0022.3565/86/2362.0350S02.oo/0THE JOURN"L or PH..R COLO<:Y..,..D EXPF.H'''ENT''L THER"PEl'TIC~Copyrilht C 1986 by The American SocIPt~. (or Ph"rm"("olo~' IInd Experimental Tht"rBPf'utu""

Vol. 236. ~(" ZPrintt'd u, t.S A

Adenosine Affects Sympathetic Neurotransmission at MultipleSites in Vivo1

GARY E. EVONIUK,2REID W. VON BORSTEL and RICHARD J. WURTMAN

Laboratory of Neuroendocrine Regulation, Department of Applied Biological Sciences, Massachusetts Institute of Technology,Cambridge, Massachusetts

Accepted for publication October 31, 1985

ABSTRACTWe examined the effects of adenosineand its analogs on sym-pathomimetic responses of pithed rats to electrical stimulationof preganglionic sympathetic nerves (ES) or to injections ofnicotine, phenylephrine(PE)or isoproterenol (ISO).Four physio-logicalindicesof sympatheticneurotransmissionweremeasured:blood pressure, heart rate and contractions of smooth muscle invas deferens and eyelid. Elevation of arterial adenosine levelsfrom 1.5 to 2 to 3 101Mcaused a 2- to 3-fold potentiation ofnicotine-induced increases in blood pressure, heart rate andsmooth muscle tension. Higher adenosine concentrations (3-4101M)produced a smallerpotentiation of the effects of nicotine. At2 to 3 101M,adenosine had no effect on sympathomimetic re-sponsesto ES or PE.Higherconcentrations(3-4 MM)attenuatedpressor responses to ES and PE and the contractile responsesof the vas deferens to ES: these levelsalso potentiated positive

chronotropic responses to ISO.The adenosineanalogs N-cyclo-propylcarboxamidoadenosine (N-CPCA),2-ch1oroadenosine(2-CLA) andR- and S-phenylisopropytadenosine(R-PIAand S-PIA)also reduced pressor responses to both ES and PE, with thepotency order: N-CPCA > R-PIA > 2-CLA > S-PIA. Theseanalogs exhibited this same potency series in attenuating c0n-tractile responses to ES in the vas deferens. However, all fouranalogs potentiated, at the lower doses tested, the contractileresponse of the vas deferens to PE; at higher concentrations,inhibition predominated. N-CPCA enhanced the chronotropiceffects of ISO and ES. Circulating adenosine,at levels within ornear the physiological range, or syntheticadenosineanalogs.can influence sympathetic. transmission in vivo by interactingwith at least three sites: sympathetic ganglia, sympathetic nerveterminals and sympathetically innervated end organs.

The purine nucleoside adenosine can modulate neurotrans-mission by interacting with specific receptors on pre- or post-synaptictissues (Dalyet 01.,1981).Presynaptically,adenosineinhibits the release of several neurotransmitters (Fredholm andHedqvist, 1980);postsynaptically it can either inhibit or poten-tiate the actions of neurotransmitters depending on the tissueand its concentration, particularly in tissues containing multi-ple subpopulations of adenosine receptors mediating differentprocesses (Van Calker et 01., 1979). For example, in neuraltissue low concentrations of adenosine inhibit isoproterenol.induced activation of adenylate cyclase via Al receptors,whereas higher concentrations activate adenylate cyclase di-rectly via A2 receptors, potentiating the stimulatory action ofnorepinephrine and other monoamines on cyclic AMP forma-tion (Van Calker et 01.,1979;Londoset 01.,1980;Mah and Daly,1976).Adenosine may exert opposing influences upon neuro-

Received for publication April 8. 1985.1Thil workwas aupportedby Grant BNS.S4IS761fromthe National Science

Foundation and a l1'ant from the International Life Scienct InatiIU!I'.I Recipient of FelloW8hip Grant MH 09191.01 (rom the U.S. Public Health

Service.

transmission at presynaptic VI. postsynaptic sites within thesame synaptic population. For eumple, in the isolated rabbitkidney, adenosine simultaneously reduces evoked release ofnorepinephrine from renal vascular sympathetic terminals andenhances vascular responsiveness to norepinephrine; its netinfluence is to enhance vasoconstrictor responses to renal nerveI\timulation, even though the release of norepinephrine duringnerve stimulation is decreased (Hedqvist et 01., 1978). Becauseadenosine is normally present in plasma (Pritchard et al., 1975:von Borstel et 01., 1983) and can cross the blood-brain barrier(Cornford and Oldendorf, 1975), the nucleoside could conceiv.ably act as a neuromodulator at a number of different sites.affecting neurotransmission and therein altering physiologicalfunctions, including blood pressure or heart rate.

We described recently a novel effect of adenosine on pressorresponsesto pharmacologicstimuli in the rat (von Borstelet01.,1984).Verysmall increases in plasma adenosine concentra.tions, or low doses of its synthetic analogs, potentiated theeffects of nicotine on ganglionic transmission 'dramatically.strongly enhancing such sympathomimetic effects of injertedor inhaled (in cigarette smoke) nicotine as hypertension and

ABBREVIATIONS:SNS. sympathetic nervous system: PE. phenylephnne: ISO. Isoproterenol; PIA. phenylisopropyladenosine;CPCA.~opyIcarboxamidoadenosine: 2-CLA. 2-chloroadenosine; ES. p~egangllOnlcelectrical stimulation.:au-- - - -- - - - - - -

7986

tachycardia (von Borstel et ai., 1984; 1986). This finding con-trasts with the well-established tendency of adenosine to atten-uate systemic cardiovascular responses to sympathetic nervestimulation (Lokhandwala, 1979; Horn and Lokhandwala, 1981; .

Kamikawa et al., 1980). The present studies were designed todetermine: 1) the net effect of adenosine on sympathomimeticresponses in vivo; 2) the relative contributions of the actions ofadenosine at various SNS loti to its overall effects on physio-logical functions; and 3) the concentrations of plasma adeno-sine needed to produce such effects. In these studies, we pro-duced controlled and sustained elevations in circulating aden-osine concentrations, using a slow Lv. infusion. Adenosinelevels were thereby elevated incrementally, allowing determi-nation of its relative potencies at various loci within the efferentSNS. We examined the abilities of adenosine, and of some ofits derivatives, to influence postsynaptic elements of sympa-thetic transmission (by modulating the effects of adrenoceptoragonists), neural elements (by modulating the effects of pre-ganglionic electrical stimulation), as well as ganglionic re-sponses to nicotine. Besides monitoring pressor responses tosympathetic stimulation, we have also examined three otherindices of sympathetic activity: heart rate and the contractionof two types of smooth muscle. This allowed us to compare, inseveral tissues from a single animal, the multiple and sometimesparadoxical effects of adenosine on sympathetic neurotrans-mission.

Methods

Animals. Male Sprague-Dawley rats (Charles River Breeding Lab-oratories, Inc., Wilmington, MA), weighing 250 to 350 g, were anes-the$ized with Nembutal (50-60 mg!kg i.p.); the left carotid artery wascannulated, allowing direct measurement of blood pressure and heartrate via a pressure transducer and cardiotachometer, and allowingperiodic withdrawal of blood samples for assay of adenosine content. Atriple catheter was also implanted in the right jugular vein; bolusinjections of drugs (nicotine, phenylephrine and isoproterenol) couldthereby be given without interrupting a continuous i.v. infusion ofadenosine or its diluent (0.9% saline).

For electrical stimulation of preganglionic sympathetic nerves, ratswere pithed by inserting a stainless-steel tube (l3-gauge) through theorbit and the foramen ma(!"Tlum,into t \w F.pinal column to the levf'l ofthe sixth cervical vertebra; this destroys the brainstem and effectivelysevers the spinal cord from the brain. Through this trocar, a steel rod(1 mm diameter) was inserted down the length of the spinal column.terminating in the sacral vertebrae. The portion of the rod that re-mained inside the trocar was insulated with a ai1astic sheath. Theanimal was respired artifically with room air (I ml/l00 g b.w., 50strokes/min) via a tracheal cannula for the duration of the experiment.Skeletal muscle was paralyzed with pancuronium bromide (0.02 mg/kgi.v.) or d.tubocurarine (1.0 mg/kg i.v.), and an indifferent electrode wasinserted under the skin of the right hindlimb. Monophasic pulses (0.1msec duration, 2 pulses per sec, 30 V, 5 see train duration) were appliedwith a Grass S11 stimulator.

Methods for measurement of arterial blood preasure, heart rate andsmooth muscle contractions of the eyelid and vas deferens as well asprocedures for determination of plasma adenosine concentrations aredescribed in the preceding report (von Boratel et oJ., 1986).

Drug treatments. Adenosine (5 mg/ml in 0.9% saline) was admin-istered as a continuous Lv. infusion at rates of 10 to 100 Ill/min, usingIi Harvard (Harvard Apparatus Co. Inc.. South Natick, MA) syringt.pump. Nicotine, PE, ISO and the four adenosine analogs, R-PIA, S.PIA "net N-CPCA, pneroullly provided by John W. Daly, and 2-rJ.AISiKma Chemical Co., St Louis, MO) were administered a8 i.v. bolusinjections in a volume of 0.1 ml of saline. A recovery time of at least

-- -- -

Adenosine and Sympathetic Neurotransmission 35'

10 min was allowed to elapse between successive drug injections t<ensure reproducibility of responses.

Statistics. Data were converted to percentage of control response I(i.e., before administration of adenosine or adenosine analogs); theslwere expressed as means :t S.E. and compared by analysis of variance

Results

Effect of adenosine infusion on sympathomimetic re-sponses. The basal concentration of adenosine in arterialplasma was 1.54 :t 0.16 pM. Infusion of adenosine at ratesranging between 0.15 to 0.60 mg/kg/min increased arterialadenosine significantly (P < .005) to levelsbetween 2.18:t 0.36and 4.10 :t 0.37 pM. The effects of these elevated adenosinelevels on basal blood pressure, heart rate and smooth muscletension are described in the preceding paper (von Bontel et al.,1986).Elevation of circulating adenosine levels to 2 to 3 pMcaused a 2- to 3-fold potentiation of responses to nicotine (40pg/kg Lv.)by all four of the sympathomimetic indiceseumined(figs. 1-4). The magnitude of this potentiation decreasedwhenarterial adenosine concentrations were increased beyond" pM.

Adenosine also modified sympathomimetic responses to ESand to adrenergic agonists; the magnitude and direction of itseffect (inhibitory or potentiatory) varied among the end organsstudied. Pressor responses to both ES and PE (5.0 pg/kg tv.)were attenuated equally at all adenosine concentrations tested(fig. 1); responses to both stimuli were inhibited by more than50% at adenosine concentrations of .. pM. At the concentra-tions tested, adenosine had no effect on the contraction ofeyelid muscleinduced by PE (fig.2) and caused an insignificanttrend toward attenuation (maximum 30%inhibition at concen-trations above 4 pM) of the contractile response to ES. Simi-larly, adenosine-failed to modify significantly PE-evoked con-tractions of the vas deferens, even at concentrations exceed-ing 4 pM (fig. 3), but tended to reduce responses to ES. The

DiastolicBloodPressure400

I 234 5

Arterial Adenosine(IlM)

Fig. 1. Relationship between arterial plasma adenosine concentrationand systemic pressor responses to nicotine (~, 40 Ilg/kg), phenylephrine(8. 5 ltg/kg) and ES (0. 10-30V, 2 msec, 2 pps, 5 see). Rats weretested before and during Lv. adenosine infusion at several controlledrates (0.15-0.5 mgfk9/min). Results are expressed as percentage ofresponses before adenosine infusion and reported as mean :t S.E.M.Control responses: nicotine, +25:t 5 mm Hg; ES, +75:t 4 mm Hg andPE. +49 :t 6 mm Hg from basal pressure of 67 % 6 mm Hg. One-wayanalysis of variance lOdicates Significant (P < .01) effect of arterialadenoSine concentration on pressor responses to nicotine, PE and ES,n = 6.

300fUlitC

8-litfUa:

200c;'"0CD

0100

Fig. 2. Relationship between arterial plasma adenosine concentrationand eyelid contractions in response to nicotine (t.). PE (8) and ES (0).Results are expressed as percentage of responses before adenosineInfusion and reported as mean % S.E.M. Control responses: nicotine.+0.22 % 0.05 g; ES. +0.57 % 0.06 g; and PE. +0.21 % 0.05 g. One-wayanalysis of variance indicates significant (P < .01) effect of adenosine oncontractile responses to nicotine. n = 6.

t 2 3 4 5

Arterial Adenosine(ILM)

Fig. 3. Relationship between arterial plasma adenosine concentrationand vas deferens contractions in response to nicotine (t.). PE (8) andES (0). Results are expressed as percentage of responses beforeadenosineinfusion and reported as mean :t 5.E.M. Control responses:nicotine. +11.2 %3.0 mm Hg:ES. +26.9:t 3.7 mm Hg;and PE. +13.1% 2.0 mm Hg. One-way analysis of variance indicates significant (P <.05) effect of adenoSineon response to nicotine. Two-way analysis ofvariance indicates significant (P < .05) difference between effects ofadenosineon PE vs. E5. n = 6.

effects of adenosine on vas deCerentiaresponsesto ES us. PEdiffered significantly (P < .05).

At the heart, adenosinefailed to alter chronotropic responsesto ES significantly (fig. 4), but did potentiate ISO-evoked (0.5pg!kg i.v.) tachycardia by a maximum oC75% at adenosineconcentrations of 3 I'M. Inasmuch as adenosineconcentrationsabove 2 "M tended to lower resting heart rates, it was possiblethat ita enhancement of the chronotropic effect of ISO couldhavebeendue to lowered resting rates, increasing the potentialspread between basal rates and the highest attainable "ceiling"heart rate. Therefore, the extent to which chronotropic reo

Vol 236

Hearl Role300

~ 200c:oQ.'"'"0::

2 3 4 5 6

Arterial Adenosine(101M)

Fig. 4. Relationship between arterial plasma adenosine concentrationand chronotropic responses to nicotine (t.), tSO (8.0.5 1t9lk9) and ES(0). Results are expressed as percentage of responses before adenosineinfusionand reported as mean % S.E.M. Control responses: nicotine.+24 %2 bpm: ISO. +56 %6 bpm; and E5, +27 % 4 bpm frombasalrate of 329 :t 12 bpm. On&-wayanalY5isof varianceindicatessignificant(P < .05) effect of adenosine on responses to nicotine and ISO.n = 6.

TABLE1Effectof nitroprussideinfusionon heart rete responses to ISOControl response to ISO (0.5 ,.9/1<9 Lv.). +79 % 15 bpm.

NtrqIrUSSideInfusion(,.gJkgJnin)

3.0 6.0

323 :t 19 304 % 27104 % 10 122 % 24

0.0

Basalheart rate (bpm) 334 %20Responseto ISO (% of 100 %19

control)

15.0

303 :t 25110%16

sponses to ISO and resting heart rates were correlated afteradenosine administration was determined. Although a signifi-cant negative correlation was observed,it was relatively weak(r2 = 0.47) accounting for less than half of the potentiation ofISOs chronotropic effect. Moreover, some animals displayedstrong potentiation (80-100% increases) of ISO responses inthe absence oClarge (>20 bpm) changes in resting heart rates.The effectof an i.v. infusion o(nitroprusside (3-15 ~g/kg/min),which produces vasodilation and bradycardia in pithed rats uiaa nonpurinergic mechanism, was also tested (table 1). Nitro-prusside had no significant effect on ISOs chronotopic effecteven though it lowered resting heart rates to the same extentas the adenosine infusion.

Effects of adenosine analogs on sympathomimetic re-s~nses. Four synthetic adenosine analogs were also adminis-tered in an attempt to characterize the adenosine receptor~ubtype(s)involvedin pre- or postsynapticmodificationsofsympathomimetic responses. Previous studies indicated thatan adenosine receptor resembling the A2 subtype mediates thepotentiation by adenosine of sympathomimetic responses tonicotine at sympathetic ganglia (von Borstel et oJ.,1984).Theadenosine analogs N-CPCA, 2-CLA and the R- and S- diaster-eomers of PIA were administered i.v. in graded doses 1 minbefore challenge by ES, PE or ISO. Three of the four agonistssuppressed to an equal extent pressor responses evoked by ESand PE (fig.5); N-CPCAwasmostpotent,exhibitinga thresh-old oCaction near 0.5 "g/kg and a half-muimal effect at 1 to 2"g/kg; R-PIAwas lesspotent, requiringdosesof greater than2.5 ~g/kg to inhibit pressor responses significantly and dosesof 5 to 10 ltg/kg to produce 50% attenuation; 2-CLA was less

--- --- - -- -

352 Evoniuk et al.

Eyelid Tension

400

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40

!~ 0,' \~ 0 ~0.1 1.0 10.0 1000 1000

Dose(jlg/kg)

Fig. 5. Effect of adenosine analogs on systemic pressor responses toPE(closedsymbols)and E5 (open symbols).The adenosine derivativesN-CPCA(0). R-PIA (0). 2-CLA (~) and S-PIA (~) were administered. inclosesindicated on the abscissa. 1 min before challenge by PE or ES.ResUts are expressed as percentage of control responses and reportedasmean :t 5.E.M. Controlresponses: ES. +57:t 4 mmHgand PE. +47t" mm Hg from basal pressure of 52 :t 3 mm Hg. One-way analysis ofvarianceindicates significant (P < .001) effect of N-CPCA. R-PIA. 2-CLAandS-PIAdose on pressor responses to both PE and ES. n = 5.

Vas DeferensPerfusionPressure300

0.1 1.0 10.0 1000 1000.0

Dose(jlg/kg)

Fig.6. Effect of adenosine analogs on vas deferens contractions inresponseto PE(closed symbols)and E5 (opensymbols).Theadenosinederivatives N-CPCA (0). R-PIA (0). 2-CLA (0) and S-PIA (~) weredninistered at indicated doses. Resuhs are expressed as percentage01controlresponses and reported as mean :t 5.E.M. Control responses:ES. +26:t 2.3 mmHgand PE.+10 :t 2 mmHg.One-wayanalysisofvN1ce indicates significant(P < .05 or better) effect of all drugs onIIoIhPEand E5 responses except forS-PIAvs. PE. Two-wayanalysis01varianceindicates significant differencebetween PE vs. ES for all four~.nD5.

potent than R-PIA, with a threshold dose of approximately 10,.g/kgand a half-maximal effect at doses between 25 and 50IIC/kg.SePIA was less than one-tenth as potent as R-PIA inattenuating pressor responses to ES; it potentiated pressormponses to PE slightly at doses less than 100 #o!g!kg.andIUppressedthem at doses greater than 100 #o!g!kg.

At the eyelid muscle, all four analogs reduced the contrac-IioDaevoked by ES and PE, exhibiting a potency series similarto that observed for inhibition of pressor responses (N -CPCA> R-PIA > 2-CLA > SePIA. data not shown). In contrast. atthevas deCerens. low doses of adenosine analogs significantlypotentiatedPE-evoked contractions. whereas higher doses sup-

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Adenosine and Sympathetic Neurotransmission 353

prl'ssNi th('m (fi~. 6). Responses to ES were not potentiated.l)Ut instead were attenuated by all four drugs at doses whichpotentiated the effects ofPE; thus, significant differences (P <.05) were observed between the actions of each drug on PE- us.ES-evoked contractions. The rank order of potency N-CPCA> R-PIA > 2-CLA > SePIA was observed for effects on reosponses to both ES and PE.

The same adenosine analogs were also tested for their abilityto alter changes in heart rate evoked by ISO and ES. N-CPCApotentiated the chronotropic effects of ISO and ES (figs. 7 and8); the latter effect was abolished as the dose of N-CPCA wasincreased beyond 2.5 Ilg!kg, converting to inhibition of re-sponses to ES. Of the other three drugs, only R-PIA had asi'gniftcant effect on ISOs chronotropic effect. attenuating the

. response to ISO by 50% at a dose of 50 Ilg!kg; all three

HeartRate- Isoprolerenol300

~2ooco0.'"....a:c;'"..m;r! 100

.0.1 1.0 10.0 100.0 10000

Dose (JLCJ/kCJ)

Fig.7.Effectofadenosineanalogson chronotropic responsesto isopro-terenol. The adenosine derivativesN-CPCA(e), R-PIA (8), 2-CLA (t)and S-PIA (~) were administeredat Indicated doses. Results are ex-pressed as percentage of control responses and reported as mean :t5.E.M. Control responses: ISO. +8O:t 10 bpm from basal rate of 347:t 11 bpm. One-way analysis of variance Indicates significant (P < .05)effect of N-CPCA and R-PIA on responses to ISO.n - 5.

Dose (JLCJ/kg)

Fig. 8. Effect of adenosine analogs on chronotropic responses to ESThe adenosine derivatives N-CPCA (0). R-PIA (0). 2-CLA (~) and S-PIA(~) were administered 8t indicated doses. Results are expressed aspercentage of control responses and reported as mean:t 5.E.M. Controlresponses: ES. +42 :t 7 bpm from basal rate of 347 :t 11 bpm. One.way analysis of variance indicates significant (P < .05) effect of all drugson responses to ES. n = 5.

HeartRote-EleclricalStimulation240

I

2oo

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-/l_m;r! 80

40 ,i'I

. . .1000.I 1.0 10.0 100.0

Evoniuk et al.

significantly reduced the chronotropic response to ES. exhib-iting the potency lIerie!l R-PIA > 2-rt.A > ""'.1'11\.

Discussion

Our data demonstrate that circulating adenosine and it~synthetic congeners can elicit multiple effects on the efferentcomponents ofthe sympathetic nervoussystem. Adenosinemayproduce the changes observed here by acting at sympatheticganglia, noradrenergic nerve terminals and/or sympatheticallyinnervated end organs. At ganglia and end organs, adenosinemay either facilitate or inhibit neurotransmission (von Borstelet aI., 1984; Henon and McAfee, 1983;Hedqvist et al., 1978;Clanachan et aI., 1977),whereas at noradrenergic nerve termi-nals, adenosine has been shown to attenuate neurotransmitterrelease (Wakade and Wakade, 1978;Fredholm and Hedqvist,1980;Hedqvist et aI., 1978).Effects of the adrenergic agonistsPE and ISO are mediated primarily by adrenoceptors locatedon sympathetically innervated end organs (Gilman et aI.,1980),providing an index of the direct effect of adenosine on tissueresponsiveness to adrenergic' stimulation. Responses to ESinvolve, in addition, pre- and postganglionic neural elements.Therefore, differences between the effects of adenosine onresponses to ES and PE reflect relative contributions of neuralvs. end organ processes to the net influence of adenosine onsympathetic transmission.

Adenosine potentiated the sympathomimetic effects of nic-otine at concentrations (2-3 11M)which produced only modest(less than 25%) inhibition of responses to ES and PE.. At theselower concentrations it is apparent that adenosine's potentia-tory effect, presumably mediated through ganglionicadenosinereceptors (von Borstel et aI., 1984), can override its. well-documented inhibitory influence at the sympathetic neuroef-fector junction (Lokhandwala, 1979;Sollevi et aI., 1981).Thus,potentiation of nicotine's ganglionic effects appears to be aden-osine's most potent action in vivo. As adenosine levels areelevated to and beyond 4 11M,its inhibitory action on responsesto ES and PE becomes significant, coinciding with a reductionin the potentiation of nicotine's effect.

The relative contributions of pre- and postsynaptic inhibitionby adenosine appear to vary from tissue to tissue. Equal inhi-bition of the systemic pressor responses to ES and PE suggeststhat postsynaptic mechanisms may be sufficient to account foradenosine's inhibition of vascular sympathetic transmissionduring ES. However, this does not preclude an additionalpresynaptic contribution to reduction of pressor response toES, as has been demonstrated in isolated rat vascular tissue invitro (Kamikawa et al., 1980; Kubo and Su, 1983;Enero andSaidman, 1977). The potency series N-CPCA > R-PIA > 2-CLA > S-PIA is similar to that observed for inhibition ofpressor responses to noradrenergic stimulation in the isolatedguinea-pig aorta (Collis and Brown, 1983).

At the vas deferens, unlike vascular and eyelid smooth mus-cle, the effect of adenosine analogs on PE-induced contractionwas biphasic; responses to PE were potentiated at low dosesand inhibited at higher doses (fig. 6). This could indicate thepresence of two or more subpopulations of postsynaptic aden-osine receptors, differing in both their affinities for adenosineand tbe direction of their effects on vas deferens responses toadrenergic stimuli. Although enhancement of postsynaptic re-sponses to adrenergic agonists has not been reported previouslyin the rat (Clanachan et aI., 1977), this phenomenon has been

Vol. 236

detected in the isolated guinea-pig vas deferens (Holck andMurk><, I !I7H).

Adenosine receptor subtypes have been defined according toseveral characteristics. The Al (or Ri) subtype, as characterizedin the central nervous system, inhibits adenylate cyclase, has ahigh affinity constant (10-100 nM) for adenosine and is char-acterized by the potency series R-PIA > 2-CLA,N-CPCA > S-PIA; the A2 (or Ra) subtype activates adenylate cyclase, has aloweraffinity for adenosine (2-20 11M)and is characterized byt.hepot.encyReriesN-CPCA. D-N-ethylcarboxnmiclonc!l'nollillt.> 2-CLA > R-PIA > S.PIA (Van Calker et al., 1979,Daly et0/., 1981;Londos et aI., 1980).This classificat.ionscheme mllYnot be generally applicable to all adenosine receptors; manyactions of adenosine do not appear to be mediated throughalterations in adenylate cyclase activity and peripheral recep-tors have not yet been defmed rigorously according to thecriteria established for central adenosine receptors.

Adenosine analogspotentiate sympathomimetic responses tonicotine with a potency order of N-CPCA > 2-CLA > R-PIA> S-PIA, suggesting the involvement of A2 receptors (vonBorstel et al., 1984; see also preceding report). However, therank order of potency for attenuation of pressor effects of ESand PE, and for both potentiation of responses to PE andattenuation of responses to ES in the vas deferens was N-CPCA > R-PIA > 2-CLA > S-PIA, and 2- to 10-fold higherdoses of the analogs were needed to produce these effects ascompared to doses needed to potentiate responses to nicotine.This latter order does not conform clearly to the Al vs. A2classification (which is now based solely on relative potenciesof adenosine agonists), suggesting either differencesin receptorsubtypes mediating adenosine's effects at ganglia vs. neuroef.fectorjunctions or differences in drug bioavailability at the twosites.

Adenosine's potentiation of chronotropic responses to ISOwas unexpected; in vitro, adenosine reduces cardiac norepi-nephrine release and attenuates the inotropic and chronotropiceffects of beta adrenergic stimulation (Rockoff and Dobson,1980;Rardon and Bailey, 1984;Dobson, 1980).In the presentin vivo studies, circulating adenosine concentrations of 3 11Mor more potentiated the positive chropotropic effect of ISO; inaddition, N-CPCA potentiated the effects of both ISO and ES.N-CPCA is more potent as a coronary vasodilator than theother analogs tested in this study (Evans et al., 1982); conse-quently, improved myocardial acce88 of circulating catechol-amines might explain N-CPCAs enhancement of the positivechronotropic effects of ISO and ES and could underlie thepotentiation of ISO effects produced by adenosine itself in vivo(Seitelberger et aI., 1984) (fig. 4). However, the failure of thenonpurinergic vasodilator nitroprusside to produce similar po.tentiation of ISOs chronotropic effect suggests that the poten-tiation was producedby a more specific purinergic mechanismwhich was not secondary to systemic or coronary vasodilation.

The action of adenosine in potentiating responses to nicotinebut not to ES was somewhat surprising, as both are generallypresumed to involvestimulation of nicotinic ganglionic recep-tor~. The present finding contributes to the body of evidencesuggesting that different subsets of nicotine receptors maymediate the effects of endogenous acetylcholine and of exoge-nous nicotinic agonists. Thus, at ganglia, autodesensitizationto nicoti~e does not impair postganglionic responses to ES(Bentley, 1972), and the hemicholinium analog a,a'-bis(dimethylammoniumacetaldehyde diethylacetal)p,p' -diace.

---- --- - -

1986

tylpbenyldibromideblocks gan~1ionicstimulation by nicotine.tetramethylammonium or dimethylpiperazinium withoutblockingtbe effects of ES (Wong and Long. 1968). We havefoundthat several nicotinic antagonists. including d.tubocu.rarine,can block sympatbomimetic effects of nicotine (in thepresenceor absence of adenosine) at doses which have littleeffecton responses to ES (unpublished data). Adenosinepoten-tiates sympathomimetic responses to other nicotinic agonistsincludinglobeline,dimethylphenylpiperazinium iodide, sebacylcholineand tetramethylammonium chloride. indicating its in-teractionwith nicotine reflects a general effect, one not peculiarto nicotine itself (von Borstel et al., 1986).

In summary, these data indicate that adenosine modulatesaympathetic neurotransmission through actions at multiplelites including ganglia, presynaptic noradrenergic nerve ter-minalsand postsynaptic end organs receiving sympathetic in-nervation.The net effectof an increase in circulating adenosinelevelson sympathetic transmission reflects the aggregate of itsindividual actions at these anatomical loci, as well as thepoesibleemtence of different subpopulations of adenosinerecepton within some of these sites. The multiplicity of aden-osine'seffects within the sympathetic nervous system suggeststhat the nucleoside and its congeners, as well as compoundsthat inhibit their actions (like caffeine), may possess a muchmore subtle array of effects than are currently attributed tothem.

IerereDc:es

B£NTL&Y.G. A.: Pharmacological studies on the hypogastric ganglion of the ratand guinea pig. Br. J. Pharmacol. 44: 492-509. 1972.

CLANACHAN.A. S.. JOHNS. A. ANDPATON.D. M.: Presynaptieinhibitory actionsol adenine nucleotides and adenosine on neurotransmisaion in the rat vasdelerens. Neuroscience 2: 59;-602.1977. .'

CoLUS,M. ANDBROWN.C.: Adenosine relaxes the aorta by interacting with anA2 receptor and an intracellular site. Eur. J. Pharmacol. 96: 61-69, 1983.

CoRNroRD, E. M. ANDOLDENDORr.W. H.: Independent blood.brain barriertrlDlport systems lor nucleic acid precursors. Biochim. Biophys. Act.8 394:211-219. 19;5.

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SeDd repriDt requeata to: Dr. Gary Evoniuk. Room £25-604, Department ofApplied Biolocica1 Sciences. M chU81tts Institute of Technology, Cambridge.MA 02139.

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