The hepatic α1-adrenoceptor

TIPS

38O

The hepatic'

I

II

II I

s-adrenoceptor

George Kunos

Departmentsof Pharmacology& Therapeutic~and MedMne, McGillUnilwrsio'.Montreal,Quebec, Canada. l'he nature of the hepatic wadrenoceptor has been described only relatively recen@; originally it was thought to represent a septsate class of 'metabolic" adrenoceptor. George Kunos discusses this problem of clas~fication, and how it has been overcome in this article. The quantitative analysis of drug-receptor interactions in the liver has been complicated by the ability of liver cells to rapidly take up mad metabolize drugs. When using very dilute hepatocyte suspenskms, which minimizes this complication, the glycogenolytic effect of catecholamines in the adult, male rat is found to be mediated by aradrenoceptors, which have pharmacological properties identical to those of arreceptors in o,:her tissues. In-vitro studies of the isolated, solubilized a r receptor of rat liver have/dentified several proteins that contain the ligand binding site. O f these, a protein with a tool. wt of 80 000 is most likely the complete receptor binding subunit, from which lower tool. wt fragments may be generated by proteolysis, arAdrenoceptor responses of the liver are mediated by a calciumsensitive cascade triggerea by a transient rise in cytosolic calcium, which is followed by extrusion of calcium from the cells. T~,,e nature of the link between receptor activation and calcium mobilization from intracellular pools is not quite clear, and in the liver the role of phosphoinositide breakdc~,wn has not yet been definitely settled. It is also unclear whether or not hepatic o~:rreceptors are regulated by guanine nucleotides. A selective reduction in the a t-~drenoceptor response of the rat liver is associated with a reciprocal increase in [3-reo~'ptoractivity in a number of conditions, many of which represent a lower level of cellular differentiation. Such changes can develop rapidly and may be mediated by changes in the activity of membrane phospholipase A 2.

Cyclic AMP, the second messenger of I~. adrenergic responses, was discovered in a study of the effects of adrenaline on hepatic gly~genolysis. It is therefore ironic that, until recently, hepatic adrenergic receptors could not be easily classified as a or 13. At least part of this difficulty arose because of attempts to analyse the effects of catecholamines in intact animals. Catecholamine-induced hyperglycemia is a complex response involving not only direct effects on the liver, but also effects on insulin and glucagon secretion by the pancreas, glucose uptake and lactate production by muscle, iipolysis in adipose tissue as well as other minor effects. Another confounding factor is that the nature of the adrem.~e,.ptor involved in liver glycogenolysis appears to vary not only with species, but also with the age, sex and physiological condition of the animal, as discussed below. Although Sutherlar, d and his coworkers realized that in some species adrenaline may activate phosphorylase through a cAMP-independent mechanism, the role of a-adrenergic receptors in this effect was only documented later, in studies in the perfused rat liver. The

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September 1984

generally used concentration (40 mg wet cell weight per ml of buffer) most of the radioactivity disappears from the medium within the first rain (Fig. IA). The secondary rise in radioactivity is due to the release of inactive metabolite(s), as indicated by the finding that the cell-free medium removed at 15 rain had no ablocking activity when tested in isolated rat aortic strips. Interestingly, uptake of ['~Hladrenaline by the cells is much slower (Fig. IB). Since receptor inhibition requires prolonged incubation with antagonists, while activation of phosphorylase by agonists is maximal within 20 s, this may explain why antagonists but no', agonists have been paradoxically ineffective in liver preparations with a high tissue to medium ratio. Crucial for further quantitative studies was our finding that depletion and inactivation of antagonists could be minimized by using very dilute suspensions of hepatocytes (Fig. 1). When [3H]POB was incubated with cell suspensions of 1 mg wet weight m l - I the cell-free medium removed at 15 rain reduced the maximal noradrenalineinduced contraction of rat aortic strips by 32%, which was similar to the block caused by POB preincubated with buf10C

possibility that this a-receptor-mediated g]ycogenolysis was secondary to the hypoxia caused by hepatic vasoconstricN tion was discounted when similar aadrenergic effects were documented in liver slices or in isolated hepatocytes. However, difficulties in classification still remained: unusually high concentrations of a-adrenergic antagonists were required to inhibit the hepatic effects of catecholamines, and the relative effectiveness of different antagonists was not the same as for other, areceptor-mediated events. Thus, it was 60 suggested that hepatic a-receptors were atypical, or that they represented an entirely separate class of 'metabolic' ;~'enoceptors. A solution to this problem first emerged when Haylett and .~enkinson suggested that the low potency i .... 1"o (, of a-receptor antagonists in the liver may be due to a saturable, high capacity Fig. I. Effectof concentrationof rat livercells on uptake system that effectively removes free lisand concenwmion in the medium, tow concentrations of the drugs from A: [JH]phenoxybenzamine (3 nM) was incubated the medium. Experiments in my labora- witha concentrated(40mg wetcellweightper ml of or dilme (1 mg ml- l)hepal(~te smix'mion tory have provided direct evidence for buffer) o~3~C, underan aunosphereof 5% C0:,195%02. 'this suggestion I. When [aH]phenoxy- At varinm times, aliquo~ were removed, rapidly benzamine ([31-1]POB), a radiolabelled centrifugedand theradioactivityin the medium was a-receptor antagonist, is incubated with determined. N = 3. B: Similar expe~vnents with isolated rat liver cells suspended at the f n l ~ O ,~). N = J.

!

a

1" ~

,

, (~pOe.

eM

38 !

TIPS - September 1984

fir only. Further studies with such dilute cell suspensions provided Schild-plots for various reversible a-blocking drugs with slope factors near unity 2, and the pA2 values for blocking adrenalineinduced phosphorylase activation were not different from the pA2 values of the same drugs for blocking 'classical' areceptor responses in various smooth muscle preparations (Table I). This dispels the notion of a special kind of hepatic a-receptor. The more than 3 000-fold difference between the inhibitory potency of prazosin and yohimbine indicates that glycogenolysis is mediated by the atreceptor subtype. Accordingly, clonidine was found to have very low potency as an agonist, but it was reasonably potent (pA2 = 6.3) as a competitive inhibitor of the effect of adrenaline. Furthermore, the finding that very low concentrations of POB reduced the maximal response to adrenaline without a significant rightshift of the dose-response curve t indicates the absence of 'spare' at-receptors for phosphorylase activation in the rat liver, which is similar to the situation with other a-receptor systems. Molecular properties of the a t - r e c t o r of rat liver The well defined pharmacology of its adrenergic response, the convenience with which drug effects can be measured in hepatocytes, and the unusually high density of cq-receptors (0.5-1.0 pmoi rag-t membrane protein), identified by the subsite selective figand ([3H]prazosin), make the rat fiver an ideal tissue for exploring the molecular nature of atreceptors. Several groups have attempted to isolate and characterize the at-receptor binding protein from rat liver. The approaches used were covalent labelling of the cq-receptor ~bllowed by SDSpolyacrylamide gel electrophoresis (SDSPAGE) t'3'4, or purification of solubilized receptors by affinity chromatography 5. We have justified the use of the irreversible ligand, [3H]POB (specific activity: 45-55 Ci retool-l) by demonstrating its high potency (half-maximal block and binding at 1 riM) and Otrselectivity in the rat liver t, When used at concentrations of 0.5-1 riM, the binding of [3H]POB is unaffected by relevant concentrations of c~2-or ~:adrenergic, serotonin, histamine H t or muscarinic antagonists ~. This contradicts the notion of notorious nonselectivity of POB, based on findings where much higher concentrations of this alkylating agent have been used. When liver plasma membranes are prepared in the presence of protease

TABLE i. Comparison of the inhibitory potency of vanous Q-receptor block,. T drug.~ in rat hver cells and in various smooth muscle preparatious

pmzosin

liver

other

10.05

9.89 (rat portal vim}

phenoxMaenzamine

9.0

9o (cat ~ n )

dihydroergocryptine yohimbine

8.19 6.48

8.72 (rabbit uterus) 627 (rabbit .orta)

Values shown are pA2 values, except for lOB. where -log !(',~, i, given Vah~cs in li~er were determined from Schild.plots of inhibition of adrenaline-induced phosphor)last a~li~ =tit,n. using dilute suspensions of liver cells (see Ref. 2). Values for smooth muscle are taken fnnn the literature, except for DHEC. which is unpublished finding of the author.

inhibitors and are labelled with 0.5 nM [3H]POB in the presence and absence of 0.1 pM prazosin, two specifically labelled proteins with mol. wts of 80 000 and 58 000 are identified by SDS-PAGE I. Omission of protease inhibitors throughout, or in some stages of the purification, results in the decrease or disappearance of the 80 000 protein, and the appearance of additional, specifically labelled proteins with lower mol. wts (15 00030 000) I. This strongly suggests that the receptor binding protein is represemed by the 80 000 band, from which lower tool. wt fragments may be generated by proteolysis. Indeed, fimited enzymatic digestion of the isolated 80 000 protein yielded labelled fragments of tool. wts 58000-62000, 45000, 40000, 27000 and 18 000 (Ref. 6). In the fight of these findings, the affinity-purified 59 000 protein which displays at-receptor specific bindings, and a [3H]POB-labelled liver membrane protein with a tool. wt ,3f 44 800 (Ref. 3) may represent proteolytic fragments of the at-receptor. However. even extreme measures to prevent proteolysis by preperfusing the intact liver with a mixture of protease inhibitors and then cooling the organ to 0°C before homogenizing it reduced, but did not completely prevent, specific labelling of the 58 000 tool. wt protein (G. Kunos, unpublished observation). Thus, one cannot exclude the possibility that a 58 000-59 000 tool. wt protein may be an integral part of the at-receptor, or it may be generated/n vivo in the intact membrane, in.vivo regulation of receptors by limited proteolysis is not unlikely in view of findings of a trypsin-induced interconversion of high and low affinity forms of the at-receptor in purified liver plasma membranes'. The presence of an 80 (10t)moi. wt or=receptor binding protein in rat liver and the possible proteolytic origin of several smaller binding proteins including a 52 000--55 000 and a 42 000 tool. wt species has recently been confirmed by the use of a novel, radio-iodinated photoaffinity probe, a structural analogue of prazosin 4. The affinity of this

ligand f•r a t-re~ptors is somewhat higher than that of [3H]POB (0.13 n.,,t), while the nonspecific binding of the two ligands is comparable. The iodinated probe has the distinct, advantage of much higher specific a~ivity, but more information is needed on its pharmacological propertie:, particularly as tested under photolysing c(mditions. By using this li?~and. Le~,;v:nz, Caron. and their co-workers suet,idea in identi~'ing the ~tt-receptor binding protein in a number of other tissues where receptor density is considerably lovt-.r than in the rat liver. A major protein of tool. wt 78 00085 000 has been detected in all preparations studied, whereas the size and proportion of smaller labelled species varied~. These observations stron#y support the candida~' of an 80 000 mol. wt protein as the major component of the a m-receptor, and further confirm the similarity between the hepatic receptor and at-receptors in other tissues. Radiation-inactivation of rat fiver a t-receptors yielded a protein of mol. wt lCf}tOk suggesting that in this organ the receptor may exist a~ a dimer in the intact membrane ('. Mechanisms involved in a radrenergic ttSlmmes in the liver Mechanisms of ct-adrenergic effects have been recently revzewed', so only certain salient points and recent developmerits will be discussed. As mentioned above, at-adrener~c effects are generally independent of cAMP. Although the underlying mechanisms are not yet clear, and often controversial, there is little doubt that calcium plays a ke~ role in most of these responses, a rAdrenergic agonists as well as certain peptide hormones such as va.~pressin and angioteusin li activate phosphorylase, the rate limiting step in hepatic glycogenclysis, via a transient and rapid increase in cyt,~olic calcium. Exton and his co-workers have found that in rat liver cells preloaded with a fluorescent indicator, the in.crease in cytosolic calcium is evident within 1 s of the addition of o-adrenergic agonists, and at maxi-

T I P S - September 1984

382 mal stimulation it represents an increase from a basal level of 0.2-0.6 gM of free cytosolic calcium. Recent evidence indicates that receptor activation first leads to a rapid release of calcium from a plasma membrane or endoplasmic reticulum pool. This initial rise in cytosolic calcium is responsible for triggering a calcium-sensitive cascade leading to the breakdown of glycogen. Net calcium influx from the

e.g. the simil~ level of phosphorylation of phosphorylase, may account for the similar effects of these hormones on glycogenol)sis, whereas phosphorylation of unique substrates predicts subtle differences in their biochemical actions. a-Adrenergic stimulation can cause a variety of effects in the liver, including increased amino acid transport, increased respiration, changes in potassium fluxes, increased ureagenesis and glycol)sis,

gi,~e

IIII I I

/

extracciluim" space does not appear to occur in the initial phase of an-receptor activation. Most of the calcium released into the cytosol is rapidly extruded from the cells by a calcium-sensitive ATPase located in the plasma membrane, Although most of the calcium released from hepatocytes is of mitochondrial origin, it is not yet clear whether mitoch¢:ndrial calcium release contributes to the initiation or the maintenance of the receptor response, or serves only to buffer cytosolic calcium levels reduced by the activi'~ of the membrane Ca 2+ATPa:-.e. Once the effect of the agonist is terminated, calcium is quickly reaccumulated, which probably accounts for the influx of extracellular calcium noted in some studies, The most likely site of action of calcium in the glycogenolytic cascade is at the level of phosphorylase kinase. In rats deficient in this enzyme, a-adrenergic agonists still mobilize calcium in livei cells, but the activation of phosphorylase is impaired7. Calmodulin has been identified as the delta subunit of phosphoryla~ kinase in skeletal muscle, and a similar component in purified rat liver phosphorylase kinase may be the locus for the calcium-sensitive regulation of this enzyme. A study of the phosphorylafion of proteins in rat hepatocytes by various glycogenolytic hormones indicates that the cAMP-linked hormone, glucagon, and the calcium-linked hormones, noradrenaline, vasopressin, and anglotensin 11, act on separate but overlapping substrates s. The overlaps,

inactivation of glycogen synthase and increased gluconeogenesis. The role of increased ~'gosolic calcium in triggering many of these effects has been documerited, although a notable exception is the nc~radrenafine-induced gluconeogenesis 'from oxidized substrates (fructose, dillydroxyacetone) in the rat liver, which is independent not only of cAMP but also of calcium. However, the link between at-receptor activation and calcium mobilization is not yet clear. Mitchell has proposed that hydrolysis of phosphoinositides is the primary event leading to a rise in cytosolic calcium, alReceptor mediated breakdown of phosphatidy~inositol has been clearly documented in many tissues including the rat fiver, but a bone of contention is whether this phospholipid effect triggers or follows the changes in calcium fluxes. in some tissues the phosphatidyfinositol respon~ is independent of calcium, but in rat liver it could be prevented by careful removal of extracellular calcium, and in the presence of calcium the time course of the effect is too slow to account for calcium release or phosphorylase activation 9. The breakdown of phosphatidylinositoi-l,4-diphosphate leads to the generation of inositol-l,4,5-triphosphate, which may act as an intracellular calcium ionophore. Although this effect develops rapidly in response to calciumlinked activators, in rat fiver cells it depends on extraceilular calcium and requires much higher concentration of agonist than the increase in phosphorylase or calcium release 9. These findings

question the role of inositol phosphofipids in mediating receptor-induced changes in cellular calcium, at least in the rat liver. It could be argued, however, that calcium dependence of a response does not necessarily mean that it is mediated by changes in cytosolic calcium, or that a small calcium-independent effect in a plasma membrane compartment is masked by calcium-dependent breakdown of phosphoinositides in other, larger cellular pools. Clearly, further studies are needed to clarify these issues, as well as the role of other potential mobilizers of cellular calcium, such as the recently proposed polyamines. The independence of oq-adrenergic effects from the adenylate cyclase system has been widely assumed, Nevertheless, in calcium-depleted rat hepatocytes, ct-adrenergic stimulation leads to increased cAMP accumulation "p. The fact that a similar effect can be detected in adult male rats even at normal levels of extracellular calcium t°'n~ suggests that this mechanism may be of physiological importance. Thus, separation of cAMP and calcium-linked pathways may not be absolute, and the two pathways may be linked under certain conditions, possibly at the level of guanine nucleotide regulatory proteins, Regulation of hepatic ctrreceptors When rat liver cells are exposed to high concentrations of calcium-linked activators, the glycogenolytic response gradually declines. This 'desensitization" in unrelated to the specific receptors involved, and is probably due to depletion of a common calcium pool. It is not ag(!nist specific, although responses to cAMP-linked activators remain unaffected, and it is rapidly reversed by removal of the agonist and replenishment of calcium stores. Guanyi nucleotides are known to regulate adenylate cyclase-coupled receptors, and their possible role in regulating hepatic al-receptors has been examined using the selective ligand, [3H]prazosin. Guanyl nucleotides failed to influence the binding of [~H]prazosin or its displacement by agonists 2'12"13, although the slope of the Hill plot was consistently less than unity 2. However, one group has reported a guanine nucleotide-induced rightshift of the adrenaline displacement curve and an increase of the slope factor to unity t4. The reason for the difference between these observations is not clear. Earlier reports of guanine nucleotide sensitivity of tritiated catecholamine binding to rat fiver plasma membranes may have been

77PS -Sei, u, m b e r 19~4 due ~o the a,-receptor component in catecholamine binding. Hetewlogous regulation of hepatic adrenergic receptors has attracted considerable attention. As pointed out above, the relative contribution of B2and at-rzceptors to the hepatic glycogenolyti¢ response is species-dependent; the a-receptor dominates in the adult male rat, while 13.receptors assume greater importance in some other species, inclvding man. Even within the same animal, the nature of the receptor is not immutable. A number of conditions that have been shown to cause a shift from a t- to 13-receptor dominance in the T A B L E il. Conditions associated with a shift fn~m a~ to 13,-receptor mediated glycogenolysis in rat liver

Hypothyroidism* Adrenalectomy* Partial hepatectomy* Cholestasis" Female v. male rats Young v. old rats Treatment with chemical carcinogens

Treatment with pertussis toxin PrimaD' culturing of hepatocytes in conditionsmarked with *, increaseddensityof B and decreased density of cq-receptor binding sites have also been detected. rat liver are listed in Table !!. in some of these, corresponding inverse changes in the density of at and 13-receptor binding sites have also been demonstrated. An increase in 13- and decrease in a-receptormediated glycogenolysis is characteristic of all of these conditions, although the decrease in the a-receptor response was small or undetectable in some studies where young female rats were used, in which the contribution of at-receptors is already reduced. The decrease in aadrenergic responses is selective, as the similar effect of vasopressin is not influenced either in adrenalectomized n-sor in hypothyroid rats ~. A recent report of a non-~lective decrease in the glucosereleasing efli:cts of various calciumdependent activato~ in female hypothyroid rats t~' is difficult to interpret, as stimulation of phosphatidylinositol breakdown, another calcium-dependent effect in the rat liver'~, was not influenced by hypothyroidism in the same animals n~'. It should be also emphasized that glucose release is not a reliable indicator of glycogenolysis for agents that may also influence glycolysis. The decreased o~-adrenoceptor response is associated with a decrease in the density but not tlhe affinity of [3H]prazosin-labelled a a-receptors in the liver of adrenale¢~omized n4, hypo-

3~3 thyroid:'t~, partially hepatectomized t 7. cholestatic iT. and young v. old rats t-~. The absence of a change in a-receptor density after adrenalectomy in earlier studies may be related to the use of ligands that label both an- and a,-receptor is, where a potential increase in the density of the latter may have offset a decrease in the former. However. changes at the level of the coupling of receptors may a l ~ contribute to the reduced al-adrenergic response, as suggested for hepatocytes of adrenalectomized rats t4. cr for isolated liver cells after short term in-vitro incubation TM. A common denominator for conditions associated with decreased atand increased 13-receptor activity in the liver (Table !1) may be the decreased level of cellular differentiation. Thus, a common mechanism related to cell differentiation may be involved in the inverse regulation of adrenergic receptors, as suggested earlier t'~. Of the experimental models listed in Table !!. rat liver cells in primary culture appear to be the most attractive for further study, as the change in receptor response develops very rapidly (4-8 h), and under in-vitro conditions nt. We recently found that incubation of isolated rat liver cells in a serum-free buffer leads to a conversion of the adrenergic activation of phosphorylase from an at- to a 13adrenergic response within 4 h, without a parallel change in the density or affinit-' of ['~H]prazosin-labelled a r or [-~HICG!/12177-labelled 13-receptors ns. This change in the adrenoceptor rcspon~ is associated with no change in the glycogenolytic response to the calcium-linked activator, vasopressin, and a decreased response to the cAMP-linked hormone. glucagon. In cells preincubated for 4 h, a further ~ min incubation with lipomodulin, and endogenous inhibitor of membrane phospholipase A , _'o. reverse.~ the adrenergic activl,tion of phospho~'lase from a 13-to an a,- receptor response. whereas in freshly isolated cells lifmmodulin does not affect the predominant a t-receptor response. Conversely, exposure of freshly isolated cells to melittin, an activator of phospholipase A,. results in the suppression of the effect of the a-receptor agonist, pheny|ephrine, and the emergence of a response to isoproterenol within 30 min TM. These findings strongly suggest that changes in membrane phospholipase Az activity are involved in controlling the expression of the adrenergic receptor phenotype, probably by influencing the coupling of at- and 13-receptors in an inverse, reciprocal manner. Corresponding changes in the density of receptors

under some conditions may be parallel or ~conda~' events. W~tether phospholipase A . influences the coupling of receptors directly, or exerts its influence indirectly by controlling the formation of a specific prostaglandin or leukotriene intermediate, is currently under study. A c k ~ t ! thank Dr Robert J. Lefkowitz for showing me the manu~ript of Ref. 4 before its publication. Work from the author's laborato D' is supported by a grant front the MR(" of Canada. Reading list I Kunos. G.. Kan. W. H.. Greguski. R. and Venter. J. C. (1983) 7. Bin/ Chem. ~ 8 . 326-332

2 Preiksaitis.H. G.. Kan. W. H. and Kunos.G (1982) 1. Biol. Chem. 257. 4321-4327 3 Guellaen, G., Go~hardt, M . Baroukl. R and Hanoune. J. (1982,)Biochem. Pharmacol

31.2.~!7-2820 4 Leeb-Lundberg,L. M. Dickinson. K E J.. Heald. S. L.. Wikberg, J. E. S.. Hagen. P.-O., DcBernardis, J. F.. Winn. M. Arendsen. D. L.. Letkowitz,R ]. and Caron. M. G. (1984)J. Biol. Cht'm. !~9. ~7g-~h'7 5 Graham. R. M., Hess. H.-J and Homo. C. J. (1982)J. Biol. Chem. 257. 15174-15181 6 Venter. J. C., Eddy. B.. Schaber, J., Lilly, L and Fraser. C. M. (1c~8."~) in Derelopmental Pharmacolo~" (McLeod. S M. Okey, A. B and Speelberg.S. P,. eds), pp. 183-206,Alan Liss, New York 7 Exton. J. H. (1981~ Mol. ('ell. Endocnnol 23. 33-264 8 Garrison, J. C. and Wagner. J. D (19S2b 1. Biol. Chem 257. 13135-13143 9 Rhodes, D., Prpi~. V., E.~on, J. H. and Blackmore. P. F. (1983) I. Btoi Chem 258, 2770-2773 10 Blair. J. B., James. M. ,~:. and Foster. J L (19"/9)J, Biol. ('hem. ~g4. 7579-75~4 !! Okajima, F. and Ui, M. (1982) Arch. Biochem. Biophys. 213.658--608 12 Hoffman, B. B.. Mullikin-Kilpatrick.D. and Lefko~itz, R. J. (1980) J Biol. Chem. ~ 5 . 464_¢,-,1652 13 Noguchi. A. {1983) Endocrmnoio,o 113, 672,-67(I 14 Goodhardt, M., Ferry, N., Geynel, P. and Hanoune. J. (1982)J. Biol. ('hem. 257, 11577-115&1 15 Chart, T. M.. Blackmore,P. F. Steiner. K E and Exton, J. H. (107q)J Bu,l. Chem Z~. 242~2433 16 Corvera, S. and Gan.aa-Smnz. J. A (!~831 FEBS Len. 152. :m6-:~8 17 Aggerbeck, M., FerD., N. Zafram. E-S. B~llon, M. C., Barouki. R and Hanoune. J (1983) J. CIin. Invest. 71,476---k~1o 18 Kunos. G. (1980) Trends Phamxacol St'i I. 282-2~g4 19 Hirata, F. (lq~l) J. Bud.

('hem. Z%,

772~-7733 ~l Kunos, G., Hirata. F.. Ishac, E. J. N. and Tchakarov, L. Proc. Natl Acad. Sci. USA {in press) George Kunos was born and eduoued in Hungary. where he graduawd m medicine in ! ~ from the Semmelwei~ Medical L'mversio'. Budapest. h: 1071 he moved to Canada and gained a Ph.D. m pharmacology under Mark Nickerson in 1073. at McGill Universio" itt Montreal. He ts currently Professor of Medicine and Pha,nacoiogy at McGil! Utmversit~." Facul~." of Medicine.