BIOSYNTHESIS OF PROSTAGLANDIN E BY RAT SUPERIOR CERVICAL GANGLIA’ J. G . WEBB,D. A. SAELENS’and P. V. HALUSHKA Department of Pharmacology, Medical University of South Carolina, Charleston, SC 29403. U.S.A. (Received 21 October 1977. Accepted 24 January 1978)
Abstract-The biosynthesis of immunoreactive prostaglandin E (iPGE) was examined in homogenates of rat superior cervical ganglia and in isolated intact ganglia incubated in vitro. Ganglia homogenates produced iPGE from exogenous arachidonic acid. Prostaglandin synthesis by the homogenates was inhibited by the prostaglandin synthetase inhibitors, eicosatetraynoic acid, indomethacin and sodium meclofenamate and was stimulated by norepinephrine and dopamine. Whole ganglia incubated in Krebs-bicarbonate solution also synthesized iPGE which was released into the incubation bath in a time-dependent manner. As observed in the homogenates, norepinephrine and dopamine enhanced iPGE formation by the intact tissue. Phospholipase A also stimulated iPGE synthesis by the whole ganglia. The effect of phospholipase A was antagonized by dibutyryl cyclic AMP but not by dibutyryl cyclic GMP. The results suggest that neuronally synthesized prostaglandins may be available for modulating adrenergic neuron function and that endogenous neuronal constituents such as catecholamines and cyclic AMP may influence the activity of the prostaglandin synthetase system.
PROSTAGLANDINS are synthesized by numerous mam- prostaglandins (HEDQVIST, 1973) and, conceivably, the malian tissues and have been proposed t o act as hor- prostaglandins released during sympathetic nerve mones for the modulation of a variety of cellular func- stimulation may arise from postjunctional sites. Howtions. The interactions of prostaglandins with ever, it is also possible that the adrenergic neurons adrenergic neurons appear particularly significant. themselves may b e a source of endogenous prostaFor example, stimulation of sympathetic nerves has glandins. Indeed, STlARNE (1972) has suggested from been demonstrated to release prostaglandin E at indirect evidence that t h e prostaglandins which inet al., 1968; G~LMOREfluence norepinephrine release from the guinea pig neuroeffector junctions (DAVIES et a!., 1968) and exogenously administered prosta- vas deferens may originate from prejunctional nerve glandins of the E series have been shown t o inhibit endings. I n the present study, we support this possibithe release of both norepinephrine and dopamine-fl- lity by demonstrating in rat sympathetic ganglia the hydroxylase from adrenergic nerve endings (HED- presence of a system for the biosynthesis and release QVIST, 1970a; HEDQVIST& WENNMALM, 1971; JOHN- of prostaglandin E. SON et al., 1971). In addition, inhibitors of prostaglandin biosynthesis have been found to enhance the MATERIALS AND METHODS outflow of norepinephrine in response t o nerve stimuet al., 1971; CHANHet al., 1972; lation (HEDQVIST FREDHOLM & HEDQVIST, 1973). It has therefore been postulated that endogenously synthesized prostaglandins of the E series provide an inhibitory feedback mechanism for neurotransmitter release and thereby modulate the activity of adrenergic neurons (HEUQVIST, 19706; HEDQVIST, 1973). Though the studies cited above support a potential role for prostaglandins of the E series in the regulation of adrenergic neuron function, the source of such prostaglandins is not clear. Direct stimulation of previously denervated neuroeffector tissues can release
Prostaglandin E synthesis by homogenates. Adult male Sprague-Dawley rats (220-250 g) were killed by cervical dislocation. The superior cervical ganglia were rapidly excised and homogenized (2 gangW500 ~ l at) %4‘C with a ground glass homogenizer in 0.1 M-phosphate buffer, pH 7.8, containing reduced glutathione (50pg/ml), hydroquinone ( 5 pgiml) and sodium arachidonate (10 pgiml). All solutions were freshly prepared and the arachidonic acid was stored in In, acetic acid in hexane at -20-C prior to use. Samples of homogenate were incubated at 37 C in air with shaking, generally for a 2 min period. The reaction was terminated by the addition of 90, formic acid to give a final pH of 3.5. Zero-time values were determined hy acidifying selected samples immediately following I Preliminary reports of this study were presented at the homogenization. Values reported represent the difference 1977 Spring Meeting of the Federation of the American between incubated and zero-time samples. Societies for Experimental Biology and at the Sixth Winter Determination of imniunoreactice prostuglandin E (iPG€l Conference on Prostaglandin Research, 1977. [3H]Prostaglandin E, was added to each acidified sample * Present address: University of Houston, College of for determination of recoveries. The samples were then Pharmacy, Houston, TX 77004, USA. extracted with 5 mi of ethyl acetate and the organic layer Abbreviations used: iPGE, immunoreactive prosta- removed and evaporated to dryness under N2 at 35 C. glandin E; cyclic AMP, adenosine 3’,5‘-monophosphate: Samples were redissolved in 300 111 of benzene-ethyl acecyclic GMP. guanosine 3‘,5’-monophosphate. tate-methanol (60:40:10) followed by 700111 of benzene-
~~~
13
J . G. WEBB,D. A. SAELENS and P. V. HALUSHKA
14
eth!l acetate (60:40).The entire I ml solution was added to a silicic acid column (0.5g). The column was washed S i t h 1 ml of benzene followed by 5.3 ml of benzene-ethyl acetate (60:40). Prostaglandins of the E series were eluted with 14 ml of benzeneethyl acetate-methanol (60:40:3) and the eluate evaporated to dryness under N,. Prostaglandin E was determined by radioimmunoassay after conversion to prostaglandin B using 0.1 N-potassium hydroxide in methanol (ALEXANDERet 01.. 1975). The term iPGE will be used since the antibody employed in the assay reacts with both prostagtandin B I and B,. However. there is no significant cross-reactivity with other prostaglandins (Table 1). TABLE1. SPECIFICITY OF
Prostaglandin PGB, PGEl PGFI, 6-0x0-PGF,, PGD, Thromboxane B, 15-0x0-PGB,
ANTIBODY
Picograms required for 50”; displacement of 0’ ’H-PGB, Cross-reactivity 170 I x 103 5 x los 3.6 10” 4.4 x 104 > 5 x lo5 1.8 x 10’
100 17 0.03 0.47 0.38 < 0.03 0.09
Time (min.)
FIG.1. Time course of iPGE synthesis by ganglia homogenates. Incubation and assay conditions were as stated in Materials and Methods. Each point represents the mean of two determinations. RESULTS
Prostaglandin E synthesis by homogenates
The time course of i P G E production from exogenous arachidonic acid by homogenates of rat superperiments. with isolated intact ganglia. superior cervical ior cervical ganglia is shown in Fig. 1. Under the ganglia were excised from rats after cervical dislocation incubation conditions employed, the rate of i P G E and dissected free of their capsules. Sets of at least 8 ganglia were incubated at 37’C in an atmosphere of 95% 0,- synthesis was linear with time for up to 2min and 50, CO, in 2 ml of Krebs-bicarbonate solution at pH 7.4. then declined with further time of incubation. A linear The composition of the bath solution was NaCl 6.60g, relationship was also observed between protein concentration and i P G E production (Fig. 2). In all subKCI0.35& CaCI, 0.28g. KH2P04 0.16& MgSO,.7 H,O 0.29 g, NaHCO, 2.10 g and dextrose 2.08 g in one liter of sequent experiments with the ganglia homogenates, distilled water. In the experiments with dopamine, nor- incubations were conducted for 2 min with 15C250 epinephrine and catechol, asorbic acid (0.20gD) was also pg of protein in 5 0 0 ~ 1of reaction mixture. included in the incubation bath and the pH adjusted to Varying the arachidonic acid concentration of the 7.4. Ascorbic acid had no effect on the control rate of reaction mixture over a 30-fold range altered the rate iPGE production. At the end of an incubation period, the of i P G E biosynthesis (Fig. 3). A Lineweaver-Burk bath solution was acidified with 9% formic acid, extracted and analyzed for iPGE as described above. Protein derermination. Proteins were determined using 5the method of Lowry and co-workers (LOWRYet al., 1951) with bovine serum albumin as standard. Materials. Prostaglandin E, [5,6,8,1 l,12q14,15-3H(N)] ,c 4 (117Ciimmol) and prostaglandin El [5,6-’H] (SO-llOCi/ 5 V mmol) were purchased from New England Nuclear CorE poration (Boston, MA). Arachidonic acid (>990< pure) was b 3obtained from Applied Sciences Laboratories, Inc. (State w College, PA), phospholipase A (Crotalus adamanteus) from 2Aldrich Chemical Co. (Milwaukee, WI), 1-norepinephrine -i0 bitartrate from Calbiochem (San Diego, CA), Biosil A (2CU400 mesh) from Bio-Rad Laboratories (Richmond. E > CA). N 6 . 02.-dihutyryl adenosine 3’.5’-monophosphate. N’, 0’-dibutyryl guanosine 3’,5’-monophosphate, and 3hydroxytyramine hydrochloride from Sigma Chemical Co. (St. Louis. MO). 0.1 0.2 0.3 0.4 0.5 Prostaglandins were kindly supplied by Dr. JOHNPIKE Protein (mg/ml) and DI. U w AXENof Upjohn Col. (Kalamazoo, MI), indomethacin by Merck Sharp and Dohme Research Labora- FIG.2. Effect of protein concentration on the rate of iPGE tories (Rahway. NJ). eicosatetraynoic acid by Hoffman La- formation by ganglia homogenates. Samples were incuRoche Inc. (Nutley. NJ) and sodium meclofenarnate by bated for 2min and each point represents the mean of Parke Davis and Co. (Detroit. MI). two determinations. Prosraylandin E synthesis hy whole ganglia. For the ex-
2
IS
Prostaglandin synthesis by sympathetic ganglia lr. 141
I
,
,
5
10
I5
,
!
20
25
#
30
,
35
I
,
,
I60
Arachidonic Acld OrM)
FIG. 3 Effect of substrate concentration on the rate of iPGE synthesis by ganglia homogenates. Insert: Double reciprocal plot of the data. Each point represents the mean of two determinations. 100-
plot of these data is shown in Fig. 3 where the line was fitted to the experimental points by linear regression analysis (r = 0.99). A kinetic analysis resulted in an apparent K , for the prostaglandin E synthetase ~ ~ an estimated V,,, of 15.4 pmol/ system of 1 4 . 9 and min/mg protein. Prostaglandin production by the homogenates could be completely inhibited by sufficient concentrations of established prostaglandin synthetase (EC 1.14.99.1) inhibitors (Fig. 4). Under the conditions of these experiments, indomethacin (ICso = 1.1 x M) was a less potent inhibitor than either sodium meclofenamate (IC50= 1.7 x 1 0 - 6 ~ )or eicosatetraynoic acid ( I c5 0 = 1.2 x 10-6M). Norepinephrine and dopamine, catecholamines which are present in sympathetic ganglia, stimulated iPGE production when added to the homogenates (Fig. 5). The effect of each catecholamine was maxiM. The maximum increase achieved with mal at dopamine (315 f 11% of control) was significantly greater ( P 0.01) than that obtained with norepinephrine (221 f 14% of control). To observe the stimulatory effects of these catecholamines in the homogenate preparation, it was necessary to omit hydroquinone from the initial homogenizing media.
-=
Prostaglandin E synthesis by whole ganglia
u Eicorotelroynoic Acid &-A Medofenornote 0
80 -
When freshly removed ganglia were incubated in uitro in Krebs-bicarbonate solution, iPGE was released spontaneously into the incubation bath in a time-dependent manner (Fig. 6). The rate of iPGE synthesis was linear for 10min and then decreased gradually with further time of incubation. In subsequent experiments, the standard incubation period was 10 min. Intact ganglia preincubated at M " C with indomethacin (5.5 x lo-' M) prior to incubation at 37°C in the Krebs-bicarbonate solution failed to produce any detectable levels of iPGE (unpublished observation). The rate of iPGE formation by the
I Indornethocm
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$ 40-
20-
I
4
I50 100
/@ b
Norepmephrtne Dopamine
50-
i
1O - 6 M
I0 - 5 ~
IO ~ M
10%
Cotecholornine Conc.
FIG.5. Effect of norepinephrine and dopamine on the rate of iPGE synthesis by ganglia hornogenate. Incubation and assay conditions were as stated in Materials and Methods except that hydroquinone was omitted from the reaction mixture. Each point represents the mean of threefour determinations.
J. G. WEBB, D. A. SAELENS and P. V. HALUSHKA
16
T I00
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075
XI
050 u-
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5
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Phospholipose A
30
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Time (rntn.1
FIG.6. Time course of iPGE production by isolated whole ganglia incubated in i,i?ro as described i n Materials and Methods. Prostaglandins were extracted from the Incubation media following the periods of incubation. Each point represents the mean +s.E.M. with the number of observations shown in brackets.
whole ganglia was much less than that observed for the cell-free homogenates. This difference is most likely due t o the presence of the co-factors. reduced glutathione and hydroquinone, and the excess substrate, arachidonic acid, added to the homogenates. As was observed in the homogenates (Fig. 5), norepinephrine and dopamine also stimulated iPGE synM, each thesis by the intact ganglia (Fig. 7). At catecholamine produced a 2-fold increase in iPGE output. There was no significant difference between the effect of dopamine and that of norepinephrine on iPGE production. The addition of catechol to the incubation media had no effect on the rate of iPGE formation.
FIG.8. Effect of phospholipase A on iPGE production by isolated whole ganglia in Citro. Each bar represents the mean ~ s . E . M . with the number of observations shown in brackets. **Denotes a significant difference from control at P < 0.01. The addition to the incubation bath of phospholipase A, an acyl hydrolase preparation that releases fatty acids from membrane phospholipids, markedly increased iPGE synthesis by the intact ganglia in a concentration-dependent manner (Fig. 8). At the highest concentration tested (50 unitsiml), a 3-fold stimulation of iPGE formation was observed. Conversely, M) appeared to diminish dibutyryl cyclic AMP the rate of iPGE biosynthesis by the whole ganglia (Fig. 9). However, under standard incubation conditions a reduction in iPGE production was difficult to quantitate since the control rate of synthesis was low. Therefore, the effect of dibutyryl cyclic AMP on iPGE production by phospholipase A treated ganglia was examined (Fig. 9). Phospholipase A (25 unitsiml) approximately doubled iPGE output. Dibutyryl cyclic AMP (lo-' M) significantly reduced iPGE synthesis k
250
f
1 I0 - 5 ~
0
[33 0%
Dopornine
Norepinephrine
Catechol
FIG.7. Effects of norepinephrine, dopamine and catechol on iPGE production by isolated whole ganglia incubated iu rirro. Each bar represents the mean ~ s . E . M . with the number of observations shown in brackets. The effect of each drug was compared against matched control preparations studied on the same day. *Denotes a significant difference from control at P i0.05. "Denotes a significant difference from control at P < 0.01.
Prostaglandin synthesis by sympathetic ganglia
I Jnits/ml
25 Unitslml 2
Phospholipose A DBcAMP
I0 - 3 ~
10-3~
17
2 Jnits/ml 10-?M
FIG. 9. Effect of dibutyryl cyclic A M P (DBcAMP) on iPGE production by isolated whole ganglia in oirro. Each bar represents the mean ~ S . E . M .with the number of observations shown in brackets. *Denotes a significant difference from phospholipase A treated ganglia at P i0.05.
by the phospholipase A stimulated preparation. In contrast, dibutyryl cyclic GMP ( lo - * M) had no effect on iPGE production when examined under the same conditions. DISCUSSION Prostaglandins of the E series are released at sympathetic neuroeffector junctions and have potent effects on the activity of adrenergic neurons (HEDQVET, 1973). The present study demonstrates the presence of a prostaglandin synthetase system in rat sympathetic ganglia and supports the concept that adrenergic neurons themselves may be a source of endogenous prostaglandins which could be available for the modulation of neuron function. In ganglia homogenates, the rate of iPGE formation from exogenous arachidonic acid was linear with protein concentration but with time for only 2min at 37°C. The rapid decline in the rate of iPGE synthesis is in agreement with previous studies of cell-free et al., 1971; PACE-ASCIAK, preparations (TAKEGUCHI 1972; TAI et al., 1976) and may reflect the accumulation of endogenous inhibitors or the proposed selflimiting nature of the cyclo-oxygenase component of prostaglandin synthetase systems (SMITH& LANDS, 1972; LANDSet a/., 1974). The apparent K , of 14.9pM estimated for the ganglion prostaglandin E synthetase system is similar to those calculated for preparations et a/., 1971) from bovine seminal vesicle (TAKEGUCHI and rat and rabbit kidney medulla (LIMAS& LIMAS, 1977; TAIet a/., 1976). That the observed conversion of arachidonic acid to iPGE by the homogenates was an enzymatic process was demonstrated by the fact that iPGE synthesis could be completely inhibited by eicosatetraynoic acid and the non-steroidal antiinflammatory drugs, indomethacin and sodium meclofenamate. In addition, norepinephrine and dopamine stimulated iPGE synthesis which is consis* c 31
I--8
tent with the demonstrated ability of catecholamines to serve as co-factors for prostaglandin synthetase systems (TAKEGUCHI et a/., 1971; PACE-ASCIAK,1972; WLODAWER& SAMUELSSON, 1973). Thus, the composite of these data indicates that the prostaglandin synthetase system of the ganglia homogenates appears to be very similar to the synthetase systems described for most other tissues. The study of iPGE biosynthesis from exogenous arachidonic acid in ganglia homogenates does not prove the capability of intact cells to produce and release prostaglandins nor allow for an analysis of factors which regulate prostaglandin synthesis from endogenous substrate. For this reason, iPGE production was also examined in isolated whole ganglia incubated in uitro in Krebs-bicarbonate solution. Incubation of the intact ganglia resulted in the release of iPGE into the incubation bath in a time-dependent manner. If the freshly removed ganglia were preincubated at 4°C in media containing indomethacin prior to incubation at 3TC, no measurable iPGE was detected in the incubation bath indicating that the observed release of iPGE from the intact ganglia reflected de novo prostaglandin synthesis. Furthermore, since no exogenous substrate was included in the incubation media, the iPGE synthesized by the whole ganglia must have arisen entirely from endogenous sources of substrate, presumably fatty acids liberated from membrane phospholipids by the actions of one or more acyl hydrolases. The 3-fold increase in iPGE synthesis produced upon addition of phospholipase A to the incubation bath supports this view and is consistent with the proposal that substrate availability may be a rate-limiting factor for the biosynthesis of prostaglandins by intact cellular systems (LANDS & SAMUELSSON, 1968; VONKEMAN& VAN DORP,1968; HAVEet al., 1973). As observed in the homogenates, norepinephrine and dopamine stimulated iPGE formation by the
18
J. G. WEBB. D. A. SAELENS and P. V. HALUSHKA
whole ganglia. At M a 4W5". increase in iPGE data suggest that endogenous neuronal constituents synthesis was observed, while at lo-' M each cat- (i.e. catecholamines, cyclic AMP) may influence or echolamine produced approximately a 2-fold increase regulate the activity of this prostaglandin synthetase in the rate of iPGE formation. Though these concen- system. trations seem high. it should be noted that a similar range of catecholamine levels has been reported for Acknowledgements-This research was supported in part threshold activation of adenylate cyclase (EC 4.6.1.1) by USPHS Grant GM 20387 and South Carolina State in isolated preparations of intact rat superior cervical Appropriation for Research. P. V. Halushka is a recipient et al., 1973) and in slices of bovine of a Pharmaceutical Manufacturers Association Foundaganglia (CRAMER sympathetic ganglia (KEBABIAN & GREENGARD, 1971; tion Faculty Development Award in Clinical Pharmacology. The authors gratefully acknowledge the technical TOMASI et a/., 1977). Dopamine is a proposed neuroassistance of Ms. C. SAELENS and Ms. G. DONNAN and transmitter for ganglionic interneurons LIB^, 1970; the excellent typing of the manuscript by Ms. M. TRUESLIBET& OWMAN, 1974) and norepinephrine release DELL. within sympathetic ganglia has been observed in response to preganglionic stimulation (NOON ef al., REFERENCES 1975). It is therefore conceivable that either of these catecholamines may be released during neuronal ac- ALEXANDERR.W., KENT K. M., PISANO J. J., KEISERH. R. tivity and reach a sufficient concentration at synaptic & COOPER T. (1975) J. elin. Invest. 55, 11741181. P. H., JUNSTAD M. & WENNMALM A. (1972) Acta junctions t o influence the rate of ganglionic prosta- CHANH physiol. scand. 86, 563-567. glandin synthesis. H., JOHNSON D. G., HANBAUER I., SILBERSTEIN The mechanism by which catecholamines stimulate CRAMER S . D. & KOPIN1. J. (1973) Brain Res. 53, 97-104. iPGE synthesis by whole ganglia is not clear. PerDAVIES 8. N., HORTON E. W. & WITHRINGTON P. G. (1968) haps, under certain conditions, co-factor availability Br. J . Pharmac. 32, 127-135. may limit the rate of prostaglandin production. HowB. & HEDQVIST P. (1973) Acta physiol. scund. FREDHOLM ever, the observation that catechol had no effect on 87, 57G572. iPGE formation indicates that in the intact cell sys- GERRARD J. M., PELLER J. D., KRICKT. P. & WHITEJ. G. tem the entire catecholamine molecule may be necess(1977) Prostaglandins 14, 39-50. ary to stimulate iPGE production and release. Poss- GILMORE N., VANE J. R. & WYLLIEJ. H. (1968) Nature, ibly, catecholamines may enhance iPGE formation by Lond. 218, 1135-1 140. stimulating membrane phospholipase acitivity as has GULLISR. J. & R o w C. E. (1976) J . Neurochem. 26, 1217-1230. been recently described in preparations of synaptic S. & JACQUEMIN C. (1973) FEES Lett. membranes from cerebral cortex of guinea pig HAVEB., CHAMPION 30, 253260. (GULLIS& ROWE, 1976). HEDQVIST P. (1970a) Life Sci. 9, 269-278. Cyclic A M P is a normal endogenous component HEDQVIST P. (1970b) Acta physiol. scand. 79, Supplement of adrenergic nerve cells and the levels of cyclic AMP 345,140, in sympathetic ganglia can be altered by pregang- HEDQVIST P. (1973)in The Prostaglandins (RAMWELLP. W., lionic stimulation (MCAFEEet a/., 1971) and by phared.) pp. 101-131. Plenum, New York. macological agents (KALIX& Roca 1975; KEBABIAN HEDQVlST P.,S T J k N E L. & WENNMALM A. (1971) Acta et al., 1975). It was therefore of interest to determine physiol. scand. 83, 430-432. A. (1971) Acta physiol. scand. P. & WENNMALM the effect of the cyclic AMP analogue dibutyryl cyclic HEDQVIST 83, 156162. AMP on the rate of iPGE production by the whole R., AXELROO D. G., THOAN. B., WEINSHILBOUM ganglia. Dibutyryl cyclic AMP appeared to diminish JOHNSON J. & KOPIN I. J. (1971) Proc. natn. Acad. Sci. U.S.A. the rate of iPGE synthesis in unstimulated ganglia 68, 2227-2230. and clearly inhibited iPGE production when synthesis P. & ROCH P. (1975) Naunyn-Schmiedebergs Arch. was stimulated by the addition of phospholipase A, KALIX Pharmacol. 291, 131-137. though, under these conditions, lo-' M dibutyryl cycKALISKERA. & DYERD. C. (1972) Eur. J. Pharmac. 19, lic AMP was required for a significant effect. Similar 305-309. inhibitory effects of dibutyryl cyclic AMP on prosta- KEBABIAN J. W.. BLOOMF. E., STEINER A. L. & GREENCARD glandin synthesis have been observed in renal medulP. (1975) Science 190, 157-159. lary interstitial cells (KALISKER& DYER, 1972) and KEBABIAN J. W. & GREENCARD P. (1971) Science 174, 13461349. also in thrombin-stimulated platelets where cyclic P. R., ROME L. & VANDERAMP appears to inhibit the activity of endogenous LANDSW.E. M., LE TELLIER HOEK J. Y. (1974) in Prostaglandin Synthetase Inhibitors et phospholipase A (MINKESer a/., 1977; GERRARD (ROBINSON H. J. &VANE J. R., eds.) pp. 1-7. Raven Press, al., 1977). New York. In summary, the results from the present study LANDS W. E. M. & SAMUELSSON B. (1968) Biochim. biophys. demonstrate the ability of adrenergic nerve cells or Acta 164, 426-429. closely associated satellite cells to synthesize prostaLIBETB. (1970) Fedn Proc. Fedn Am. Socs. exp. B i d . 29, glandin E which could then be available to modulate 1945-1956. neurotransmitter release or perhaps some other LIBETB. & OWMAN C. (1974) J. Physiol., Lond. 237, aspect of adrenergic neuron function. In addition, the 63%62.
Prostaglandin synthesis by sympathetic ganglia
19
LIMASC. J. & LIMASC. (1977) Am. J . Phpiol. 233, SMITHW. L. & LANDSW.E. M. (1972) Biochemistry 11, H87-H92. 3276-3285. LOWRY0.. ROSEBROUGH N. J.. FARRA. L. & RANDALL STJARNE L. (1972) Acta physiol. scand. 86, 574-576. R. J. (1951) J. h i d . Chrm. 193, 265-275. TAI H., TAIC. L. & HOLLANDER C. S . (1976) Biochem. MCAFEED. A,. SCHORDERET M. & GREENGARD P. (1971) J . 154, 257-264. Srierice 71, 1156-1158. TAKEGUCHI C., KOHNOE. & SIH C. J. (1971) Biochemistry N., CHI M.. ROTH G., RAZ A,, MINKESM.. STANFORD 10, 2372-2376. NFEDLEMAN P. & MAJERUS P. W. (1977) J . d i n . Inresr. TOMASI V., BIONDIC., TREVISANI A,, MARTINI M.& F’ERRI 59, 449-454. V. (1977) J . Neurochem. 28, 1289-1297. NOONJ. P., MCAFEED. A. & ROTHR. H. (1975) NnunynVONKEMAN H. & VANDDRPD. A. (1968) Biochim. biophys. Schmiedebrrys Arch. Pharmar. 291, 139-162. Acta 164, 430-432. PACE-ASCIAK C. (1972) Biochim. biophys. A r m 280, WLODAWER P. & SAJWELSSON B. (1973) J . b i d . Chem. 248, 161-171. 5673-5678.
Abstract-The biosynthesis of immunoreactive prostaglandin E (iPGE) was examined in homogenates of rat superior cervical ganglia and in isolated intact ganglia incubated in vitro. Ganglia homogenates produced iPGE from exogenous arachidonic acid. Prostaglandin synthesis by the homogenates was inhibited by the prostaglandin synthetase inhibitors, eicosatetraynoic acid, indomethacin and sodium meclofenamate and was stimulated by norepinephrine and dopamine. Whole ganglia incubated in Krebs-bicarbonate solution also synthesized iPGE which was released into the incubation bath in a time-dependent manner. As observed in the homogenates, norepinephrine and dopamine enhanced iPGE formation by the intact tissue. Phospholipase A also stimulated iPGE synthesis by the whole ganglia. The effect of phospholipase A was antagonized by dibutyryl cyclic AMP but not by dibutyryl cyclic GMP. The results suggest that neuronally synthesized prostaglandins may be available for modulating adrenergic neuron function and that endogenous neuronal constituents such as catecholamines and cyclic AMP may influence the activity of the prostaglandin synthetase system.
PROSTAGLANDINS are synthesized by numerous mam- prostaglandins (HEDQVIST, 1973) and, conceivably, the malian tissues and have been proposed t o act as hor- prostaglandins released during sympathetic nerve mones for the modulation of a variety of cellular func- stimulation may arise from postjunctional sites. Howtions. The interactions of prostaglandins with ever, it is also possible that the adrenergic neurons adrenergic neurons appear particularly significant. themselves may b e a source of endogenous prostaFor example, stimulation of sympathetic nerves has glandins. Indeed, STlARNE (1972) has suggested from been demonstrated to release prostaglandin E at indirect evidence that t h e prostaglandins which inet al., 1968; G~LMOREfluence norepinephrine release from the guinea pig neuroeffector junctions (DAVIES et a!., 1968) and exogenously administered prosta- vas deferens may originate from prejunctional nerve glandins of the E series have been shown t o inhibit endings. I n the present study, we support this possibithe release of both norepinephrine and dopamine-fl- lity by demonstrating in rat sympathetic ganglia the hydroxylase from adrenergic nerve endings (HED- presence of a system for the biosynthesis and release QVIST, 1970a; HEDQVIST& WENNMALM, 1971; JOHN- of prostaglandin E. SON et al., 1971). In addition, inhibitors of prostaglandin biosynthesis have been found to enhance the MATERIALS AND METHODS outflow of norepinephrine in response t o nerve stimuet al., 1971; CHANHet al., 1972; lation (HEDQVIST FREDHOLM & HEDQVIST, 1973). It has therefore been postulated that endogenously synthesized prostaglandins of the E series provide an inhibitory feedback mechanism for neurotransmitter release and thereby modulate the activity of adrenergic neurons (HEUQVIST, 19706; HEDQVIST, 1973). Though the studies cited above support a potential role for prostaglandins of the E series in the regulation of adrenergic neuron function, the source of such prostaglandins is not clear. Direct stimulation of previously denervated neuroeffector tissues can release
Prostaglandin E synthesis by homogenates. Adult male Sprague-Dawley rats (220-250 g) were killed by cervical dislocation. The superior cervical ganglia were rapidly excised and homogenized (2 gangW500 ~ l at) %4‘C with a ground glass homogenizer in 0.1 M-phosphate buffer, pH 7.8, containing reduced glutathione (50pg/ml), hydroquinone ( 5 pgiml) and sodium arachidonate (10 pgiml). All solutions were freshly prepared and the arachidonic acid was stored in In, acetic acid in hexane at -20-C prior to use. Samples of homogenate were incubated at 37 C in air with shaking, generally for a 2 min period. The reaction was terminated by the addition of 90, formic acid to give a final pH of 3.5. Zero-time values were determined hy acidifying selected samples immediately following I Preliminary reports of this study were presented at the homogenization. Values reported represent the difference 1977 Spring Meeting of the Federation of the American between incubated and zero-time samples. Societies for Experimental Biology and at the Sixth Winter Determination of imniunoreactice prostuglandin E (iPG€l Conference on Prostaglandin Research, 1977. [3H]Prostaglandin E, was added to each acidified sample * Present address: University of Houston, College of for determination of recoveries. The samples were then Pharmacy, Houston, TX 77004, USA. extracted with 5 mi of ethyl acetate and the organic layer Abbreviations used: iPGE, immunoreactive prosta- removed and evaporated to dryness under N2 at 35 C. glandin E; cyclic AMP, adenosine 3’,5‘-monophosphate: Samples were redissolved in 300 111 of benzene-ethyl acecyclic GMP. guanosine 3‘,5’-monophosphate. tate-methanol (60:40:10) followed by 700111 of benzene-
~~~
13
J . G. WEBB,D. A. SAELENS and P. V. HALUSHKA
14
eth!l acetate (60:40).The entire I ml solution was added to a silicic acid column (0.5g). The column was washed S i t h 1 ml of benzene followed by 5.3 ml of benzene-ethyl acetate (60:40). Prostaglandins of the E series were eluted with 14 ml of benzeneethyl acetate-methanol (60:40:3) and the eluate evaporated to dryness under N,. Prostaglandin E was determined by radioimmunoassay after conversion to prostaglandin B using 0.1 N-potassium hydroxide in methanol (ALEXANDERet 01.. 1975). The term iPGE will be used since the antibody employed in the assay reacts with both prostagtandin B I and B,. However. there is no significant cross-reactivity with other prostaglandins (Table 1). TABLE1. SPECIFICITY OF
Prostaglandin PGB, PGEl PGFI, 6-0x0-PGF,, PGD, Thromboxane B, 15-0x0-PGB,
ANTIBODY
Picograms required for 50”; displacement of 0’ ’H-PGB, Cross-reactivity 170 I x 103 5 x los 3.6 10” 4.4 x 104 > 5 x lo5 1.8 x 10’
100 17 0.03 0.47 0.38 < 0.03 0.09
Time (min.)
FIG.1. Time course of iPGE synthesis by ganglia homogenates. Incubation and assay conditions were as stated in Materials and Methods. Each point represents the mean of two determinations. RESULTS
Prostaglandin E synthesis by homogenates
The time course of i P G E production from exogenous arachidonic acid by homogenates of rat superperiments. with isolated intact ganglia. superior cervical ior cervical ganglia is shown in Fig. 1. Under the ganglia were excised from rats after cervical dislocation incubation conditions employed, the rate of i P G E and dissected free of their capsules. Sets of at least 8 ganglia were incubated at 37’C in an atmosphere of 95% 0,- synthesis was linear with time for up to 2min and 50, CO, in 2 ml of Krebs-bicarbonate solution at pH 7.4. then declined with further time of incubation. A linear The composition of the bath solution was NaCl 6.60g, relationship was also observed between protein concentration and i P G E production (Fig. 2). In all subKCI0.35& CaCI, 0.28g. KH2P04 0.16& MgSO,.7 H,O 0.29 g, NaHCO, 2.10 g and dextrose 2.08 g in one liter of sequent experiments with the ganglia homogenates, distilled water. In the experiments with dopamine, nor- incubations were conducted for 2 min with 15C250 epinephrine and catechol, asorbic acid (0.20gD) was also pg of protein in 5 0 0 ~ 1of reaction mixture. included in the incubation bath and the pH adjusted to Varying the arachidonic acid concentration of the 7.4. Ascorbic acid had no effect on the control rate of reaction mixture over a 30-fold range altered the rate iPGE production. At the end of an incubation period, the of i P G E biosynthesis (Fig. 3). A Lineweaver-Burk bath solution was acidified with 9% formic acid, extracted and analyzed for iPGE as described above. Protein derermination. Proteins were determined using 5the method of Lowry and co-workers (LOWRYet al., 1951) with bovine serum albumin as standard. Materials. Prostaglandin E, [5,6,8,1 l,12q14,15-3H(N)] ,c 4 (117Ciimmol) and prostaglandin El [5,6-’H] (SO-llOCi/ 5 V mmol) were purchased from New England Nuclear CorE poration (Boston, MA). Arachidonic acid (>990< pure) was b 3obtained from Applied Sciences Laboratories, Inc. (State w College, PA), phospholipase A (Crotalus adamanteus) from 2Aldrich Chemical Co. (Milwaukee, WI), 1-norepinephrine -i0 bitartrate from Calbiochem (San Diego, CA), Biosil A (2CU400 mesh) from Bio-Rad Laboratories (Richmond. E > CA). N 6 . 02.-dihutyryl adenosine 3’.5’-monophosphate. N’, 0’-dibutyryl guanosine 3’,5’-monophosphate, and 3hydroxytyramine hydrochloride from Sigma Chemical Co. (St. Louis. MO). 0.1 0.2 0.3 0.4 0.5 Prostaglandins were kindly supplied by Dr. JOHNPIKE Protein (mg/ml) and DI. U w AXENof Upjohn Col. (Kalamazoo, MI), indomethacin by Merck Sharp and Dohme Research Labora- FIG.2. Effect of protein concentration on the rate of iPGE tories (Rahway. NJ). eicosatetraynoic acid by Hoffman La- formation by ganglia homogenates. Samples were incuRoche Inc. (Nutley. NJ) and sodium meclofenarnate by bated for 2min and each point represents the mean of Parke Davis and Co. (Detroit. MI). two determinations. Prosraylandin E synthesis hy whole ganglia. For the ex-
2
IS
Prostaglandin synthesis by sympathetic ganglia lr. 141
I
,
,
5
10
I5
,
!
20
25
#
30
,
35
I
,
,
I60
Arachidonic Acld OrM)
FIG. 3 Effect of substrate concentration on the rate of iPGE synthesis by ganglia homogenates. Insert: Double reciprocal plot of the data. Each point represents the mean of two determinations. 100-
plot of these data is shown in Fig. 3 where the line was fitted to the experimental points by linear regression analysis (r = 0.99). A kinetic analysis resulted in an apparent K , for the prostaglandin E synthetase ~ ~ an estimated V,,, of 15.4 pmol/ system of 1 4 . 9 and min/mg protein. Prostaglandin production by the homogenates could be completely inhibited by sufficient concentrations of established prostaglandin synthetase (EC 1.14.99.1) inhibitors (Fig. 4). Under the conditions of these experiments, indomethacin (ICso = 1.1 x M) was a less potent inhibitor than either sodium meclofenamate (IC50= 1.7 x 1 0 - 6 ~ )or eicosatetraynoic acid ( I c5 0 = 1.2 x 10-6M). Norepinephrine and dopamine, catecholamines which are present in sympathetic ganglia, stimulated iPGE production when added to the homogenates (Fig. 5). The effect of each catecholamine was maxiM. The maximum increase achieved with mal at dopamine (315 f 11% of control) was significantly greater ( P 0.01) than that obtained with norepinephrine (221 f 14% of control). To observe the stimulatory effects of these catecholamines in the homogenate preparation, it was necessary to omit hydroquinone from the initial homogenizing media.
-=
Prostaglandin E synthesis by whole ganglia
u Eicorotelroynoic Acid &-A Medofenornote 0
80 -
When freshly removed ganglia were incubated in uitro in Krebs-bicarbonate solution, iPGE was released spontaneously into the incubation bath in a time-dependent manner (Fig. 6). The rate of iPGE synthesis was linear for 10min and then decreased gradually with further time of incubation. In subsequent experiments, the standard incubation period was 10 min. Intact ganglia preincubated at M " C with indomethacin (5.5 x lo-' M) prior to incubation at 37°C in the Krebs-bicarbonate solution failed to produce any detectable levels of iPGE (unpublished observation). The rate of iPGE formation by the
I Indornethocm
2
6 60r
$ 40-
20-
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4
I50 100
/@ b
Norepmephrtne Dopamine
50-
i
1O - 6 M
I0 - 5 ~
IO ~ M
10%
Cotecholornine Conc.
FIG.5. Effect of norepinephrine and dopamine on the rate of iPGE synthesis by ganglia hornogenate. Incubation and assay conditions were as stated in Materials and Methods except that hydroquinone was omitted from the reaction mixture. Each point represents the mean of threefour determinations.
J. G. WEBB, D. A. SAELENS and P. V. HALUSHKA
16
T I00
-ED r
075
XI
050 u-
: Y
025
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5
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30
15
Time (rntn.1
FIG.6. Time course of iPGE production by isolated whole ganglia incubated in i,i?ro as described i n Materials and Methods. Prostaglandins were extracted from the Incubation media following the periods of incubation. Each point represents the mean +s.E.M. with the number of observations shown in brackets.
whole ganglia was much less than that observed for the cell-free homogenates. This difference is most likely due t o the presence of the co-factors. reduced glutathione and hydroquinone, and the excess substrate, arachidonic acid, added to the homogenates. As was observed in the homogenates (Fig. 5), norepinephrine and dopamine also stimulated iPGE synM, each thesis by the intact ganglia (Fig. 7). At catecholamine produced a 2-fold increase in iPGE output. There was no significant difference between the effect of dopamine and that of norepinephrine on iPGE production. The addition of catechol to the incubation media had no effect on the rate of iPGE formation.
FIG.8. Effect of phospholipase A on iPGE production by isolated whole ganglia in Citro. Each bar represents the mean ~ s . E . M . with the number of observations shown in brackets. **Denotes a significant difference from control at P < 0.01. The addition to the incubation bath of phospholipase A, an acyl hydrolase preparation that releases fatty acids from membrane phospholipids, markedly increased iPGE synthesis by the intact ganglia in a concentration-dependent manner (Fig. 8). At the highest concentration tested (50 unitsiml), a 3-fold stimulation of iPGE formation was observed. Conversely, M) appeared to diminish dibutyryl cyclic AMP the rate of iPGE biosynthesis by the whole ganglia (Fig. 9). However, under standard incubation conditions a reduction in iPGE production was difficult to quantitate since the control rate of synthesis was low. Therefore, the effect of dibutyryl cyclic AMP on iPGE production by phospholipase A treated ganglia was examined (Fig. 9). Phospholipase A (25 unitsiml) approximately doubled iPGE output. Dibutyryl cyclic AMP (lo-' M) significantly reduced iPGE synthesis k
250
f
1 I0 - 5 ~
0
[33 0%
Dopornine
Norepinephrine
Catechol
FIG.7. Effects of norepinephrine, dopamine and catechol on iPGE production by isolated whole ganglia incubated iu rirro. Each bar represents the mean ~ s . E . M . with the number of observations shown in brackets. The effect of each drug was compared against matched control preparations studied on the same day. *Denotes a significant difference from control at P i0.05. "Denotes a significant difference from control at P < 0.01.
Prostaglandin synthesis by sympathetic ganglia
I Jnits/ml
25 Unitslml 2
Phospholipose A DBcAMP
I0 - 3 ~
10-3~
17
2 Jnits/ml 10-?M
FIG. 9. Effect of dibutyryl cyclic A M P (DBcAMP) on iPGE production by isolated whole ganglia in oirro. Each bar represents the mean ~ S . E . M .with the number of observations shown in brackets. *Denotes a significant difference from phospholipase A treated ganglia at P i0.05.
by the phospholipase A stimulated preparation. In contrast, dibutyryl cyclic GMP ( lo - * M) had no effect on iPGE production when examined under the same conditions. DISCUSSION Prostaglandins of the E series are released at sympathetic neuroeffector junctions and have potent effects on the activity of adrenergic neurons (HEDQVET, 1973). The present study demonstrates the presence of a prostaglandin synthetase system in rat sympathetic ganglia and supports the concept that adrenergic neurons themselves may be a source of endogenous prostaglandins which could be available for the modulation of neuron function. In ganglia homogenates, the rate of iPGE formation from exogenous arachidonic acid was linear with protein concentration but with time for only 2min at 37°C. The rapid decline in the rate of iPGE synthesis is in agreement with previous studies of cell-free et al., 1971; PACE-ASCIAK, preparations (TAKEGUCHI 1972; TAI et al., 1976) and may reflect the accumulation of endogenous inhibitors or the proposed selflimiting nature of the cyclo-oxygenase component of prostaglandin synthetase systems (SMITH& LANDS, 1972; LANDSet a/., 1974). The apparent K , of 14.9pM estimated for the ganglion prostaglandin E synthetase system is similar to those calculated for preparations et a/., 1971) from bovine seminal vesicle (TAKEGUCHI and rat and rabbit kidney medulla (LIMAS& LIMAS, 1977; TAIet a/., 1976). That the observed conversion of arachidonic acid to iPGE by the homogenates was an enzymatic process was demonstrated by the fact that iPGE synthesis could be completely inhibited by eicosatetraynoic acid and the non-steroidal antiinflammatory drugs, indomethacin and sodium meclofenamate. In addition, norepinephrine and dopamine stimulated iPGE synthesis which is consis* c 31
I--8
tent with the demonstrated ability of catecholamines to serve as co-factors for prostaglandin synthetase systems (TAKEGUCHI et a/., 1971; PACE-ASCIAK,1972; WLODAWER& SAMUELSSON, 1973). Thus, the composite of these data indicates that the prostaglandin synthetase system of the ganglia homogenates appears to be very similar to the synthetase systems described for most other tissues. The study of iPGE biosynthesis from exogenous arachidonic acid in ganglia homogenates does not prove the capability of intact cells to produce and release prostaglandins nor allow for an analysis of factors which regulate prostaglandin synthesis from endogenous substrate. For this reason, iPGE production was also examined in isolated whole ganglia incubated in uitro in Krebs-bicarbonate solution. Incubation of the intact ganglia resulted in the release of iPGE into the incubation bath in a time-dependent manner. If the freshly removed ganglia were preincubated at 4°C in media containing indomethacin prior to incubation at 3TC, no measurable iPGE was detected in the incubation bath indicating that the observed release of iPGE from the intact ganglia reflected de novo prostaglandin synthesis. Furthermore, since no exogenous substrate was included in the incubation media, the iPGE synthesized by the whole ganglia must have arisen entirely from endogenous sources of substrate, presumably fatty acids liberated from membrane phospholipids by the actions of one or more acyl hydrolases. The 3-fold increase in iPGE synthesis produced upon addition of phospholipase A to the incubation bath supports this view and is consistent with the proposal that substrate availability may be a rate-limiting factor for the biosynthesis of prostaglandins by intact cellular systems (LANDS & SAMUELSSON, 1968; VONKEMAN& VAN DORP,1968; HAVEet al., 1973). As observed in the homogenates, norepinephrine and dopamine stimulated iPGE formation by the
18
J. G. WEBB. D. A. SAELENS and P. V. HALUSHKA
whole ganglia. At M a 4W5". increase in iPGE data suggest that endogenous neuronal constituents synthesis was observed, while at lo-' M each cat- (i.e. catecholamines, cyclic AMP) may influence or echolamine produced approximately a 2-fold increase regulate the activity of this prostaglandin synthetase in the rate of iPGE formation. Though these concen- system. trations seem high. it should be noted that a similar range of catecholamine levels has been reported for Acknowledgements-This research was supported in part threshold activation of adenylate cyclase (EC 4.6.1.1) by USPHS Grant GM 20387 and South Carolina State in isolated preparations of intact rat superior cervical Appropriation for Research. P. V. Halushka is a recipient et al., 1973) and in slices of bovine of a Pharmaceutical Manufacturers Association Foundaganglia (CRAMER sympathetic ganglia (KEBABIAN & GREENGARD, 1971; tion Faculty Development Award in Clinical Pharmacology. The authors gratefully acknowledge the technical TOMASI et a/., 1977). Dopamine is a proposed neuroassistance of Ms. C. SAELENS and Ms. G. DONNAN and transmitter for ganglionic interneurons LIB^, 1970; the excellent typing of the manuscript by Ms. M. TRUESLIBET& OWMAN, 1974) and norepinephrine release DELL. within sympathetic ganglia has been observed in response to preganglionic stimulation (NOON ef al., REFERENCES 1975). It is therefore conceivable that either of these catecholamines may be released during neuronal ac- ALEXANDERR.W., KENT K. M., PISANO J. J., KEISERH. R. tivity and reach a sufficient concentration at synaptic & COOPER T. (1975) J. elin. Invest. 55, 11741181. P. H., JUNSTAD M. & WENNMALM A. (1972) Acta junctions t o influence the rate of ganglionic prosta- CHANH physiol. scand. 86, 563-567. glandin synthesis. H., JOHNSON D. G., HANBAUER I., SILBERSTEIN The mechanism by which catecholamines stimulate CRAMER S . D. & KOPIN1. J. (1973) Brain Res. 53, 97-104. iPGE synthesis by whole ganglia is not clear. PerDAVIES 8. N., HORTON E. W. & WITHRINGTON P. G. (1968) haps, under certain conditions, co-factor availability Br. J . Pharmac. 32, 127-135. may limit the rate of prostaglandin production. HowB. & HEDQVIST P. (1973) Acta physiol. scund. FREDHOLM ever, the observation that catechol had no effect on 87, 57G572. iPGE formation indicates that in the intact cell sys- GERRARD J. M., PELLER J. D., KRICKT. P. & WHITEJ. G. tem the entire catecholamine molecule may be necess(1977) Prostaglandins 14, 39-50. ary to stimulate iPGE production and release. Poss- GILMORE N., VANE J. R. & WYLLIEJ. H. (1968) Nature, ibly, catecholamines may enhance iPGE formation by Lond. 218, 1135-1 140. stimulating membrane phospholipase acitivity as has GULLISR. J. & R o w C. E. (1976) J . Neurochem. 26, 1217-1230. been recently described in preparations of synaptic S. & JACQUEMIN C. (1973) FEES Lett. membranes from cerebral cortex of guinea pig HAVEB., CHAMPION 30, 253260. (GULLIS& ROWE, 1976). HEDQVIST P. (1970a) Life Sci. 9, 269-278. Cyclic A M P is a normal endogenous component HEDQVIST P. (1970b) Acta physiol. scand. 79, Supplement of adrenergic nerve cells and the levels of cyclic AMP 345,140, in sympathetic ganglia can be altered by pregang- HEDQVIST P. (1973)in The Prostaglandins (RAMWELLP. W., lionic stimulation (MCAFEEet a/., 1971) and by phared.) pp. 101-131. Plenum, New York. macological agents (KALIX& Roca 1975; KEBABIAN HEDQVlST P.,S T J k N E L. & WENNMALM A. (1971) Acta et al., 1975). It was therefore of interest to determine physiol. scand. 83, 430-432. A. (1971) Acta physiol. scand. P. & WENNMALM the effect of the cyclic AMP analogue dibutyryl cyclic HEDQVIST 83, 156162. AMP on the rate of iPGE production by the whole R., AXELROO D. G., THOAN. B., WEINSHILBOUM ganglia. Dibutyryl cyclic AMP appeared to diminish JOHNSON J. & KOPIN I. J. (1971) Proc. natn. Acad. Sci. U.S.A. the rate of iPGE synthesis in unstimulated ganglia 68, 2227-2230. and clearly inhibited iPGE production when synthesis P. & ROCH P. (1975) Naunyn-Schmiedebergs Arch. was stimulated by the addition of phospholipase A, KALIX Pharmacol. 291, 131-137. though, under these conditions, lo-' M dibutyryl cycKALISKERA. & DYERD. C. (1972) Eur. J. Pharmac. 19, lic AMP was required for a significant effect. Similar 305-309. inhibitory effects of dibutyryl cyclic AMP on prosta- KEBABIAN J. W.. BLOOMF. E., STEINER A. L. & GREENCARD glandin synthesis have been observed in renal medulP. (1975) Science 190, 157-159. lary interstitial cells (KALISKER& DYER, 1972) and KEBABIAN J. W. & GREENCARD P. (1971) Science 174, 13461349. also in thrombin-stimulated platelets where cyclic P. R., ROME L. & VANDERAMP appears to inhibit the activity of endogenous LANDSW.E. M., LE TELLIER HOEK J. Y. (1974) in Prostaglandin Synthetase Inhibitors et phospholipase A (MINKESer a/., 1977; GERRARD (ROBINSON H. J. &VANE J. R., eds.) pp. 1-7. Raven Press, al., 1977). New York. In summary, the results from the present study LANDS W. E. M. & SAMUELSSON B. (1968) Biochim. biophys. demonstrate the ability of adrenergic nerve cells or Acta 164, 426-429. closely associated satellite cells to synthesize prostaLIBETB. (1970) Fedn Proc. Fedn Am. Socs. exp. B i d . 29, glandin E which could then be available to modulate 1945-1956. neurotransmitter release or perhaps some other LIBETB. & OWMAN C. (1974) J. Physiol., Lond. 237, aspect of adrenergic neuron function. In addition, the 63%62.
Prostaglandin synthesis by sympathetic ganglia
19
LIMASC. J. & LIMASC. (1977) Am. J . Phpiol. 233, SMITHW. L. & LANDSW.E. M. (1972) Biochemistry 11, H87-H92. 3276-3285. LOWRY0.. ROSEBROUGH N. J.. FARRA. L. & RANDALL STJARNE L. (1972) Acta physiol. scand. 86, 574-576. R. J. (1951) J. h i d . Chrm. 193, 265-275. TAI H., TAIC. L. & HOLLANDER C. S . (1976) Biochem. MCAFEED. A,. SCHORDERET M. & GREENGARD P. (1971) J . 154, 257-264. Srierice 71, 1156-1158. TAKEGUCHI C., KOHNOE. & SIH C. J. (1971) Biochemistry N., CHI M.. ROTH G., RAZ A,, MINKESM.. STANFORD 10, 2372-2376. NFEDLEMAN P. & MAJERUS P. W. (1977) J . d i n . Inresr. TOMASI V., BIONDIC., TREVISANI A,, MARTINI M.& F’ERRI 59, 449-454. V. (1977) J . Neurochem. 28, 1289-1297. NOONJ. P., MCAFEED. A. & ROTHR. H. (1975) NnunynVONKEMAN H. & VANDDRPD. A. (1968) Biochim. biophys. Schmiedebrrys Arch. Pharmar. 291, 139-162. Acta 164, 430-432. PACE-ASCIAK C. (1972) Biochim. biophys. A r m 280, WLODAWER P. & SAJWELSSON B. (1973) J . b i d . Chem. 248, 161-171. 5673-5678.
