PIA, phenylisopropyladenosine; 6-OHDA, 6-hydroxydopamine; WT, wild-type;Gpp(NH)p, 5-guanylylimidodiphosphate.
2615
would be observed when assays are carried out in the presence of saturating Mg2 concentrations. However, we found no effect of ethanol on carbachol-induced inhibition of adenylate cyclase activity in several brain areas, even when a wide range of Mg2 concentrations was used (33). Direct evidence that ethanol does not act via G was obtained in studies of WT and cyc S49 cells, in which ethanol-induced inhibition of forskolinstimulated adenylate cyclase activity (which had previously been observed in other cells at high concentrations of ethanol) (34) was not affected by treatment of cells with pertussis toxin, a treatment that blocked the inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides (19). There is one report that ethanol reduced the inhibition of cerebral cortical adenylate cyclase by adenosine (35). This effect of ethanol was observed only “under conditions favoring activation of G5,” i.e., low GTP and high Mg2. Although the authors suggested an effect of ethanol on G1, such an effect is not necessary to explain the data. That is, if inhibition of adenylate cyclase activity is mediated by the interaction of f3/-y with a5, and if ethanol enhances the activation (dissociation) of G5 as proposed, then G-mediated adenylate cyclase inhibition might seem to be reduced in the presence of ethanol. ETHANOL ON
AND
G PROTEINS:
POLYPHOSPHOINOSITIDE
ACUTE
EFFECTS
METABOLISM
Another second messenger system that, in a number of instances, has been shown to be regulated by a G protein is receptor-coupled polyphosphoinositide (P1) metabolism (36). The G protein (or proteins) reported to couple receptors to phosphoinositide phosphodiesterase (phospholipase C) can be pertussis toxin-sensitive or insensitive, and the identity of this G protein (or proteins) has not been established. As for G1-mediated inhibition of adenylate cyclase, ethanol at pharmacologically relevant concentrations seems to have little effect on receptor-coupled P1 metabolism. For example, in slices of mouse cortex, hippocampus, or striatum, 500 mM ethanol was required to significantly decrease the efficacy of carbachol to stimulate PT metabolism, and ethanol had no effect on the response to norepinephrine (NE) (37). In slices of rat cortex, hippocampus, hypothalamus, or striatum, ethanol was reported to inhibit NE-stimulated P1 metabolism, but consistent results were obtained only at concentrations
of 300-500
Vol. 4
ETHANOL, G, ACUTE EFFECTS
June 1990
AND
CALCIUM
CHANNELS:
It has been proposed that G5 may not only couple stimulatory receptors to adenylate cyclase, but may also directly activate dihydropyridine-sensitive calcium channels in heart and skeletal muscle membrane preparations (44). When the different forms of a8 that arise from alternative splicing of a mRNA were synthesized in Esc/zerichia coil by recombinant DNA techniques, both forms were found to increase adenylate cyclase activity and to activate cardiac calcium channels (3). The activity of voltage-gated calcium channels in cardiac and skeletal muscle is also regulated indirectly by G5 through receptor-coupled stimulation of cyclic AMP
TABLE
1. G protein functions
G prOtein’ Identity G,
Adenylate
G,2 Transducin G011
and
Stimulation
Cardiac voltage-sensitive calcium channel Adenylate cyclase (inhibition) cGMP phosphodiesterase Adenylate cyclase
NT
and/or
Potassium
?
G1
Polyphosphoinositide metabolism
G0
Voltage-sensitive calcium channels7
G,
? ‘Five newly discovered described
to ethanol
known
ethanol
G,,
The FASEB Journal
function
(stimulation)
Identity
been
Response
cyclase
G13 (Gk)
recently
and effects of ethanol
Effector
mM (38).
The effect of ethanol has also been examined in peripheral cells, in some instances under conditions (i.e., permeabilized cells) in which stimulation by guanine nucleotides can also be measured. Although ethanol (50 and 100 mM) was reported to decrease the efficacy of thrombin to stimulate P1 metabolism in platelets of rabbits (39), in human platelets and rat hepatocytes, only very high (200-400 mM) concentrations of ethanol were found to increase guanine nucleotide-stimulated and basal phospholipase C activity, respectively (40, 41). 2616
Thus, ethanol, even at concentrations that exceed the range of in vivo pharmacological relevance, appears to have only minor effects on G protein-mediated inhibition of adenylate cyclase or on P1 metabolism. Overall, the data support the hypothesis that G5-mediated stimulation of adenylate cyclase activity is a signal transduction system that is uniquely sensitive to ethanol, despite considerable similarity in the structure and function of various G proteins (4). This sensitivity may derive from particular properties of 0s itself (e.g., lack of myristoylation) (42) or from lipid-protein interactions that are characteristic of G5 in certain environments (e.g., these interactions may vary in different brain areas). As indicated in Table 1, however, the family of identified G proteins is growing rapidly, and the effect of ethanol on the function of other G proteins, particularly those believed to regulate ion channel function (see below), remains to be investigated.
by low
concentrations
None NT NT
function
not
channel2
known NT NT None (inhibition or stimulation only by high ethanol concentrations) NT
NT G protein (43).
a subunits NT,
not
in the brain
have
tested.
HOFFMAN
AND
TABAKOFF
formation and phosphorylation of the calcium channel protein (45). Based on the data indicating that ethanol selectively affects G, it might be postulated that ethanol would enhance voltage-dependent calcium currents in the heart, both by increasing agonist-activated adenylate cyclase activity and by promoting the direct action of on the channels. However, there are few or no data available regarding the effects of ethanol on calcium currents or influx in cardiac tissue (Table 1). Most work on the effects of ethanol on voltage-dependent calcium flux has been done in synaptosomal preparations from various brain areas and on neuronal cells in culture, where ethanol, at relatively low concentrations, inhibits fast-phase calcium influx through voltage-gated channels (46). In neuronal cells, a number of neurotransmitters and neuromodulators (e.g., GABA, dopamine, opioids, acetylcholine, adenosine, somatostatin) also inhibit calcium currents, and a pertussis toxin-sensitive G protein is involved in this inhibition (45). Although the identity of this protein has not been established, the brain and many other systems studied contain large quantities of G0, and this 0 protein has been suggested to play a role in the inhibition of calcium flux by various agonists (6). Thus, it seems possible that in the brain, ethanol could also affect the function of G0, and this possibility remains to be tested. On the other hand, it has been suggested that the primary effect of ethanol in synaptosomal preparations is to increase the release of calcium from intracellular stores, which then results in a decrease in depolarization-induced calcium influx (47). Therefore, ethanol may affect calcium influx in brain via an indirect mechanism, and a comparison of the effects of ethanol on voltage-dependent calcium influx in heart and brain would be of interest.
ETHANOL ON
AND G PROTEINS:
ADENYLATE
If ethanol
acutely
increases
cyclase activity
the
stimulation
in vivo, it might
from ethanol-fed
of brain
be expected
mice restored
dopamine-
stimulated adenylate cyclase activity to levels seen in control mice (49). Thus, normal activity was dependent
ETHANOL
AND GUANINE
NUCLEOTIDE
BINDING
of ethanol.
adenylate
cyclase
The
changes
activity
in dopamine-
reversed
rapidly
after ethanol withdrawal, which may explain why some investigators did not observe changes in response to chronic ethanol treatment (52). There was no change in fluoride-stimulated adenylate cyclase activity in striatal tissue of ethanol-treated rats (49). Chronic ethanol exposure also affects NE-stimulated adenylate cyclase activity in the cerebral cortex (50, 53-55). Early studies with mice and rats showed a decrease in this activity at the time of ethanol withdrawal (54, 55), and this change was paralleled by changes in antagonist binding to cerebral cortical j3adrenergic receptors (56). Later, more detailed studies have shown that in mice chronically fed ethanol in a liquid diet, there was no change in basal cerebral cortical adenylate cyclase activity or in activity of the digitoninsolubiized catalytic unit of adenylate cyclase, but at the time of ethanol withdrawal, the response of adenylate cyclase to stimulation by guanine nucleotides, isoproterenol, vasoactive intestinal peptide, and forskolin (which acts on the G5-adenylate cyclase complex), as well as by ethanol added in vitro, was significantly decreased (53, 57). As in the striatum, the response to fluoride was unchanged (P. T. Nhamburo, P. L. Hoffman, and B. Tabakoff, unpublished results). In addition to the changes in adenylate cyclase activity, binding studies showed a loss of high-affinity 3-adrenergic receptor agonist binding, with no change in j3-adrenergic receptor antagonist binding (58). This change, in combination with the decreased sensitivity of adenylate cyclase to various agonists and to agents acting through G, is indicative of an uncoupled receptor-adenylate cyclase system, comparable to changes that occur during heterologous desensitization (48). The data are consistent with a qualitative or quantitative defect in the function of brain G5 after chronic treatment of animals with ethanol.
Neural
that there would be an adaptive change (desensitization) in receptor-coupled adenylate cyclase activity after chronic ethanol exposure or ingestion. Because the primary site of action of ethanol appears to be G, such an adaptation could be similar to changes that occur during agonist-induced heterologous (as opposed to homologous) desensitization (48). In striatal tissue of mice and rats, dopamine-stimulated adenylate cyclase activity was reduced after chronic ethanol exposure (49-51), and in rats the ability of GTP to modify apomorphine binding to striatal tissue was also reduced (51). In vitro addition of ethanol to striatal membrane
preparations
stimulated
EFFECTS
CHRONIC
CYCLASE
Brain adenylate
on the presence
PROTEINS
cells in culture
The effect of chronic ethanol exposure on receptorcoupled adenylate cyclase systems has also been examined in cultured cells. In neuroblastoma N1E-115 cells, ethanol acutely potentiated cyclic AMP production in response to prostaglandin E1 (PGE). When the cells were incubated in 100 mM ethanol for 7 days, the response to PGE was significantly decreased, and addition of ethanol to the assay medium restored the response to the control value (59). A similar change was observed in NO 108-15 cells. Ethanol acutely enhanced cyclic AMP stimulation in response to an adenosine analog (phenylisopropyladenosine, PIA). After chronic exposure of the cells to ethanol (200 mM), a decreased response to PIA was observed, and this decreased response could be returned to the control level by addition of ethanol in vitro (20). The change in these cells, similar to that in the cerebral cortex of ethanol-fed mice, appeared to resemble heterologous desensitization, since the response to agonists acting at both PGE and adenosine receptors was reduced after chronic ethanol exposure (60).
2617
MECHANISM ETHANOL
OF
THE
CHRONIC
ACTION
OF
It was proposed that the mechanism of the ethanolinduced change in adenylate cyclase in NG 108-15 cells involves a decrease in the synthesis of a3, as both the quantity of mRNA for a5 (measured by Northern and slot blots) and the quantity of a3 protein (measured by Western blots) were decreased in membranes of cells that had been chronically exposed (48 h) to ethanol (100 mM). Also, the activity of a5 extracted from ethanolexposed cells was lower than that from control cells, as estimated from reconstitution assays in S49 cyC cells (60). The decrease in all parameters measured was 25-30%, which suggested that in NG 108-15 cells, the quantity of a5 is the rate-limiting step in activation of adenylate cyclase. In the NG 108-15 cells, there was no change in a. In other cell lines, however, the mechanism underlying ethanol-induced changes in the response of adenylate cyclase to agonists may vary. For example, exposure of NIE-1l5 cells to 200 mM ethanol for 48 h produced a decreased cyclic AMP response to PGE and PTA but not to cholera toxin. In this cell line, a large (threefold) increase in a was found (by Western blot analysis). If the cells were incubated for a longer time with ethanol, however, a 60% decrease in a3 was also observed. In a third cell line, N18TG2, ethanol exposure had no significant effect on agonist-stimulated cyclic AMP production or on the quantity of either G protein (61). These studies emphasize the fact that different cell types can respond to chronic ethanol exposure in different ways, and even if the final functional change (e.g., decreased stimulation of adenylate cyclase) is the same, the mechanisms underlying the change may vary. In fact, recent studies have suggested that both acute and chronic effects of ethanol on cyclic AMP levels in NG 108-15 cells may be attributed to an ethanol-induced accumulation of extracellular adenosine (62). Whether such a mechanism applies to other cell types remains to be investigated. Another example of this diversity of responses is the report that chronic exposure of PC12 cells to ethanol did not alter agonist or guanine nucleotide-stimulated adenylate cyclase activity, but increased the sensitivity to ethanol added in vitro (63). If individual cell types can respond differentially to the chronic presence of ethanol, it is perhaps not surprising that different areas of the brain would show differential changes after chronic, in vivo ethanol ingestion by animals, and certain of these changes may differ from those seen in transformed cells in culture. As discussed above, the ethanol-induced changes in cerebral cortical adenylate cyclase activity were compatible with a quantitative or qualitative change that decreased the activity of a3. Measurement of a5 by Western blot analysis, using antibodies raised against a synthetic peptide with the COOH-terminal sequence of a5 (64), did not reveal any significant decrease in either form of a (46 or 52 kDa) in cerebral cortical tissue of mice that had ingested ethanol chronically U. P. Whelan, P. L. Hoffman, and B. Tabakoff, unpublished results). On 2618
Vol. 4
June 1990
the other hand, there was a significant and selective decrease in cholera toxin-induced [32P]ADP-ribosylation of a protein from cortical membranes of the ethanol-fed mice that migrated like a5 on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (57). These data are consistent with the possibility that, in the cerebral cortex, chronic ethanol exposure results in a posttranslational modification of a3 that alters its interaction with receptor and effector, and also alters the ability of the protein to be ADP-ribosylated. The ribosylation site, and the domains within the protein that are involved in binding of guanine nucleotides, as well as in receptor, /3/-y subunit, and effector interactions with a are becoming known (3), and so future studies should be aimed at examining possible modifications of these sites. Adenylate
cyclase
activity
in all brain
regions
does
not respond identically when animals are fed ethanol chronically. Changes in /3-adrenergic agonist-stimulated adenylate cyclase activity and a loss of high-affinity agonist binding were found in hippocampus as well as cerebral cortex of ethanol-fed mice (65). However, no change in response to agonist was found in mesolimbic or cerebellar tissue of these animals (49, 65). A lack of change in cerebellar adenylate cyclase of rats after chronic ethanol exposure has also been reported (52). This regional variation may reflect differences in the properties of G5 or G5-adenylate cyclase coupling or in the lipid microdomains surrounding the proteins. .A more detailed examination of the brain regional differences was obtained by an autoradiographic examination of high-affinity forskolin binding in the brains of mice chronically fed ethanol (66). The binding observed at nanomolar concentrations of forskolin reflects the interaction of forskolin with the a5-adenylate cyclase complex (see ref 66). Decreased basal and Gpp(NH)p-stimulated
forskolin
binding
were
observed
in many but not all areas of the brain of ethanol-fed mice (Fig. 2). The results in appropriate brain areas agreed with the studies of adenylate cyclase activity and agonist binding. Although chronic ethanol exposure produced increases in a’ in some cultured cell lines, there was no change, after chronic ethanol ingestion, in pertussis toxin-catalyzed ADP-ribosylation of mouse cerebral cortical membrane proteins or in a1 as measured by Western blotting. Furthermore, in the striatum, we found no change in opiate-induced inhibition of adenylate cyclase activity in mice fed ethanol chronically (29). Chronic ethanol ingestion also had little effect on agonist-stimulated polyphosphoinositide metabolism in the brain (37, 67), and the changes that were observed could be attributed to alterations in receptor number rather than to G protein modification (37). Results from studies of the brain support the postulate that chronic ethanol ingestion produces a regionspecific alteration in the properties and function of G5, while having little or no effect on other identified G proteins. A determination of the mechanism (or mechanisms) underlying this selective effect of ethanol may provide insight not only into the mode of action of ethanol, but also into the changes underlying heterolo-
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pretreated with 6-hydroxydopamine (6-OHDA) to deplete the level of norepinephrine in the brain, they did not develop functional tolerance to ethanol after chronic ethanol ingestion (see ref 46). However, daily intracerebroventricular injections of forskolin during the period of ethanol ingestion overcame the 6-OHDAinduced block of tolerance development (68). Thus, the changes that occur in agonist-stimulated adenylate cyclase activity as a result of ethanol ingestion might be related to the initiation of ethanol tolerance and/or physical dependence. Changes in adenylate cyclase activity may also be associated with cognitive deficits seen after chronic alcohol consumption in humans, because, for example, norepinephrine-stimulated adenylate cyclase activity has been suggested to be important for long-term synaptic potentiation, a potential mechanism for memory. A postmortem study of human brains revealed a loss of high-affinity binding sites for the 13-adrenergic agonist, isoproterenol, in the cerebral cortex of alcoholics. As in the animal studies, no difference was seen in the cerebellum (69). In this study, alcoholics were divided into two groups: those who had measurable blood alcohol levels at the time of death, and those who did not. The difference in isoproterenol binding was observed only in the former group, suggesting that, as in animals, the alteration in brain G5 in humans is reversible after a period of abstinence.
CONTROL
CLINICAL A MARKER Adenylate
platelets
ETHANOL-FED of high-affinity forskolin binding in the ethanol-fed mice. C57BL/6 mice were fed ethanol in a liquid diet for 7 days (lower panel) or control diet (top panel). [3H]Forskolin binding was assessed by quantitative autoradiography and found to be lower in most brain areas of ethanolfed mice. This finding was compatible with a quantitative or qualitative change in a, in brains of the ethanol-fed mice. Reprinted with permission from ref 65. Figure
2. Autoradiograph
brain of
control
and
gous desensitization
and the pathways
G protein
in various cell types.
function
PHARMACOLOGICAL ETHANOL-G PROTEIN
for regulation
SIGNIFICANCE INTERACTION
of
OF THE
With respect to the possible pharmacological significance of ethanol-induced changes in G5 function, it has been reported that noradrenergic stimulation of brain adenylate cyclase activity is necessary for the development of functional tolerance to ethanol (68). When mice were ETHANOL
AND
GUANINE
NUCLEOTIDE
BINDING
PROTEINS
STUDIES: ADENYLATE FOR ALCOHOLISM cyclase
activity
and lymphocytes,
CYCLASE
can also be readily
which
would
AS
assayed
in
be accessible
tissues for use in clinical testing procedures. In a study of 10 alcoholics and matched controls, basal and adenosine receptor-stimulated cyclic AMP levels were reduced in the lymphocytes of alcoholics, and there was also a reduction in ethanol-stimulated cyclic AMP accumulation (70). In a larger study of alcoholics and matched controls, adenylate cyclase activity in platelet membranes was measured (71). There was no change in basal adenylate cyclase activity, but stimulation of adenylate cyclase activity by Gpp(NH)p, fluoride ion (in contrast to the results in the brain), and PGE was significantly reduced in the alcoholics (71). The differences in platelet adenylate cyclase activity were not associated with age, race, smoking, or illicit drug use, and there was no significant correlation with the duration of problems with alcohol. More recently, these results have been confirmed in platelets from several other populations of alcoholics and controls (72). In the initial study of platelet adenylate cyclase, the observed changes were long-lasting, with lower fluoridestimulated adenylate cyclase activity observed in platelets of alcoholic subjects who had reported abstention from tions result
alcohol for 1-4 years. in platelet adenylate of chronic ethanol
Thus,
cyclase ingestion,
although
activity they
the
could
altera-
be a
apparently
reversed much more slowly after cessation of alcohol consumption than changes seen in the brain tissue of alcoholics (69). Alternatively, it is possible that the 2619
long-lasting
differences in platelet adenylate cyclase acinherent characteristics of alcoholics. Some circumstantial evidence for this possibility was obtained in a study of Swedish alcoholics, where a factor that significantly influenced an individual’s platelet
tivity
represent
adenylate
cyclase
activity
was the
number
of first-
relatives who were alcoholics (72). There is also evidence that the lower cyclic AMP levels in lymphocytes of alcoholics represent a genetically influenced trait. When lymphocytes obtained from alcoholics and control subjects were kept in culture in the absence of ethanol for four to six generations, differences in basal cyclic AMP levels were found, and the lymphocytes originally obtained from the alcoholics were more sensitive to the effect of chronic ethanol exposure on cyclic AMP levels that those from controls (73). The role of degree
genetic
factors in influencing
adenylate
cyclase activity
in platelets and lymphocytes, and the possible use of adenylate cyclase as a marker of a genetic predisposition to alcoholism, are currently under investigation. The differences in platelet adenylate cyclase activity in alcoholics cannot be adequately explained by quantitative differences in a8. Initial studies U. P. Whelan, P. L. Hoffman, and B. Tabakoff, unpublished results) in which a3 was quantitated by slot blots did not reveal a significant reduction in the amount of a5 coincident with lower fluoride-stimulated adenylate cyclase activity. On the other hand, there has been a preliminary report of a decreased amount of a3 in lymphocytes of alcoholics (I. Diamond, communication at conference of Research Society on Alcoholism, June 1989); further work is needed to clarify the differences between alcoholics and control subjects. CONCLUSIONS The interaction of ethanol with G proteins provides a demonstration of the specificity of action of ethanol in the CNS. The finding that chronic ethanol ingestion produces a brain region-specific change in function of a particular G protein, which is one member of a family of similar proteins, suggests a mechanism by which ethanol may exert certain pharmacological effects, including the development of tolerance and dependence, as well as neurotoxicity reflected by cognitive deficits. The characteristics of G5-mediated signal transduction may not only provide an indicator of chronic ethanol consumption, but may also allow for
development alcoholism.
of markers
for a genetic
predisposition
to
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67. Gonzales, R. A., and Crews, F. T. (1988) Effectsof ethanol in vivo and in vitro on stimulated phosphoinositide hydrolysis in rat cortex and cerebellum. Alcoholism: Clin. Exp. Res. 12, 94-98 68. Szab#{243}, 0., Hoffman, P. L., and Tabakoff, B. (1988) Forskolin promotes the development of ethanol tolerance in 6-hydroxydopamine-treated mice. Lfe Sci. 42, 615-621 69. Valverius, P., Borg, S., Valvenius, M. R., Hoffman, P. L., and Tabakoff, B. (1989) fl-Adrenergic receptor binding in brain of alcoholics. ExpeL NeuroL 105, 280-286 70. Diamond, I., Wrubel, B., Estnn, W., and Gordon, A., (1987) Basal and adenosine receptor-stimulated levels of CAMP are reduced in lymphocytes in alcoholic patients. Proc. NatL Acad.
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Sci. USA 84, 1413-1416 71. Tabakoff, B., Hoffman, P. L., Lee, J. M., Saito, T, Willard, B., and De Leon-Jones, F. (1988) Differences in platelet enzyme activity between alcoholics and nonalcoholics. New EngL j Med. 318, 134-139 72. Tabakoff, B., and Hoffman, P. L. (1989) Genetics and biological markers of risk for alcoholism. In Genetic Aspects of Alcoholism (Kiianmaa, K., Tabakoff, B., and Saito, T, eds) pp. 127-142, The Finnish Foundation for Alcohol Studies, Helsinki 73. Nagy, L. E., Diamond, I., and Gordon, A. (1988) Cultured lymphocytes from alcoholic subjects have altered CAMP signal transduction.Proc. NaIL Acad. Sci. USA 85, 6973-6976
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