Eur. J Biocheni 2.j4, 466-471 (1995) 0 FEES 1995

The mechanism of action of hepatic sympathetic nerves on ketone-body output from perfused rat liver The effect of the interaction of noradrenaline with ATP on the release of /3-hydroxybutyrate

Tetsuya YAMAMOTO'.', Masaru IWAl I, Shigeru KIMURAL and Tdkashi SHIMAZU I I Department of Medical Biochemistry, Ehime University School of Mcdicinc, hpm

Second Department of Surgery, Ehinie IJniversity School of Medicine, Japan

(Received 7S September 1995) -. EJR 95 15h3/1

The regulatory mechanism of ketone-body output by the hepatic sympathetic nerves was studied in rat liver perfuscd iiz siti~. Enrichment of thc perfusion medium with 1 mM octanoate increased the basal ketone-body output from the liver up to 1.5 p i 0 1 . inin I 1 g liver -'. Under thcsc conditions, electrical stimulation of the hepatic nerves (20 V, 20 Hz, 2 ms) decreased the output of both aceloacetate and /3- hydroxybutyrate, and was accoinpanied by an accumulation ol P-hydroxybutyrate in thc liver. The effects of nerve stimulation were inhibited by the a,-antagonist bunamsin (10 pM). However, noradrenaline, a typical sympathetic neurotransmitter, at a conccntration of 1 11 M decreascd (he output of acetoacetate but did not affect @-hydroxybutyrate output. Prostaglandin F,, at a concentration of 10 pM produced an effect similar to treatment with noradrenaline, without a dccrcase in /l-hydroxybutyrate output. ATP at 50 pM, however, decreased the output of both acetoacetate and 11-hydroxybutyrate and incrcased the tissue con- centration of /I-hydroxybutyrate, mimicking the effect of nerve stimulation. Moreover, in thc presence of 0.2 pM ATP, a concentration that produced neither metabolic nor hemodynainic changes, noradrcnaline (1 pM) was shown to decreasc the p-hydroxybutyrate oulput. These results indicate the p ment of ATP in the action of hepatic sympathetic nerves on ,&hydroxybutyrate output presumably through its interaction with noradrenaline.

Keywords: perfused liver; ketone body ; sympathetic nerve ; noradrenaline ; ATP,

Liver is innervated by the sympathetic and parasympathetic nerves, which consist of the nerve plexus around the hcpiitic artery and portal vein, and which are concerned with the meta- bolic and heinodynamic rcgulation of the liver [I -41. In per- fused rat liver, the stimulation of the perivascular nerves CBUSCS

metabolic changes such as an increase in glucose production accompanied by a reduction of perfusion flow 15, 61, and a decrease in bile acid secretion and bile flow 171. Since these changes during ncrve stimulation were inhibited by an n-blocker 131, and nerve stimulation incrcased noradrcnaline overtlow into the effluent 16, 81, the effects of hepatic nerve stimulation were considered to be mediated mainly by noradrenaline through u , - adrenergic receptors.

It has previously been rcported that hcpatic nerve stimulation decreases the output of ketone bodics, acetoacetatc and /r'-hydro- xybutyrate, from the liver perfused in situ [9]. In our preliminary experiments [1Ol, however, the infusion of noradrenaline into the portal vein did not totally mimic ncrve action. Noradrenaline decreased the output of acetoacetate but not the output of /!- hydroxybutyrate. These observations suggest the involvement of some factors other than noradrenaline in hepatic nerve action on ketone-body output from perfuscd rat liver.

The present study, therefore, examined the difference be- tween the effects of hepatic nervc stirnulation and exogenoudy infused noradrenalinc on ketone-body output. The effects of sev-

Corr~/~~)~)nd~,riLe to M. Iwai, Department of Medicd Biochemistry, Ehirnc University School of Mcdicine, Shigcnobu, Ehi mc, Japan 791 -02

~. --

era1 cmdirlates for transmiller suhqtances were also examined and the rcsults were comparcd with those of nerve stimulation.

MATICRIALS AND METHODS

Materials. All chemicals were analytical grade. n-Octanoic acid, noradrenaline hydrochloride, ATP, adenosine, prostaglan- din F,(? rind BSA wcrc purchased from Sigma. Surainiii and bunamsin hydrochloride were gifts from Rayer AG and Eisai Co. Ltd., respectivcly. Other reagents were from Wako Pure Chemicals (Osaka).

Animals. Mule Sprague-Oawley rats, weighing 170-230 g, were kcpt at 25 2 1 "C on a 12-h light-dark cycle (lights on from 6 am to 5 pin) with frec access to watcr and laboratory chow (MF, Oriental Yeast Co.). The experimental prot~col was ap- proved by the Animal Studies Committee of Ehimc University.

Liver perfusion. All experiments were started between 10 mi and 12 am. Animals were anesthetized by intraperitoncal injection of sodium pentobarbital (SO nig/kg body mass). The livers were perfused in situ via the portal vein without rccircula- tion in a 37°C cabinet. The perfusion medium was Krehs- Hcnseleit bicarbonate buffer, pH 7.4, containing 5 mM glucose, 2 mM lactate, and 0.2 mM pyruvate, and was equilibrated with 95% 0, and 5 % CO,. Livers were prcviously perfused for 20 min before starting the experiments. 7 min before giving il stimulus, the perfusion medium was enriched with 'I mM octa-

467

Time (min)

Fig. 1. Alterations in ketone-body output and perfusion flow induced by hepatic nerve stimulation or noradrenaline infusion. Livcrs were perlused in .vitm without recirculation as dcscribed in the Materials and Merhods sccfiun. Thc nerve plcxus around thc pcirhl vciii and thc hcpilric: artcry was stimulated for 32-37 rnin of perfusion (nerve stirtiulalion: 20 V, 20 HI. 2 ins). Noradrenalirle (NA) wah inlused for 32-37 tnin 01 pcrfusion at a concentration of 1 pM. Bunamsin, :in n,-antagonisr. was infuhed at a ct.mcentratim of 10 p M . V;ilues arc Itir nieatis L SEM for. ltlrer or four rats. NS, nerve stimiulation. NA, noradrenalinc.

noate i n 0.5% RSA in order to enhance ketonc-body producrion. 'Ihc perfusion pressure was constant at about 10 cm H,O with a flow rate of 4 in1 . min ..I . g liver-' under basal conditions. The flow rate was measured by fractionating the effluent.

Nerve stimulation and infusion of chemical substances. The hepiitic nerves were stimulated electrically (20 Hz, 20 V. 2 ms) with u bipolar platinum-wire electrode placed around both the portal vein and hepatic artery. Noradrenaline was first dis- solved in 0.9%~ NaCl containing 1 rnM ascorbic acid, and diluted with the perfusion incdium containing 0.1 % BSA to obtain the final concentration of 1 pM. ATP, prostaglandin F,,,, bunamsin, and surarnin wcre dissolved in the perfusion inedium to obtain final concentrations of 51) pM (or 0.2 pM), 10 pM, 10 pM, and 800 pM, respectively. Adenosine was first dissolved in 1 M HUI, iind subscquently diluted with the perfusion buffer to obtain the final conccntration of 50 pM.

Assay of ketone bodies. Acetoacctate and P-hydroxybutyr- ate in the effluent were measured with standard enzymic rneth- ods [ 11 J aftcr deproteinization with 10% perchloric acid. To de- termine the tissue concentration of ketone bodies, portions or perfused liver were rapidly frozen in liquid N, by freeze clamp- ing 2.5 inin after thc onset of stimulation; samples were then crushcd in 6% perchloric acid ( I 2 ml/g tissue mass). After cen- trifugation at 3000 rpm for 5 i i i in in a swing-rotor (TS-7; Tomy, Tokyo), thc supcrnatant was neutralized with solid K,CO, a( O0C, and ketonc bodies wei-e assayed as dcscribed above. The tissue concentration of ketone bodics was estimated using the ratio of water distribution within livcr salnples as described by Fafournoux et al . [12].

Statistical analysis. The effects of various sti tnuli were evaluated by uniilysis of the variance followed by a Newman- Keuls' test for rnultiple comparisons. The difference was consid- ered to be significant if P was lcss than 0.05.

RES U I ,TS The effects uf hepatic nerve stimulation and noradrenaline infusion on ketone-body output. TL) enhance h e basal activity of ketone-bcody production, 1 rnM octatioate was added as II sub- stratc into thc perfusate 7 min before the addition ol' stimuli. After thc addition of octanoate, lhc outpui of aceloacetale and /l-hydroxyhutyrite increased up to :ihout 0.5 yrrid . tnin ' . g liver..' and 1 pio l + min- ' . g liver respectively (Fig. 1) . The addition of octannate did not change thc basal rate of perfusion ilow.

As shown i n Fig. I , electrical stimulation of the hepatic nerves decreascd the output of both acetoacetate and /3-hydroxy- butyrate and was accornpanicd by ;I rcduction of the flow ratc; the results iirc similar to thosc reporied previously hy Beuers ct al. [9]. The effects of nerve stimulalion on ketone-body output were prevented almost completely by the (1,-antagonist buna- zosin (13) (Fig. 1).

The infusion of noradrenuline, a lypical neurotransmitter of the sympathetic nerves, into the portal vein iit a concentration of 1 pM decreased the flow rate und acetoacetate output in a nian- ner similar to that obtained in response to nerve stimulalion. Howcver, noradrenaline infusion did not tlccrerrse the output of /~-hydroxybutyratc (Fig. I ) .

The effects uf prostaglandin F,,x, ATP, and adenosine on ketone-body output. Thc infusion of prostaglandin FZ,, (PGF,,,) at 10 pM dccreascd the flow rate (Fig. 2 ) and increased glucose output fi-om the liver (data not shown) 1141 i l l ii manner sirriilur t o the respoiisc to tiervc stirnulation. With regard to kctone-body output, however, PGF,!,, could decrease the output of acctoacc- kite but not /3-hydr-oxybutyrutc output (Fig. 2 ) . In this regard, the effects of I'GFZ,, were very similar to those of noradrenaline but werc diffcrcnt from the effects of iiervc stirnulation (Fig. 1).

468 Yamamoto et al. (Eur. J. Biodzem. 234)

Time (min)

Fig.2. Changes in ketone-body output and perfusion flow induced by prostaglandin F,,,, ATP or adenosine. Livers were perfused as described i n the legend uf Pig. 1. Prostaglandin F,,x, ATP, and adenosine were infused for 32-37 inin of perfusion at concentrations of 10, 50 and SO pM, rcspecrively. Values tire the means i SEM for three rats. PGF,,, prostaglandin F>(,.

Table 1. Effect of suramin on the balance of ketone-body output induced by nerve stimulation or ATP infusion in pertiised rat liver. The changes in ketone-body output were measured with (+) or without (-) 800 pM surarnin and are expressed as areas under the curve (AUC). Values arc the means f SEM for three or four rats.

Ketone body Keionc-body balance with

no siimulus nerve stirnulation ATP (SO pM)

5 - - + + AllC 1 pmol - g liver-'

Acetoacetate 0.07 L 0.04 -0.08 ? 0.04 - 1.19 t 0.18 -1.07 Z 0.16 - 1.60 t 0.20 -0.33 ? 0.24" /%Hydroxybuiyrate 0.02 * 0.04 0.05 * 0.04 - 1.27 +I 0.19 -0.72 Z 0.16 .- 1.28 t 0.32 0.46 2 0.09,'

" Significantly different from the valucs without suratnin (P<O.OS J

I n contrast, thc infusion of ATP into the portal vein at a concentration of SO pM decreased the output of both acetoace- [ate and /I-hydroxybutyrate in a iniiiiner similar lo that obtaiiied after hepatic nerve stimulation (Fig. 2). Since ATP is metabo- lized in the liver, it might be possible that the effects of ATP are due to its metabolite. The ineasurement of ATP concentration in the influent and effluent revealed that approximately 80% of the infused ATP was metabolized by a single passage through the liver (data not shown). However. adenosine, a major metabolite of ATP, at 50 pM did not cause any change i n ketone-body out - put (Fig. 2) .

Tissue concentration of ketone bvdies during nerve stirnula- tion and infusion of noradrenaline or ATP. In addition, to cxamine whether the output of kclone bodies reflccts changes in the intracellular concentration of ketone bodies, their tissue

coiicentrations were assayed after application of differcnt stim- ul i . The tissue concentration of /~-hyciroxybutyratc in perfused liver was increased by about 30% and 35%) of the control value (0,313 -t0.024 pmol + g liver I ) by nervc stimulation and ATP infusion, respectively, but it did no( change significantly after noradrenaline infusion. The concentration of acetoacetate, how- ever, did not change appreciably with these stimuli (data not shown).

'Che effect of suramin on the changes in ketone-body output induced by nerve stimulation or infusion of ATP. Table 1 shows the changes in ketone-body output induced by nerve stimulation or ATP with and without suramin, a P,-receptor antagonist [15-17], which are expressed as areas under the curve. The infusion of surainin alone at a concentration of 800 pM did not affect ketone-body output or the flow rate. The

1 .o

0.5

0

4.0

3.0

30 40 Time (min)

Fig.3. The effect of the interaction of noradrenaline with ATP on ketone-hody output in perfused rat liver. Livers were perfused as dc- scribed in thc legend of Fig. 1. Noradrenaline and ATP werc infused at concentralions of 1 FM and 0.2 pM, respectively. Values are the means 5 SEM for three rats. NA, noradrenaline.

cffects of ATP on ketone-body output (Table 1) and flow rate (data not shown) were almost totally nbolishcd by suramin, ex- cept that the b-hydroxybutyrate output was cxceeded in the pres- ence of suramin (Table 1 ) Suraniin also caused a tendency to suppress the decreasc in hetonc-body output in response to nerve stimulation (Table 1 ).

The interaction of noradrenaline with ATP on P-hydroxybu- tyrate output from perfused liver. When the concentration of ATP was lowered to 0.2 pM i n the perfusion experiments, it had no apparent effects on the tlow rate and ketone-body output (data not shown). In the prcsence of this low concentration of ATP, however, noradrenaline elicited an apparent decrease in the output of /Lhydroxybutyrate together with a decrease in ace[- oacetate output (Fig. 3), thereby totally mimicking the effects of nerve stimulation.

DISCUSSION

The prcscnt study demonstrates the pu noradrcnali tie with ATP in mediating thc e pathctic nerves on ketone-body output from perfused livcr of rats. It was shown that clcctrical stimulation cif hepatic sympil- thGtiC IlGrveS caused the dGCEaSed oulpul of both XetOaCehle and /1'-hydroxybutyrate. Although noradrenaline could deurcase the acetoacctutc output but not the P-hydroxybutyrate outptkt, in the presence of a low concentriition (0.2 pMj of ATP il exhibited effects on both acctoacetate and P-hydroxybutyrate output. thereby coniplctcly mimicking the action of hepatic sympathetic llerves.

In the present study, the effects of nerve stirilulation and the infusion of several transmitter substances on ketone-body output were examined by supplcinenting with octailoiitc to enhance the basal activity uf kctone-body production in the liver. Since octa- noate is not utilized directly for trincylglycerol synthesis 1 l X ] and it enters into the mitochondria independently of the carnitine system [19], the effects of nerve stimulation on ketone-body pro- duction can he analyzed without consideration of these nleta- bolic processes. Indeed, enrichnient of the perfusate with I mM octanuatc increased ketone-body release about 1 5-fold. (Jnder thesc conditions, hepatic nerve stimulation resulted in a dc- creased output of both acetoacetatc iind lI-hydlnxyhutyrate ac- companied with the reduction in flow rate (Fig. 1). It was pre- viously shown that the decrcasc i n ketone-body output during sympathetic nerve stimulation is not due to thc reduction of pcr- fusion flow, since preventioii of the hernodynamic change with sodium nitroprusside did not affect the decrease i n ketone-body output caused by ncrve stimulation 191.

With the use of perfused rat liver, it has also been reported that metabolic effects of hepatic nerve stimulation are incdiated mainly by noradrcnnline [3]. Hcpatic nerve action on ketonc- body output was almost completely inhibited by the (1,-antago- nirit bunazosin (Fig. 1) . However, infusion 01 1 pM noradrena- line could not totally rnimic the effect of nerve action on kctone- body oitlput (Fig. 1 ) . This dose of noradrenaline is rclatively high cnmpnred with thc physiolopicul concentration, but i t has been used iis the dosc that mimics thc metabolic and hel-riody- namic effccts of hepatic nerve stimulation in perfused rat livcr 16, 20-22 1. Although noradrenaline at a lower concentration of 0.1 pM n o longer mimicked the hepatic nerve action on oxygen consumption in perfused rat liver 1221, Bcucrs et al. [ C l l pre- viously noted that 0.1 @I noradrenaline did tint decrease /&hy- droxybutyratc output like 1 pM noradrenaline. Therefore. the differen1 effects observed after nerve stirnulation and noradrena- line infusion on /j-hydroxybutyrate output are common irrespec- tive of the concentration of noradrenaline used, though thc dif- ference has not been discussed satisfactorily i n the previous re- port [91.

In the present study, changes in ketone-body output werc also exiimined with prostaglandin F,, iind ATP (Fig. 2). Prosta- glandins are produced in sinusoidal cells of the livcr 1231 and are considered to be mediators or modulators or licpatic nervc action on glucose metabolism 114. 20, 241. However, the el'fcct of prostaglandin Fj,L on ketone-body output was essentially simi- lar to that of noradrenaline (Figs 1 and 2) . ATP is also known to act as a cotransmitter of noradrenaline in sympathetically iti- nervated tissues of various species [251. I n perfused rat livcr, ATP caused an increase i n the output of glucose arid liictnte and a decrease in tlow ratc, which arc very simil:ir to the effects observcd after iicrve stimulation [20, 26, 271. With regnrd to ketone-body output from the liver. infusion of SO pM ATP also mimicked the effects of nerve stitnulation (Fig. 2). Moreover, thc action of ATP appeared to be niediated by the P,-type purinergic

470 Yarnamoto ct al. (Etrr: J Htochvn. 234)

receptor [28], because its antagonist suramin reversed the ATP- induced decrease i n ketone-body output (Table 1 ), and because a PI -receptor agonist, adenosine, had no apparent effect on ketone- body output (Fig. 2) .

The decreased output of ketone bodies in response to ncrve stirnulation, noradrenaline, prostaglandin F,?,,, and ATP may re- tlect the decrease in ketone-body synthesis in hepatocytcs. All these stimuli decreased consistently the output of acetoacetate. I n addition, noradrenaline was shown to inhibit ketogenesis in isolated hepatocytes 1291. However, judging froin the distinct effects of different stimuli on /I-hydroxybutyrate output (Figs I and 2), the decreased output of ketonc bodies caused by nerve stimulation cannot be simply explained by the inhibition of ketone-body synthesis. The measurement of tissue con- centrations of ketone bodies after application of different stimuli indicates that the decrease in /I-hydroxybutyrate output from perfused liver in response to nerve stimulation or ATP infusion was, at least in part, duc to the inhibition of its secretion froin hepatocytes, since the tissue concentration of 13- hydxoxybutyrate increased aftcr nerve stimulation or ATP infusion. These results also suggest that therc might bc a releasing mechanism of P-hydroxybutyratc different from that of acetoacetate. Although the detail of this rnechanism has not yet becn clarified, it is possihly related to the difference in the ratio of’ the intracellular to extracellular distribution of ketone bodies

Considering these results logether, it is possible that there are separate mechanisms regulating the output of acetoacetatc and p-hydroxybutyrate. Possibly, the decrease i n acetoacetate output is caused mainly by the change in synthetic late. How- ever, the output of P-hydroxybutyrate is regulatcd, at least in part, by its secretion or release from hepatocytes, and ATP might be involved in this process. This regulatory process docs not seem to depend on tlow change, since noriidrenalinc and prosta- glandin F,,, reduced the trow rate but did not altcr the jl-hydroxy- butyrate output.

Although our results suggest the involvement of ATP in he- patic nerve action on ketone-body output, the effects of nerve stirnulation were almost conipletcly abolished by thc u,-antago- nist (Fig. 1). The most likely explunation for this discrepancy is cotransmission and interaction of noradrenaline with ATP. In- deed, in the presence of ii physiologically low concentration (0.2 pM) of ATP, noradrenaline that could not affect /I- hydroxybutyrate output (Fig. l), could cause an apparent decrease in 8-hydroxybutyrate output (Fig. 3). The existence of such an interaction between ncrradrcnalitle and ATP could ex- plain thc inhibitory effect of thc cx,-aritagonist on hepatic nerve stimulation, arid supports the involvemcnl (of ATP in the action of hepatic sympathetic ilerves on ketone-body output in rat liver.

1121.

The authors gratefully acknowledge the gifts ol‘ surainin Irorn Uayer AG (Imwkusen, Ceiinany) m d hunazosin from Eisni Co. 1,td. (Osaka, Japan). This work was supported in par! hy a Grant-in-Aid for scientific research from the Ministry ol Rducariun, Science and Culture of Japan.

REFERENCES 1. Shimazu, T. (1987) Neuronal regulation of hepatic glucose rnetaho-

lisni in Iuammals, Diah / r , r . MLJtub. ih). 3, 185- 206. 2 . Shimazu, ‘1, Rr Iwai, M. (1953) The hypothalamus, syiiipathctic

nerves and regulation of pcripheral glucose metabolism, in N o w ,furictiond uspwts Cf tho sul)rrrLhW.~rriatic nut:leus ur rlie h y p o r h d uwius (Nakngawa, II. . Oomura. Y . & Nagai, K.. eds) pp. 93-103, John Libbey Rr. Co., London.

3. C;ardeiiianii, A., Piisclicl, C. & Jungermann, K. (1992) Nervous con- trol of liver metabolism and hemodynamics, Em .I. Biochrm. 207,

4. Lault. MI. W. (1983) Afferent and effei-en1 neural roles in liver I\lnc- tion, Prog. Neurmbiol. 21 , 323-348.

5 . Hartmnnn, H., Beckh, K. & Jungermann, K. (1982) Direct control of glycogen metabolism in the perfused rat livcr by the sympathetic

Biochrm. 12.3, 521 -526. 6. Iwai. M., Miyashita, T. & Shimam, ’r. (1 99’1) Inhibition of glucose

production during hepatic nerve stimulation iii regeneraring rat liver perfused irt siru: possible involvement of gap junctions in the aclion of sympatheric ncrvcs, Elm J. Rh:hern. 200, 61)- 74.

7. Hcckli, K. & Arnold, R. (1991) Regulation 01- hile sccretion by sympathetic nerves in wt liver, h i . .I. Physiol. 261, C775- ci780.

X. Beckh, K., Blaks, H. J. & Jungei-mann. K. (1982) Activation of glycogenolysis and norepinephrine overflow in the perfused rat liver during repetitive perivascular nerve stimulation, FEBS Lrx

9. Heuers, U., Beckh, K. & Jungermann, K. (1986) Control ol‘ketogcn- esis i n the perlused rat liver by thc sympathetic innervation, ELK .I. Biochrm. 158, 19-24.

10. Ymamoto, T., Iwai, M., Kiinura, S. & Shimnzu, T. (1992) Effect of hepatic sympathetic activation on the kctone-body output fi-orn perfused rat liver, Jpti. J. Biochem. Soc. 64, 934.

1 1. Kientsch-lhgel, R. 1. Kc Siess, E. A. ( 1 985) o-(-)-3-Hydroxybulyrate and aceroacctate, in Mrthorls qf’ mzvmrrtic nnrr/vsi.5 (Hergineyer, H. U., ed.) 3rd cdn, vol. 8, pp. 60-69, V C H Vei-lagsgesellschaft in b H, Weinheirri.

12. Pafournoux, P., Dernignk, C. & RirnBsy, C. (1987) Mcchaiiisms in- volved in ketone body release by rat livcr cells: influence of pH and hic:irbonatc. Am. J. Phy.rio1. 252, G200-G208.

13. Suxuki, H., Ishikawa, S., Nagao, T., Komori, K., Ibengwe, J. K. & Fujioka, M. (1987) Effects of hunazosin on electrical responses of smooth muscle cells of the guinea-pig mesenteric artery and vciii to perivascular nervc stimulation and 10 noradrenaline, Gerl. Phwrtioccd. 18, 171 -177.

14. Iwai, M., Gardemmn, A., Piischel, C;. & Jungermann, K. (1988) Potential role for prostaglandin F,,,, D,, E2 and thromhoxme A, in mediating the metabolic and hemodyiiarnic actions of sympatlieric ncrves in perfused rat liver, Errr J . /liodiern. 175, 45 -SO.

15. Hertog, A. D., Nclenians, A. & Akker, V. D. (1989) The inhibitory aclion of suramin on [he P,-purinergic recepror response i n siiioorh muscle cells of guinea-pig taenia cacci. Eur. .I. Phnrmrc- col. 166, 53 1-534.

16. IIoyle, C. 11. V., Knighr, C;. E. & Rurnstoch, C;. (1990) Surainin nntagonizcs response to P,-purinerpic rcccptor agonists and ~ L I -

rinergic nerve stimularion in the guinea-pig urinary bladder a i d taeiiia caeci, Br: J . Phnrnirrcol. 99, 617-62 I.

17. Inoue. K.. Nakazawa, K., Ohara-lmaizumi, M., Oh:tiii;i, T., hjiinuri, K . & Tukanakn, A. (1 991) Selective and compelitivc nntagonism by suriiinin of ATP-stimulated catccliolaminc-secretion froin PCl2 phaeochrc~mocytori~~ cells, Br ./. niar?wr’n~. 102, 581 - 5x4.

1 X. Frirx, I . B. ( I!)hl) Fncrors intluencing the r a m of long-chain Catty acid oxidation and synthesis in inarnmaliiln systems, Physiol. R P I ~ . 41, S2-129.

19. McGai-ry. J. D. & Foster, D. W. (1980) Kegtilation of hepatic thlly acid oxidation and ketone body producrion, Anrin. KN Biodwm. 49, 395-420.

20. Twai, M. & Jungermann, K. (19x7) Possible involvement 01- eicosa- rioids in the actions of synipathetic hepatic ncrves on carbohydrnte mctabolisrn and hemodynatnics in perfused rat liver, FhXS Lrll. 221, 155-160.

21. J i , S., Tkckh, K. & Jungermann, K. (1984) Regulatioli of oxygen corisuriiption and microcirculation by a-sympathetic nerves in iso- lared pcrfused rat liver. PEBS Lr’tt. 167, 1 17- 122.

22. Beckh, K., Otto, R., J i , S. k Jungemann, K. (1985) Control of oxygen uptake, rnicrocirculation and glucose release by circulat- ing noradrenaline iii perfused rat liver, Rio l . Chrnt. Hoppe-Sev1r.r 366, 671 - 678.

399-41 1.

140, 261-265.

23.

24.

2s.

26.

Yarnatnoto et i l l . (Eitr J . Bioc./te/rr. 234) 47 1

Birmelin, M. & Decker, K. (1984) Synthesis oiprostanoids and cy- clic nuclcotides by phagouytosing wt Kupffer cclls. 6ur: J . Biw chew. 142, 219-225.

Shimazu, T. & Usami, M. (1982) Further studies on the mechanism 01- phosphorylase activation in rabbit liver in response to splarrch- tiic ncrve stimulation, J. f h y s i n l . 329, 231 -242.

vcln Kugelgen, I. & Starke, K. ( 1 991 ) Noradrenaline-ATP co-trnns- mission in the sympathetic t iervoi ib system, Trmd.s Plrirrmor~d Sci. 12, 319-324.

Buxtonc, D. B., Robertson, S. M. & Olson, M. S. (1986) Stimulation of glycogcnolysis by adenine nucleotidcs in the perrrised rat livrr. Hiiwhrrn. J . 237, 113-780.

21

28.

29.

HIussingcr. D., Siehle, T. & Geroh, W. ( 1987) Actions ol-exti-acellu- lar II'W and ATP itr perfused rat liver. A ccrtriparntive study, Eft?: .I. N i o c . h m . 167, 65 -1 1.

Rurnstnck, G. &L liennedy, C. (1985) Is there ;I basis lor distinguish- ing two types of I>,-purinoceptor '! Gm. Phrrrniai~ol. 16, 433 - 440.

Notnunr, T., Nomura, Y., Txhibann, M., Nomura, H., Ilkai, K.. Yokoyanin, K. & Hagino, Y. (1 991 1 tr,-AdJttnt.rglC regululion of ketogenesis iii isolated hepatocytcs, Biochhr. Bioph,y ,r . Arln 1092, 94 - 100.