Hormone-sensitive magnesium transport and magnesium regulation of adenylate cyclase

T I P S - February 1984 IIII

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site is on the Mg ~'-guan~l nucleotide coupling wotem (G/F or N ) and is responsible for ahenng the ability of N to regulate both agonist affinity~ for the hormone receptor (R) and the intet-,a:tion of the catalyt|c sub-

Hormone-sensitive magnesium transport and magnesium regulatio t of adenylate cyclase

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Mg 2* regulation of aden.vlate cyclase Modulation of cyclase activation is an established effect of free Mg ~" on the receptor-cyclase complex ~~ ~. Several laboratories have demonstrated the abiliq, of free Mg ~* to activate adenylate cyclase activity and to alter the affinity of agonist binding to fl-adrenergic ~nd other receptors (Fig. ! ). StcllaCech and ~:ecendy demonstrated that two distinc~ and independent Mg ~" sites mediate these effects. One Mg="

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Deparm~ent of Pharmacology. School o] Medicine. (me Western rewrve t :ntrersa~'. ( let,eland. O I l 4411~. U,%,,|.

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Michael E. Maguire Free Mg a+ ion modulates the activation of hormone-sensitive adenylate cyclase in a wide variety of cell types independent of the hormonal agent responsible for activating or inhibiting the enzymeL This sensitivity to Mg ~÷ suggests that the cyclase might be responsive to changes in mtracellular free Mg~+ concentration. During studies to investigate this possibility, a hormotw,-sensitive Mg ~" transport system was discovered in the murine $49 lymphoma cell, wherein Mga+ influx is inhibited by activation of/]-adrenergic recep tots. In contrast, Mg ~+ effiux as well as transport of other ions, including Ca ~+, are unaffected by receptor activation. We have since shown, hormone-sensitive Mg~+ transport in three other cell lines and in response to two other hormones (PGE~ and adenosine). Regulated Mg ~+ transport has also been reported in rat adipocytesa and in rat pancreatic islet cells8. The $49 lyrephoma cell Mg n+ transport system has three unusual properties of interest with regard to regulation of adenylate cyclase activity, First, even though Mg ~+ influx is inhibited by the same population of ft..receptors that stimulate adenylate cyclase, cAMP does not mediate the effect of hormone on Mg ~* transport. Second, we have shown that the effect of hormone on adenylate cyclase desensitizes quickly, within 2 mitt, while the effect on Mg ~+ influx desensitizes much more slowly, over 40-60 rain, Finally, and of most interest, the hormone-sensitive Mg ~+ transport system is coupled to a small, subcytoplasmic pool of Mgs÷ containing less than 2% of totat cell Mg ~+. It is to be noted that none of these properties reported for Mg~+ are shared by or, for the most part, inhibited by Ca *+ or Ca *+/cat. modulin. Out current working hypothesis i~ that this small pool of Mg t+, sensitive to hormonal regulation, is involved in the chronic regulation of the sensitivity e.f

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probably must be ¢~cupiedh~ Mg ~" for the ncces.--.,ary initial interaction belgc.en R and

[ ndcr h,i~,,ii cllndllion,. ~hc /~.,I,,[ ,t~.i~', ~. tlon of adeny]at¢ c,,clase bs free M g ~" at thb, site is I-3 m M in $49 cells S i m d ~ values have been obtained in other s)stems. fill ( Ilri,addu,. ~n ;n~ t,ih~,r:ll,,i~ demonstrated thai the.~ .li-lgz" ~iles arc accessible only from the internal lc)toplasmic) membrane face, through studie~ with sealed membrane v~icles ~a. Thus. the

Mg =" needed for cyclase activation is that inside the cell• This reqmres that the internal free Mg*" concentration available to the receptor-cyclase complex be sufficientl.~ high to activate the enzyme. Hogever. m the large majority of cell types tested to date, the cytoplase:ic free Mg:" concentration is 0. !--0.4 m',t, markedly less than the I-3 mM required for half-maximal basal activation of aden)late cyclase. In collab~-ADRENERGIC RECEPTOR BINDING ~,,~----o--~

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Fig. I Mg'* aclivationof adenylateo'claseand enhancement of agonm affin~':/or~.adrenergic receptors. The left panel shows the activation o f adenylate cyclase b)' fret Mg s°. The ordinate is the fold increase in V ~ o f the cyclase. It is calculated from mMliple kmenc plots of acdvily at a giren .free Mg s+ concentration and various MgA TP concentrat&,ns from 0.01-3 rest. The effect of Mg s" on hormone.stimulated activily in 549 cells cannot be determined by this type o f plol because the affinity ofthe S49cyclase for free Mg ~+is too high (see Refs I and 0 for aiscussionL The right panel shows the effect of !0 m,u Mg:" on the abili~." of the ~gonLvt ¢-~-zsoproterenol mid the antagonL~t ( -)-propranoloi to inhibit [miliodohydroxybenzylpindolol binding to ~.receptors. The Mg*" effect is clearly specific for agonists, and the Mg'+ concentration for half.maximal effect is 1 ~ m u L~ |qigi, Elst~'wcr ,¢a.~ct~,"gP ~ I ~ .

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MINUTES FiR. 2. Effect o / ( -)-isoproterenol ( IN E, open symbols) on Mg'÷ transport in $49 cells. Transport was measured ruing ~Mg:" and a microcemriJhgation assay as described TM. Panel A shows the effect o f 10 ~ INE on MR.+ influx oflriplicate samples. The SD o f each set oflriplic.dte$ is less than the size of the points. In Panel B. $49 cells wen. incubated for 3 h with :~Mg:'. cooled on ice.for 30 mi,. washed nvice in ice.cold buffer and the final cell pellet resuspended in buffer at 370C with or without i0 ~ ¢ INE. Single aliqums were taken at the times shown.

oration ~vithDr Joyce Jentofl we have used 3~p NMR measurement of the phosphate resonances of cytosoli,: ATP in intact cells to estimate the amount of ATP chelated with Mg z'. Knowledge of this parameter, coupled with the affinity constant for MgATP fommtion, allows calculation of the free cytosolic Mga+ concentration independently of the absolute ATP or Mga+ concentrations. We currently estimate a maximal value in $49 cells of 0.3 mM free Mg*+, with a value of 0.1 mM more likely. This implies that under resting conditions, the available cytoplasmic Mg *+ concentration is not sufficient to activate adenylate cyclase to its maximal extent. However, the mechanism of hormonal activation of adenylate cyclase clearly involves a marked increase in enzyme affinity for free Mg*+, a dec'tease in the Kºto about 0.1--O.2 mM; in $49 cells the shift may be even more pro. nonnced, to about 0.01 mM Otefs 1 and 6). The hormone-induced shift in Mg *+ sen. sifivity allows the cyclase complex to achieve a much higher rate of cAMP synthesis than would otherwise be obtained. These Mga+ effects contrast sharply with guanyl nucleotide modulation of adenylate cyclase. The K, for GTP activation is about 0.001 mM while the intracellular GTP concentration is at least 0.1 raM; as might be expected from these relative concentra. tions, hormonal activation does not alter cyclase sensitivity to GTP. Moreover, in most cell systems, GTP by itself is a poor activator of catalytic activity 0ass than 2-fold) while Mgs+ activates 10- to40-fold. While GTP is clearly required for hormonal activation of adenylate cyclase, this sharp

contrast in Mgt+ v. GTP effects suggests that GTP itself is not the actual physiologicad agent within the cell which activates the enzyme. Most importantly, changes in GTP concentration are unlikely to have chronic regulatory significance. Indeed, the current GTPase hypothesis, that hydrolysis of bound GTP to GDP by N is a 'turn-off mechanism, seems more suited to acute regulation of cyclase activity. The ability of Mg *+ to activate adenylate cyclase does not, of course, show that Mg z+ is a regulatory agent, only that the mechanism is in place for Mg s+ to perform a l~olein physiological regulation of the enzyme. In this regard the vectorial access of free Mg ~+ to the ~egulatory sites on adenylate cyclase must be considered, Hormone-sensitive Mg *+ transport Our initial studies on Mgt+ 'metabolism' in the $49 cell were a characterization of basic transport properties",~'. However, J. J. Erdos and I very quickly discovered that /~.agonists inhibit the influx of Mg *+ into $49 cells but do not alter its effiux (Fig 2). The effect of (-).isopmterenol has a K, of 10 riM, slightly more potent than its activation of cAMP accumulation (30 riM), and is blocked by (-).propranolol with a K~ of 1 riM. This effect of fl.agonists on Mga+ influx is absolutely specific for Mg *+. Neither Ca *+ influx, Cat+ efflux, nor transport of Mn *+, K +, or Na + is altered by hormone. Moreover, although Ca*+ is able to inhibit Mg a+ transport slightly, the effect is kinetically non-competitive and the K~ for Ca*+ is > 5 raM, clearly non-physiological. We fLrSt assumed that an increase in

February 1984

cAMP concentration stimulated phosphorylation of a Mg2+ transport complex, thus inhibiting it. To our surprise, cAMP was shown not to mediate the effect of fl.agonists on Mg I+ influx". For example, the kinase- mutan~ of the $49 cell shows distinct inhibitiml "of M g ~+ influx upon addition of (-)-rsoproterenol. The kinase- mutant lacLs all cAMP-dependent protein kinase acJvity, through which all known effects of cAMP are mediated. Several other lines of evidence also indicate independence from cAMP mediation. As one example, 8-bromo cAMP has no effect on Mgz+ influx: however, even after $49 cells have had their intracellular cAMP levels markedly iv.creased by preincubation with this analog, (-)-isoproterenol still inhibits Mg:" influx. A further contrast between 0-receptor activation of adm~ylate cyclase and inhibition of Mgt+ influx is the time course of desensitization" (Fig. 3). After hormonal stimulation the rate of synthesis of cAMP decreases rapidly, falling to a basal rate within 2-3 rain after addition of agonist. However, fl-agonist inhibition of M7~÷ influx, while rapid, is maintained for 25- " ) rain and does not desensitize completely ,-v about 60 rain. Since efflux of Mg~+ is not affected by hormone, the most obvious effect of this relatively long decrease in influx rate would be to decrease the amount of intracellular Mga+. Cempmmentatlen of Mg '+ ff the effect of hormone is to reduce intracellular Mg 2+ concentration, it becomes important to determine both the extent and the location of that decrease. Robert Grubbs in my laboratory investigated these parameters by selective membrane permeabilization with digitonin16.". Digitonin interacts relatively selectively with cholesterol; since the cholesterol cow tent of the plasma membrane is much higher than that of other (intracellular) membranes, it is possible to select a digito. nin concentration which, at a specific incubation time and temperature, permeabilizes the plasma membrane but does not alter other membranous structures of the cell (Grubbs, R. D., Coffins, S. D. and Maguire, M. E. unpublished observations, see also Refs 16 and 17). As a result of this permeabilization, the cell's cytoplasmic contents are extruded into the medium leaving behind high molecular weight and particulate structures. Fig. 4 shows the time course of ~lease of lactic dehydrogenase, a soluble cytoplasmic enzyme, and lysosomal acid phosphatase after addition of 0.003% digitonin. Total cellular lactic dehydrogenase is released rapidly, while lysosomal integrity is minimally altered. Further controis indicate that mitochondria retain

TIPS-

75

February 1984

respiratory control even after prolonged laD]_ ,-o--o-..o incubation with these low digitonin con",, centrations, a further indication of the lack I, x of significant effect of digitonin on cell t~ ~)x smtctm~ other than the plasma membrane. ~ x Indeed, electron micrographs of digitonin- t~l 80 ~ x treated cells are indistinguishable from ~ L OX untreated cells. This technique allows us to assess the 50 ~, location of newly-transported Mgt÷, the ~ 60 x oX Mgt+ that has just been taken up into the -r | \ cell. The data of Fig. 5 show three impor- ,,~ rant results. First, Mg*+entry, measured by uptake of UMg*+. plate~us at about 90-120 < 4 0 rain, reaching an apparent isotopic equilib. rium. The mass amount of Mg"+ taken up by the $49 cell is normally about I-2 nmoles/10~cells. However. the total Mg:* ~ content of the cells is about 85 nmoles/lO7 w 20 cells. Thus, at apparent isotopic equilib. rium, the amount of exclumgeable Mg~÷ is | (1. only about 2% of total cellular Mg=+, How[ ever, to conclude that this apparent equilib. O ' t ~" rium is a true isotopic equilibrium, it is lO 20 30

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necessaryto showthat (a) efflux of isotope is occurring at an appreciable rate and (b) that the rote of influx is constant with time. Both of these conditions can be met. The effiux of Mg*+ occurs with a half.time of about 4 h. The rate ofinflux hus bcen meustiled over periods o f u p to 3h and is COil-

DESENSITIZATION AFTER ADDITION OF ISOPROTERENOL • Rote of cyclic ~.MP synthesis o Rote of nMgt* influx

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MINUTES AFTER ISOPROTERENOL ADDITION Fig. 3. Desensitization o f adenylate cyclase and Mg s÷m a t t after oddi__~iono f ] O I~M[N E to intact $49 cells. Cyclase desensitization was measured by determining the rate o f cAMP synthesis over fixed time inlen'als after addition of INE to ~aire cells. The Mg:" mtTul ran, was :!etermmed h~ the uptake ,,f mMgt+ over 15 rain periods beginning at the time indicated by each point and comparing this rate to t h e r a ~ e d e t e ~ d w i t h ~imilarpulses inthe 30 min immediately p r e c e o ~ g a d d u w n o f l N E ISee Ref. 15 for details.)

stant. Thus, since true isotopic equilibrium is achieved by 90-120 rain incubation, we

can conclude that only about 2% of total cellular Mgt+ is exchangeable", The secand conclusion from Fig. 5 t~gards the effect of digitonin on UMg*+release.All of the r~ewlyIxansponedaq~Agt+remainssoluble in the cytosol for at least 2h; none of the newly-transported cation becomes sequesteredin cell particulate structures. In other experiments, we have measured UMg*+ distribution after as much as 16 h incubation; essentially 100% of newlytransported Mg~+ is still cytosolic. Third, this behavior of MS*+is in sharp contrast to that of the other major divalent cation, Cat+, which upon enu3, into the cell is rapidly and almost completely taken up into cell particulate structures (Fig. 5). This is precisely the behaviOrconcentrationeXpectedOfisCat+

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T/P~- F e b r , a r y 1984

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Fig..~. Time course of the redistribution o f " M g '~ and 4~Ca'+ after transport into intact $49 cells. After incubation with the respective isotope for the times m,ti.raled o, the ah~ci.~sa, c~.ll afi~luot.~ were washedtwice in fi'c-o,ld bu.H'erby coltrithgatio~r m a microflege, ancl a~ ali~luot ta~¢sa [~prmca.~a~r(',wt~t o[ total upt~,ke. An equal volume of 0.003% digitonin was then added for 60 set" and the cells spun in the microfuge. An aliquot o f the supernatant was taken to determine release o f cytosolic conten~ and aJier removal o f the remaining supernatant, the pellet was counted as a measure o f the cell particulate content o f ne~ly-transported cation, (See Ref. t 6 jor details.

that can be regulated, b) control of Mga+ availability in a vectorial manner, and c) vectorial access of the regulatory agent to the system being regulated The fu'st criterion is met, with respect to Mg 2+, by a large number of systems. Our focus has been on the adenyiate cyclase complex, a system

MEDIUM

whose hormonal activation appears to he dependent on Mg ~+ and which possesses specific allosteric sites for Mga+ action. The second criterion, vectorial control of Mgz+ availability, is met by a hormonesensitive transport system, highly specific for Mg~+. This transport system provides a

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Fig. 6. Hypothetical model o f the relationship between the hormone receptor-adenylate cyclase complex and the M B *+ transportsystem. The M g t+ influxsystem isapparently intimatelyassociated with the receptor-cyclasecomplex while M g t+ efflu.ginvolvesan independent transportsystem not regulated by ho.rmone. The M g a+ influorsystem is highly specificfor M g j+ and appears to communicate with a verysmall introcellularM g z+pool comprising 2 % o r lessof totalcellularM g '+. This small pool isentwelycytoplasmicand does not readilyexchange with totalcytoplasmicor other crllularM g "÷. The physical basisfor such compertmentation is unknown at present. (See textfor details o f the hypothes~.)

means of altering Mg a÷ concentration within the cell by virtue of its effect on Mg2÷ influx but not efflux. The. amount of Mgz÷ transported is clearly insufficientto alter the total cell content of Mg~÷ significantly. However, the existence of a small subcytoplasmic compartment of Mg ~÷ suggests that total cell Mg 2÷ need not be ',altered. Since hormone alters Mg ~÷ influx ¢aly, then the content of the relevant Mg2. compartment, containing only about 2% of total cell Mg2÷, could be altered sufficiently to have a physiological effect. The third criterion is the most problematic. Vectorial access of Mg 2÷ to a regulated system (adenylate cyclase, in this case) is superficially obvious. The adenylate cyclase complex is a component of the plasma membrane, and the sites on adenylate cyclase at which Mga+ acts are accessible only from the cytoplasmic membrane face, indicating that the relevant Mg a+ pool is intracellular. Further, the hormone.. sensitive Mg2+ transport system is also a plasma membrane component and operates in such a manner as to decrease the Mga+ content of the relevant (intracellulart pool. Moreover, we have shown that the /3-adrenergic receptors which modulate adenylate cyclase activity and those which modulate Mg s+ transport have the same pharmacological characteristics. Thus, the conclusion that the Mga* pool regulated by hormone is associated with adenylate cyclase seems obvious. That is, application of a/~-agonist not only stimulates adenylate cyclase and cAMP formation but also decreases the content of a Mga+ pool associated with the same receptor--cyclase complex. The caveat, and it is an extremely important one, is that we have at present no

TIPS - February ! 984

77

direct evidence to suggest such a close con- hormonal stimulation titan control cells. nection. While an association of the recep- We are presently working with two such tor-cyclase complex and the pool of Mg =* model systems: murine $49 cells able to is reasonable, it is equally possible that two grow in Mg=+-deficient medium and different and independent but phar- splenic T-lymphocytes from rats mainmacologically identical populations of tained on an Mg=+-deficient diet. In both ~adrenergic receptors exist in a single cases our preliminary da!a indicate that homogeneous clonal cell line, each popula- such cells are markedly hyposensitive to tion mediating separate biological effects. /~.agonists. in conclusion, our recent studies Although our working hypothesi is the former, i.e. that the Mg t+ pool regulated by suggest that free Mg~÷ has an important hormone is directly associated with the role in the regulation of hormonal receptor-cyclase complex, the possibility responsiveness. Whether or not Mg ~* is a of a single receptor mediating two distinct chronic regulatory agem for Mg ~* sensieffects in the same cell is of obvious inter- tive enzymes, such as adenylate cyclase, est, not the least aspects of which would be our Mg t÷ transport studies have clearly how the receptor populations mediating shown that lymphocytes and presumably each biological effect are kept separate and other cells transport and compartment Mg :+ in a manner distinctly different from whether they are regulated independently. If this small Mg ~÷ pool is indeed the manner in which they handle Ca t+ . intimately associated with the recep. These observations alone indicate that, tor-cyclase complex, the pieces would aside from its ubiquitous role combined then he in place for a chronic regulation of with nucleotide as substrate in phosphoryl ~denylate cyclase using free Mg ~+ as the transfer reactions, free Mg =+ performs regulatory ligand. However, the funda- important roles in regulation of cell mental questions remain: does Mg *+ function. actually regulate the receptor-cyclase complex in vivo, and what function does Reading list ! Cech, S. Y.. Broaddus, W. C. and Maguire, this regulation serve. M. E. (1980) Mol. Cell Biochem. 33, 67-92 I suggest first that Mg *+ does not play a 2 Eiliott, D. A. and Ri~Ack, M. A. (1974)1. BioL regulatory role in the acute densensitizaChem. 249, 3985-3990 tion of adenylate cyclase, an event which 3 Henquin, J. C., Tamagawa, T., N©nquin, M. and occurs quite rapidly in $49 cells, within Cogneau, M. (1983)Nature (London) 301, 1-2 min after addition of agonist. At this 73-74 4 Bird, S. J. and Maguire, M. E (1978)J. Biol. point in time, the decrease in Mg ~+ influx Chem. 253, 8826-8834 is unlikely to have caused a significant 5 Garbers, D. L. and Johnson, R. A. (1975~ change in pool size, and sufficient Mg *+ is J. Biol. ('hem. Z~O. 8449-8456 still present to allow full activation of the cyclase. However, after Mg "+ influx has remained decreased for an hour or so, the Mg ~+ content of the relevant pool would be expected to have diminished markedly. At this point, the Mg t+ pool associated with adenylate cyclase might he suffb ciently depleted that a second hormonal stimulation would elicit a decreased response both from a lack of Mg =+ to activate the cyc;ase and from a lack of Mg ~+ to form the initial high affinity Mammalian Neuroendocrinology agonist--receptor complex. This working hypothesis is obviously speculative; however, it is eminently by Geoffrey W. Bennett and Saffron A. testable in several ways. For example, we Whitehead, Croom Helm, 1983. £17.93 are presently characterizing the (hardback) £8.95 (paperback) (279 pages) Mg'+ICa =+ selectivity of several divalent ISBN 0 7099 0638 2 (hardback) 0 70¢Aj cation ionophores. A minority of such 0674 9 (paperback) ionophores appear to be Mg-~+-selective under a ~ a t e conditions. If further Neuroendocrinology, the study of the data confirms this, we would be able to regulation of hormone production by induce rapid Mg *+ influx (or efflux) and the C'NS, has developed rapidly in the determine hormonal sensitivity after such years since the laboratories of Guillimain an acute change in intracellular Mg t+ and Schally isolated and characterized concentration. A second test of our the first releasing factor. This factor was. hypothesis is that chronically Mg t+- thyrotropin releasing hormone, a trideficient cells should be less responsive to peptide which is made in the hypothala-

0 lyeng~, R and Btmbaumer. L. 11982) Proc Nail Acad. Sci. USA 79, 5179-5183 7 Williams, L. T . Mulhkm. D and Lefkowitz. R. J (1978)7. B~ol C h e m 253, 2984-2989 8 Cech, S. Y and Maguire. M E (1082)Afol Pharmacol 22. 267-273 9 Maguh-e, M. E (1982)Mol Pharmacol 22. 274-28O IO Katada. T. and UI, M (1982)J. Biol. C h e m 257, 7210-7216 I I Bokoch. G M . Katada, T . Noflhup. J K . Hewktt. E. L. andGilman, A. G (1983)1 Btol. Chem. 258, 2072-2075 12 Broaddus. W. C , Vauqueltn, G. and Magutre. M. E. tlOSO)Adv. Cyclw Nucleotute Res 14.

656 13 Magulre,M. E and Erdos. J. J (1980iJ B~ol Chem. 255. 1030-1035 14 1 rtk~. J 1 andMaguirc %t [ ~ltlNll] P/It'~h,] tLtmdo~l~ 3~7 351-371 15 Ertk~, I. J. and Magulr~. M E 11980JMol Pharmacol 18, 379-~183 16 Grubbs. R D and Magmre. M. E 11982~ Magne~iwn I, 34--40 17 Zuurendonk, P. F. and Tager, J M (19"74) Biochbn. Bmphy$. Acre 333, 393-399

Tile alahor ohldlned hts P;i [) trom /rid apnl I ntt~ersirt in hlothemlstrt in 10"2 ht'h,re ~pevdtt:t: follr potldot'torol vear~ at 'he L tilt pr~llt ¢,t [ irk,'ttl~l with Alfred (; (;llman. 3 lille ICtTh lit" hf~ ht't-P ,,r., the fi,cult~ ot (a~e B',.,tent Re~etac ~ tllt('r'~l% .~chttol o¢ .~cdlclHe Bl:prt" h~' 1~ tlt It~ .4~t ~thilt' Professor o f Phannatoiog~ arid ,In ~_~tabh~hcd inte~tlgat,~r o f the Ame.tcan ttt'art A..ot tagh,tt His mator research interest b the regulat on or cell re.~pon3e "o horrnone~. In addltll,tl ill .r/It" ~,ltldtC~ t lf; M g : and MU" transport de~crtbt'd a~otc. /~t~ other re,earch inclmh'~ eh't Iron mttro~o)l'lt rt~ttah"~211on o; lndtrtdual hormone r t ' t t T I O r rrlatromolecuk'.~, p a r l l c l d a r l t daft'tiers.It vet e~fi ~r~ Ill tilt" membrane of the inl,t, tt'l/

Books

Introducing CNS control of hormone release

mus, released into tb," portal bi,x~d system that connects the hypothalamus to the anterior pituitary gland, and that stimulates release of thyrotropin and prolactin from this gland. Since this factor was isolated, four more peptidergic substances which affect pituitaD hormone release have been isolated and some of these compounds have ~ e n found not only in the hypothalamus, but in other parts of the brain and in unexpected parts of the body in addition to the brain. They have been found in single neurons with other peptides in various combinations ancl with compotmds more classically regarded as neurotmnmfitters, such as serotonin and the catecholamines. During the same period, major advances were made in the elucidation of the mechanisms cf the synthesis of peptide hormones, so we know now they