Journal o/ N',irr,x.kcrtiirrn.. 1978. Vol. 30. pp. 537 541. Perpamon Press. Printed In Great Britain
EFFECTS OF SYMPATHECTOMY O N AXOPLASMIC TRANSPORT OF SELECTED ENZYMES INCLUDING M A 0 AND OTHER MITOCHONDRIAL ENZYMES ROBERTE. SCHMIDT, C. DAVIDRoss and DAVIDB. MCDOUGAL, JR. Department of Pharmacology, Washington University School of Medicine, St. Louis, MO 631 10, U.S.A. (Received 21 April 1977. Accepted 25 July 1977)
Abstract-Axophsmic transport in guanethidine sympathectomized and control rats was investigated by monitoring the accumulations of vanous enzyme activities proximal to a ligature placed on the sciatic nerve. Sympathectomy affected the accumulations of three different mitochondrial enzymes quite differently: the accumulation of monoamine oxidase (MAO, EC 1.4.3.4) activity was inhibited 65% or more, that of hexokinase (HK, EC 2.7.1.1) activity was only inhibited 26%, while accumulation of glutamic dehydrogenase (GDH, EC I .4.1.3) activity was unaffected by sympathectomy. Accumulation of AChE (EC 3.1.1.7) activity was depressed 40%, but accumulations of the activities of the lysosomal enzyme, acid phosphatase (acid P'tase, EC 3.1.3.2), and of the cytosolic enzyme, choline acetyltransferase (CAT, EC 2.3.1.6) were unchanged. Despite impressive inhibition of MA0 accumulation, the intrinsic activity of this enzyme in sciatic nerve was unaffected by sympathectomy.The existence in nerve of isozymes of M A 0 was demonstrated using the inhibitors clorgyline and deprenyl. Transported MA0 activity was almost entirely type A; intnnsic activity was 2/3 type A and 1/3 type B. The differential response of the accumulations of the three mitochondrial enzyme activities measured was interpreted to indicate the existence, within neurons, of mitochondria with different enzyme complements.
PERIPHERAL nerve is composed of a complex mixture of somatic and sympathetic motor and sensory axons. Each axon type may be expected to differ from the others in the nature or amount of material moved by axoplasmic transport. Accumulation of particlespecific enzyme activities at a ligature applied to a peripheral nerve has been shown to be dependent on axoplasmic transport (Schmidt & McDougal, 1978). Although some transmitter-related enzymes, such as dopamine-beta-hydroxylase (3,4-dihydroxy phenylethylamine, ascorbate:oxygen oxidoreductase (hydroxylating), EC 1.14.2.1) or choline acetyltransferase (acetyl-CoA:choline 0-acetyltransferase, EC 2.3.1.6) are likely to be localized to a specific anatomical or physiologic type of neuron, it is difficult to predict the distribution among the several fiber types of enzymes whose functions are more general. We have shown that the activities of two mitochondrial enzymes, glutamic dehydrogenase (L-glutamate:NAD(P) oxidoreductase (deaminating), EC 1.4.1.3, GDH) and monoamine oxidase (monoamine:oxygen oxidoreductase (deaminating), EC 1.4.3.4, MAO), accumulate at a ligature on rat sciatic nerve. However, the patterns of the accumulation of activity were quite different (Schmidt & McDougal, 1978): M A 0 activity accumulated more rapidly relative t o intrinsic nerve activity than did GDH activity during the first two days after ligation. The involvement of M A 0 in catecholamine metabolism made it seem likely that the difference in the accumulation of the two mitochondria1 enzymes could be the result
of the preferential localization of M A 0 to adrenergic axons. Since chemical sympathectomy has been successfully used to examine the sympathetic contribution to the innervation of many organs (see THOENEN, 1972 for review) the accumulation of M A 0 and GDH activities has been studied at ligatures placed on sciatic nerves of chemically sympathectomized and control rats. The accumulations of the activities of acetyIcholinesterase (acetylcholine hydrolase, EC 3.1.1.7, AChE), choline acetyltransferase (CAT), hexokinase (ATP:D-hexose 6 phosphotransferase EC 2.7.1.1, HK), another mitochondrial enzyme, and acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2, acid Ptase) a lysosomal enzyme, were also determined.
MATERIALS AND METHODS Sprague-Dawley rats were sympathectomized using 21 daily injections of guanethidine (100 mg/kg s.c.) beginning at birth. At the age of 3 months (approx 200g) male animals were tested for completeness of sympathectomy (resultsincluded in DOUGLAS et al., 1975), and females were used in the present experiments. We are indebted to Drs. DOUGLAS and NEEDLEMAN for these animals. Control animals of the same strain and sex were selected to match the sympathectomized animals by weight. The animals were anesthetized with pentobarbital (35 mg/kg i.p.). The sciatic nerves of both legs were exposed and ligated with 6-0 silk thread in midthigh. The wound
538
R . E. SCHMIDT, C. D. Ross and D. B. MCDO~JGAL, JR
was closed and the animal allowed to survive I2 h before decapitation. Sciatic nerves were rapidly dissected free, cleaned of adherent tissue and frozen on solid CO,. After freeze drying at -35 to -40°C nerve proximal to the ligature was sectioned into three consecutive pieces from positions 0-2, 2 4 , and 4-8 mm (segments 1, 2 and 3 respectively) proximal to the tie. This last (segment 3, designated the 'intrinsic segment') under the conditions of this experiment is known to have the same specific enzyme activities as unligated nerve (SCHMIDT & MCDOUGAL, 1978). These segments were weighed on a quartz fiber balance (LOWRY & PASSONNEAU, 1972). Each segment was homogenized in 90 pl (2 mm) or 180 p I (4 mm) of 100 mM-phosphate buffer, pH 7.8, containing 0.05"; BSA. Homogenates were stored at -80 C until they were assayed for AChE (GUTHet 01.. 1964). MA0 (WURTMAN & AXELROD, 1963), GDH et 01.. 1962). acid Ptase (LOWRY et a/.. (GARCIA-BUNUEL 1954), CAT (Ross & MCDOUGAL, 1976) and HK (LOWRY et ul., 1961). The accumulation of enzyme activity in the two 2mm nerve segments proximal to the tie was calculated as follows:
1978) from the total activity of the 2mm pretie segment. Materials Clorgyline and deprenyl were gifts from Dr. NORTON NEFF. Substrates. coenzymes and enzymes used in the assays were obtained from Sigma and Boehringer and Sons. ['4C]Tryptamine bisuccinate was obtained from New England Nuclear.
RESULTS Sympathectomy produced a 20% decrease in AChE and a 13% decrease in acid P t a s e activities of intrinsic nerve segments (Table I). The activities of MAO, CAT, GDH, a n d HK of intrinsic nerve segments were not measurably affected by sympathectomy. No change in nerve segment weight per unit length was observed.
+
TABLE 1. EFFECTOF SYMPATHECTOMY ON INTRINSIC ENZYME Absolute = ( A , - A , ) W , ( A 2 - A,) W , ACTIVITY IN SCIATIC NERVE accumulation where:A,,2,3 = enzyme activity per unit dry weight of 1st Enzyme Control Sympathectomy or 2nd (2 mm) or 3rd (4 mm) nerve segment, (pmol/kg dry weight/h) 232 5 14 214 & 12 W, ,2 = weights of corresponding nerve segments. M A 0 1 p ~ 20 pM 1222 f 106 1172 f 93 In control animals the enzyme activity accumulated in (mmol/kg dry weight/h) segment 1 12 h after ligation produced an increase in the HK 326 12 291 f 11 measured activity of 150% of the activity of the intrinsic GDH 250 9 230 12 segment (No. 3) for MAO. 70% for GDH, 135% for HK, AChE 125 & 5* 156 k 4 235% for AChE. 250% for acid P'tase and 55% for CAT. Acid P'tase 84 f 4 73 f I t 41 + 2 37 2 With the exception of CAT the accumulations of enzyme CAT activities in segment 2 were always less than 20% of those (pggimm) 218 k 5 220 10 in segment 1. For CAT accumulations in these two seg- Nerve weight ments were about equal. Enzyme activities in the nerve segment located 4-8 mm M A 0 and inhibitors Sciatic nerves were ligated in uioo proximal to the ligature (intrinsic nerve segment, No. 3. for 2 days. Frozen nerve was sectioned into two segments see Methods) are given. M A 0 was assayed at 2 tryptamine proximal to the ligature: a 2 mm segment immediately concentrations, 1 p~ and 20 p ~ Each . value represents the adjacent to the tie and a 6 mm (intrinsic activity) segment mean f S.E. of 8 sympathectomized and 6 control nerves. extending 7-13 mm from the tie. A few nerves were sec- * P < 0.01. tioned distally into a two mm segment adjacent to the t P < 0.05. tie and a 5 mm segment extending 4-9 mm from the ligaOF SYMPATHECTOMY ON ACCUMULATION OF ture. Nerve segments were homogenized in 100pI of TABLE2. EFFECT ENZYME ACTIVITY AT A LIGATURE ON SCIATIC NERVE 100 mM-phosphate buffer, pH 7.8, containing 0.05% BSA. M A 0 was assayed in the presence of 20 pwtryptamine Enzyme Control Sympathectomy bisuccinate. The effect of clorgyline on M A 0 activity was determined by assaying M A 0 in the presence of increasing (pmol/h) concentrations of inhibitor without preincubation of 158 k 26 MA0 1 j i ~ 55 k 14* enzyme with inhibitor. Preincubation of enzyme with clor20 j l M 616 160 178 71t gyline without substrate for 15' at 25°C or 1 0 at 37°C (nmolih) did not enhance inhibition. Assay of M A 0 in the presence HK 186 f 6 138 & 4* GDH 75 & 10 7 0 + 11 of increasing concentrations of deprenyl was performed 160 k 9 90 f 4* using 15' preincubation without substrate at 25°C. AChE Acid P'tase 90 & 9 77 k 5 Deprenyl was used at 100 ng/ml without preincubation in CAT 8.5 & 2.1 9.6 f 1.5 an experiment testing the additivity of clorgyline and deprenyl inhibition. The extent of inhibition by deprenyl Accumulations are expressed in terms of absolute acat 100ng/ml was the same with and without 15 min pretivity accumulating at a tie after 12 h t n vim. M A 0 was incubation. assayed at 2 tryptamine concentrations, I pM and 20pM. The amount of axoplasmically transported M A 0 ac- Number of nerves is the same as in Table I with the excep cumulating at a ligature was calculated by subtracting tion of CAT accumulation in sympathectomized nerves 2 mm worth of intrinsic segment activity (corrected for a where n was I. 10% increase in amount of enzyme at the ligature induced * P < 0.01. & MCDOUGAL, t P < 0.05. by the mechanical act of ligation, SCHMIDT
+ +
+
+
+
*
Effects of sympathectomy on axonal transport
539
TABLE 3. ADDITIVITY OF CLORGYLINE AND DEPRENYL INHIBI-
3 1000 K
TION OF
M A 0 ACTIVITY
I-
z
s
Clorgyline Deprenyl Clorgyline and deprenyl (percentage inhibition)
& Unligated
50-
a
->
INTRINSIC
P W
\
.
k
1
!-
:: 0 -
TRANSPORTED \
-10- A @ o io-'O 1 0 ' ~ I O - ~ lo-'
IO-~
CLORGYLINE CONCENTRATION
I-
2 0
L,, 0 1
(M)
#
50
DEPRENYL ( n g / m l )
I+
100 1000
FIG.1. Effect of clorgyline and deprenyl on intrinsic and accumulated M A 0 activity in control sciatic nerve. M A 0 of the intrinsic (-) and pre-tie (. . . . .) segments and transported M A 0 activity (-----) of the rat sciatic nerves 2 days after tying, calculated as described in Methods, was examined in the presence of increasing concentrations of (A) clorgyline and (B) deprenyl. See Materials and Methods for details. All M A 0 activities are expressed as a percentage of the uninhibited enzyme activity. Uninhibited M A 0 activities of pre-tie and intrinsic segments and transported M A 0 activity were 4099 f 75, 1768 f 47, and 2134 & 94 pmol/h/2 mm nerve respectively in the clorgyline experiment and 4320 f 121, 1802 f 127, and 2318 f 118 pmol/h/2 mm nerve in the deprenyl experiment. Each point represents the mean f S.E. of 6 nerves. However, there were very appreciable changes in enzyme accumulation at a ligature (Table 2). The amount of M A 0 activity accumulating at a tie was decreased 65% or more by sympathectomy. Accumulation of hexokinase, another mitochondria1 enzyme, was reduced 26%, while that of still a third mitochondrial enzyme, GDH, was unchanged. The accumulation of AChE activity was decreased 40% by sympathectomy. Accumulations of the lysosoma1 enzyme acid P'tase and the soluble CAT were not altered significantly. The profound decrease in transported M A 0 activity with a minimal change in M A 0 activity of intrinsic nerve segments and the somewhat different ratios of accumulated and intrinsic M A 0 activities with different substrate concentrations suggested the possibility of several forms of M A 0 in this system. The apparent K , for tryptamine for M A 0 in sciatic nerve homogenate was about 7 PM. Both transported and intrinsic M A 0 activity are sensitive to the type
N.C. 30/3-C
. 54
nerve
0
Pre-tie Intrinsic
79 f 2 57 f 1
41 f 2
91 f 1
31 f 2 46 f 3
91 + 2 89 2
+
M A 0 activity of unligated nerve and pre-tie and intrinsic nerve segments of 2 day in uiuo ligated nerve was assayed without inhibitors, or in the presence of l o T 8 M-clorgyline, 100 ng/ml deprenyl, or 10- * M-ClOrgyline + 100 ng/ml deprenyl. Activity is expressed as percentage of inhibition compared to uninhibited control MA0 activity. Absolute M A 0 activities of unligated, pre-tie and intrinsic nerve segments were 1993 _+ 139, 3666 _+ 354, and 1444 _+ 112 pmol/h/2 m m nerve respectively. Each value represents the mean f S.E. of 6 nerves.
A M A 0 inhibitor clorgyline at very low concentrations (Fig. 1A). At lo-' M, clorgyline inhibited transported M A 0 activity completely but intrinsic segment M A 0 activity was only inhibited 60%. Inhibition by the type B M A 0 inhibitor deprenyl reached an approximate plateau of inhibition between 10 and 100ng/ml of inhibitor (Fig. 1B). Intrinsic M A 0 activity was inhibited 40% over this range and transported M A 0 only 10%. The additivity of inhibition of M A 0 activity with clorgyline and deprenyl was investigated with enzyme from unligated nerve and intrinsic and pre-tie segment M A 0 from two day ligated nerves (Table 3). Approximate additivity of inhibition occurs with enzyme from all three sources; however, some overlap of inhibition probably occurs, so that the inhibition produced by the two inhibitors together was somewhat less than the sum of inhibitions produced by each one separately. DISCUSSION The fact that there was no change in weight per unit length of nerve produced by sympathectomy (Table 1) suggests that the mass of fibers removed by sympathectomy was relatively small; however, a decrease of less than 1045% would not have been discernible. Therefore, these fibers must be relativel; rich in transported M A 0 activity, since approx 213 of the M A 0 accumulating at a ligature would be contained in less, perhaps much less, than 1/5 the mass of axons. In contrast to the transported enzyme activity, the intrinsic MAO activity associated with the sympathetic fibers did not appear particularly high. The accumulation of some M A 0 activity in sympathectomized nerves is not necessarily evidence for the incomplete removal of sympathetic fibers. The littermates of these animals were shown to have essentially no surviving peripheral sympathetic neurons (DOUGLAS et al., 1979, and M A 0 is known not to be restricted to sympathetic neurons (BLQOMet al.,
540
R. E. SCHMIDT, C. D. Ross and D. B. MCDOUGAL. JR.
1972; THOENEN, 1972). Some studies, however, three accumulating mitochondria1 enzymes provides reported the absence of M A 0 in cholinergic axons additional evidence that mitochondria in the nervous (CONSOLO et al., 1968) and the relative absence of system are not homogeneous (MCDOUGALet al., M A 0 in sensory and cholinergic cells compared to 1964; NEIDLEet al., 1969). In rat brain, M A 0 sedisympathetic ganglion cells (KOELLE& VALK, 1954). ments in a different fraction than GDH (NEIDLEet DAHLSTROM et al. (1 969) also measured the accumu- al., 1969), and in the same system mitochondria were lation of M A 0 at a ligature in sciatic nerves of con- separated into several subclasses with different relatrol and (surgically) sympathectomized rats. They tive activities against various substrates for M A 0 found no accumulation in control animals until 3 (KROON& VELDSTRA,1972). In the present instance, days after ligation, and in sympathectomized animals, since GDH, HK and M A 0 have been shown to acnone until the seventh day. The amount of accumu- cumulate as a result of axoplasmic transport (PART1978), it lation we found in normal animals at 7 days (SCHMIDT LOW et a/., 1972; SCHMiDT & MCDOUGAL, & MCDOUGAL, 1978) was about the same as that now appears that different neurons can have mitoet al. (1969), but chondria with different enzyme compositions. found in controls by DAHLSTROM we detected no measurable delay between ligation The 40% decrease in AChE transport suggests that and the onset of accumulation of MA0 activity. Some sympathetic fibers are relatively rich in transported of the discrepancy may be explainable by the diluting A G E , while the substantial decrease in intrinsic effect of the use of larger nerve segments (5mm) by A G E activity suggests relatively large amounts of et al., since the accumulation is mostly AChE in their axolemmata. It has been shown in rat DAHLSTROM confined to the first 2 m m adjacent to the ligature. superior cervical ganglion that adrenergic neurons Transport to MA0 has been observed to decrease (identified by catecholamine fluorescence) contain hisin amphibians subjected to operative sympathectomy tochemically demonstrable AChE in their cell bodies (H~KONEN , 1963). Since guanethidine has been (MCLEAN & BURNSTOCK,1972). Use of kinetic studies, selective inhibitors, immuno- shown to be specific for adrenergic neurons (JENSENet al., 1971 ; ANGEprecipitation, substrate specificity, thermal denatur- HOLM& JUUL, 1971; BURNSTOCK ation, and electrophoretic mobility has revealed the LETTI & LEVI-MONTALCINI, 1972), one would expect existence of two isozymes or isozymic classes of M A 0 that guanethidine treatment would leave cholinergic 1968; GORIDIS & NEE, 1971 ; SHm & EIDU- sympathetic axons untouched. Thus the decrease in (JOHNSON, SON, 1971; YANG et al., 1972; YANG & NEFF, 1973, AChE transport cannot be explained by destruction 1974; MCCAULEY & RACER, 1973). One form of of these neurons. AChE staining of rat sciatic nerve MAO, designated type A, is more active against the suggests that sympathetic fibers are rich in axolemmal neurotransmitters norepinephrine and serotonin, is AChE (SCHLAEPFER & TORACK, 1966). Our observaexquisitely sensitive to clorgyline, harmine, and har- tions are consistent with the notion that CAT is not maline, but is resistant to inhibition by deprenyl. concentrated in adrenergic axons; however, it should Type B enzyme is more active against phenylethyl- be stressed that even appreciable changes in CAT amine and benzylamine, is insensitive to clorgyline transport might have been overlooked in this study and sensitive to deprenyl and pargyline. Both types since the time allowed for accumulation was short of enzyme are active against tyramine and tryptamine. relative to the apparent rate of transport of this In certain experimental systems, such as the innerva- enzyme. The lack of change in accumulation of acid Ptase tion of the pineal gland by the cervical sympathetic ganglion, type A MA0 seems to be concentrated in activity in response to sympathectomy does not mean the neurons of the ganglion, including the nerves in that adrenergic axons lack fysosomes, but only that the gland (YANG et al., 1972). Type B appears to be lysosomes containing acid Ptase are not concentrated localized in the (non-neuronal) parenchymal cells of in these axons. Indeed, electron microscopic studies the pineal gland. However, the type A enzyme is of rat sympathetic axons reveal dense bodies accumu8z &ISMAN, 1972). probably not exclusively neuronal. Our experiments lating at a ligature (MATTHEWS are consistent with the neuronal localization of type A enzyme, since only intraneuronal materials would Acknowledgements-We thank Ms. MARGARETYv for be expected to accumulate by axonal transport at a skillful technical assistance. Supported by NIH grants ligature under the conditions of our experiments. GM-02016, NS-11941 and NS-06800. Transported enzyme is almost entirely type A; unligated or intrinsic segment activity seems to be composed of 213 type A enzyme and 1/3 type B enzyme. REFERENCES Intrinsic activity may be localized to Schwann cells, A N G F L E P. ~ U. & LEVI-MONTALCINI R. (1972) Proc. nutn. connective tissue, vascular tissue or non-sympathetic Acud. SCL, U.S.A. 69, 86-88. axons. Preliminary experiments with M A 0 activity BLOOMF. E., SIMSK. L., WEITSENH.A,, DAVISG. A. accumulating distal to a tie revealed it also to be & HANKER J. S. (1972) Adv. Biochem. Pharmuc. 5, mostly type A enzyme, i.e. it was inhibited 90% by 243-262. 10- 8w-clorgyline. BURNSTOCKG., EVANS B., GANNON B. J., HEATHJ. W. 8c JAMES V. (1971) Br. J . Pharmuc. 43, 295-301. The differential response to sympathectomy of the
Effects of sympathectomy on axonal transport S., GIACOBINI E. & KARJALAINEN K. (1968) Acta. CONSOLO physiol. scand. 74, 513-520. DAHLSTR~M A., JONASON J., & NORBERG K. A. (1969) Eur. J. Pharmac. 6, 248-254. DOUGLAS J. R., JR., JOHNSONE. M., JR., MARSHAL G. R., B. K. & NEEDLEMAN P. (1975) CirHEISTJ., HARTMANN culation Res. 36 and 37, Suppl. 1, 171-178. GARCIA-BUNUEL L., MCDOUGAL D. B., JR., BURCHH. B., JONESE. M. & TOUHILLE. (1962) J. Neurochem. 9, 589-594. GORIDISC. & NEFFN. H. (1971) Neuropharmacology 10, 557-564. G u m L., ALBERS R. W. & BROWNW. C. (1964) Exp. Neurol. 10, 236-250. HARKONEN M. (1964) Acta physrol. scand. 63, Suppl. 237, 3-94. JENSEN-HOLM J. & JUULP. (1971) Acta Pharmac. Toxicol. 30, 308-320. JOHNSON J. P. (1968) Biochem. Pharmac. 17, 1285-1297. KOELLEG . B. & VALK A. de T. (1954) J. Physiol., Lond. 126, 434447. KROONM. C. & VELDSTRA H. (1972) FEBS Lett. 24, 173-176. LOWRY0. H. & PASSONNEAU J. V. (1972) A Flexible Sysrem of Enzymatic Analysis. Academic Press, New York. LOWRY0. H., ROBERTS N. R., SCHULZD. W., CLOW J E. & CLARKJ. R. (1961) J . biol. Chem. 236, 28 13-2820. LOWRY0. H., ROBERTSN. R., Wu M.-L., HIXONW. S. & CRAWFORD E. J. (1954) J. biol. Chem. 207, 19-37.
541
MATTHEWS M. R. & RAISMANG. (1972)Proc. R. SOC.,Lond. B. 181, 43-79. MCCAULEY R. & RACKERE. (1973) Mol. Cell Biochem. 1, 73-81. MCDOUGAL D. B., JR., SCHIMKE R. T., JONESE. M., TouHILL E. (1964) J. gen Physiol. 47, 419-432. G . (1972) Z . Zellforsch. 124, MCLEANJ. R. & BURNSTOCK 44-56. NEIDLEA,, VAN DEN BERGC. 0. & GRYNBAUM A. (1969) J . Neurochem. 16, 225-234. PARTLOW L. M., Ross C. D., MOTWANI R. & MCDOUGAL D. B., JR. (1972) J. gen. Physiol. 60, 388405. Ross C. D. & MCDOUGAL D. B., JR. (1976) J. Neurochem. 26, 521-526. SCHLAEPFER W. W. & TORACKR. M. (1966) J. Histochem. Cytochem. 14, 369-378. SCHMIDT R. E. & MCDOUGAL D. B., JR. (1978) J . Neurochem. 30, 527-535. SHIH J.-H. C . & EIDUSONS. (1971) J. Neurochem. 18, 1221-1 227. THOENEN H. (1972) Handbook of Experimental Pharmacology (BLASCHKO H. & MUSCHOLL E., eds) Vol. XXXIII, pp. 813-844, Springer, Berlin. WURTMAN R. J. & AXELRODJ. (1963) Biochem. Pharmac. 12, 143S1441. YANG H.-Y. T., GORIDIS C. & NEFFN. H. (1972) J. Neurochem. 19, 1241-1250. YANC H.-Y. T. & NEFFN. H. (1973) J . Pharmac. exp. Ther. 187, 365-371. YANG H.-Y. T. & NEFFN. H. (1974) J. Pharmac. exp. Ther. 189, 733-740.
EFFECTS OF SYMPATHECTOMY O N AXOPLASMIC TRANSPORT OF SELECTED ENZYMES INCLUDING M A 0 AND OTHER MITOCHONDRIAL ENZYMES ROBERTE. SCHMIDT, C. DAVIDRoss and DAVIDB. MCDOUGAL, JR. Department of Pharmacology, Washington University School of Medicine, St. Louis, MO 631 10, U.S.A. (Received 21 April 1977. Accepted 25 July 1977)
Abstract-Axophsmic transport in guanethidine sympathectomized and control rats was investigated by monitoring the accumulations of vanous enzyme activities proximal to a ligature placed on the sciatic nerve. Sympathectomy affected the accumulations of three different mitochondrial enzymes quite differently: the accumulation of monoamine oxidase (MAO, EC 1.4.3.4) activity was inhibited 65% or more, that of hexokinase (HK, EC 2.7.1.1) activity was only inhibited 26%, while accumulation of glutamic dehydrogenase (GDH, EC I .4.1.3) activity was unaffected by sympathectomy. Accumulation of AChE (EC 3.1.1.7) activity was depressed 40%, but accumulations of the activities of the lysosomal enzyme, acid phosphatase (acid P'tase, EC 3.1.3.2), and of the cytosolic enzyme, choline acetyltransferase (CAT, EC 2.3.1.6) were unchanged. Despite impressive inhibition of MA0 accumulation, the intrinsic activity of this enzyme in sciatic nerve was unaffected by sympathectomy.The existence in nerve of isozymes of M A 0 was demonstrated using the inhibitors clorgyline and deprenyl. Transported MA0 activity was almost entirely type A; intnnsic activity was 2/3 type A and 1/3 type B. The differential response of the accumulations of the three mitochondrial enzyme activities measured was interpreted to indicate the existence, within neurons, of mitochondria with different enzyme complements.
PERIPHERAL nerve is composed of a complex mixture of somatic and sympathetic motor and sensory axons. Each axon type may be expected to differ from the others in the nature or amount of material moved by axoplasmic transport. Accumulation of particlespecific enzyme activities at a ligature applied to a peripheral nerve has been shown to be dependent on axoplasmic transport (Schmidt & McDougal, 1978). Although some transmitter-related enzymes, such as dopamine-beta-hydroxylase (3,4-dihydroxy phenylethylamine, ascorbate:oxygen oxidoreductase (hydroxylating), EC 1.14.2.1) or choline acetyltransferase (acetyl-CoA:choline 0-acetyltransferase, EC 2.3.1.6) are likely to be localized to a specific anatomical or physiologic type of neuron, it is difficult to predict the distribution among the several fiber types of enzymes whose functions are more general. We have shown that the activities of two mitochondrial enzymes, glutamic dehydrogenase (L-glutamate:NAD(P) oxidoreductase (deaminating), EC 1.4.1.3, GDH) and monoamine oxidase (monoamine:oxygen oxidoreductase (deaminating), EC 1.4.3.4, MAO), accumulate at a ligature on rat sciatic nerve. However, the patterns of the accumulation of activity were quite different (Schmidt & McDougal, 1978): M A 0 activity accumulated more rapidly relative t o intrinsic nerve activity than did GDH activity during the first two days after ligation. The involvement of M A 0 in catecholamine metabolism made it seem likely that the difference in the accumulation of the two mitochondria1 enzymes could be the result
of the preferential localization of M A 0 to adrenergic axons. Since chemical sympathectomy has been successfully used to examine the sympathetic contribution to the innervation of many organs (see THOENEN, 1972 for review) the accumulation of M A 0 and GDH activities has been studied at ligatures placed on sciatic nerves of chemically sympathectomized and control rats. The accumulations of the activities of acetyIcholinesterase (acetylcholine hydrolase, EC 3.1.1.7, AChE), choline acetyltransferase (CAT), hexokinase (ATP:D-hexose 6 phosphotransferase EC 2.7.1.1, HK), another mitochondrial enzyme, and acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2, acid Ptase) a lysosomal enzyme, were also determined.
MATERIALS AND METHODS Sprague-Dawley rats were sympathectomized using 21 daily injections of guanethidine (100 mg/kg s.c.) beginning at birth. At the age of 3 months (approx 200g) male animals were tested for completeness of sympathectomy (resultsincluded in DOUGLAS et al., 1975), and females were used in the present experiments. We are indebted to Drs. DOUGLAS and NEEDLEMAN for these animals. Control animals of the same strain and sex were selected to match the sympathectomized animals by weight. The animals were anesthetized with pentobarbital (35 mg/kg i.p.). The sciatic nerves of both legs were exposed and ligated with 6-0 silk thread in midthigh. The wound
538
R . E. SCHMIDT, C. D. Ross and D. B. MCDO~JGAL, JR
was closed and the animal allowed to survive I2 h before decapitation. Sciatic nerves were rapidly dissected free, cleaned of adherent tissue and frozen on solid CO,. After freeze drying at -35 to -40°C nerve proximal to the ligature was sectioned into three consecutive pieces from positions 0-2, 2 4 , and 4-8 mm (segments 1, 2 and 3 respectively) proximal to the tie. This last (segment 3, designated the 'intrinsic segment') under the conditions of this experiment is known to have the same specific enzyme activities as unligated nerve (SCHMIDT & MCDOUGAL, 1978). These segments were weighed on a quartz fiber balance (LOWRY & PASSONNEAU, 1972). Each segment was homogenized in 90 pl (2 mm) or 180 p I (4 mm) of 100 mM-phosphate buffer, pH 7.8, containing 0.05"; BSA. Homogenates were stored at -80 C until they were assayed for AChE (GUTHet 01.. 1964). MA0 (WURTMAN & AXELROD, 1963), GDH et 01.. 1962). acid Ptase (LOWRY et a/.. (GARCIA-BUNUEL 1954), CAT (Ross & MCDOUGAL, 1976) and HK (LOWRY et ul., 1961). The accumulation of enzyme activity in the two 2mm nerve segments proximal to the tie was calculated as follows:
1978) from the total activity of the 2mm pretie segment. Materials Clorgyline and deprenyl were gifts from Dr. NORTON NEFF. Substrates. coenzymes and enzymes used in the assays were obtained from Sigma and Boehringer and Sons. ['4C]Tryptamine bisuccinate was obtained from New England Nuclear.
RESULTS Sympathectomy produced a 20% decrease in AChE and a 13% decrease in acid P t a s e activities of intrinsic nerve segments (Table I). The activities of MAO, CAT, GDH, a n d HK of intrinsic nerve segments were not measurably affected by sympathectomy. No change in nerve segment weight per unit length was observed.
+
TABLE 1. EFFECTOF SYMPATHECTOMY ON INTRINSIC ENZYME Absolute = ( A , - A , ) W , ( A 2 - A,) W , ACTIVITY IN SCIATIC NERVE accumulation where:A,,2,3 = enzyme activity per unit dry weight of 1st Enzyme Control Sympathectomy or 2nd (2 mm) or 3rd (4 mm) nerve segment, (pmol/kg dry weight/h) 232 5 14 214 & 12 W, ,2 = weights of corresponding nerve segments. M A 0 1 p ~ 20 pM 1222 f 106 1172 f 93 In control animals the enzyme activity accumulated in (mmol/kg dry weight/h) segment 1 12 h after ligation produced an increase in the HK 326 12 291 f 11 measured activity of 150% of the activity of the intrinsic GDH 250 9 230 12 segment (No. 3) for MAO. 70% for GDH, 135% for HK, AChE 125 & 5* 156 k 4 235% for AChE. 250% for acid P'tase and 55% for CAT. Acid P'tase 84 f 4 73 f I t 41 + 2 37 2 With the exception of CAT the accumulations of enzyme CAT activities in segment 2 were always less than 20% of those (pggimm) 218 k 5 220 10 in segment 1. For CAT accumulations in these two seg- Nerve weight ments were about equal. Enzyme activities in the nerve segment located 4-8 mm M A 0 and inhibitors Sciatic nerves were ligated in uioo proximal to the ligature (intrinsic nerve segment, No. 3. for 2 days. Frozen nerve was sectioned into two segments see Methods) are given. M A 0 was assayed at 2 tryptamine proximal to the ligature: a 2 mm segment immediately concentrations, 1 p~ and 20 p ~ Each . value represents the adjacent to the tie and a 6 mm (intrinsic activity) segment mean f S.E. of 8 sympathectomized and 6 control nerves. extending 7-13 mm from the tie. A few nerves were sec- * P < 0.01. tioned distally into a two mm segment adjacent to the t P < 0.05. tie and a 5 mm segment extending 4-9 mm from the ligaOF SYMPATHECTOMY ON ACCUMULATION OF ture. Nerve segments were homogenized in 100pI of TABLE2. EFFECT ENZYME ACTIVITY AT A LIGATURE ON SCIATIC NERVE 100 mM-phosphate buffer, pH 7.8, containing 0.05% BSA. M A 0 was assayed in the presence of 20 pwtryptamine Enzyme Control Sympathectomy bisuccinate. The effect of clorgyline on M A 0 activity was determined by assaying M A 0 in the presence of increasing (pmol/h) concentrations of inhibitor without preincubation of 158 k 26 MA0 1 j i ~ 55 k 14* enzyme with inhibitor. Preincubation of enzyme with clor20 j l M 616 160 178 71t gyline without substrate for 15' at 25°C or 1 0 at 37°C (nmolih) did not enhance inhibition. Assay of M A 0 in the presence HK 186 f 6 138 & 4* GDH 75 & 10 7 0 + 11 of increasing concentrations of deprenyl was performed 160 k 9 90 f 4* using 15' preincubation without substrate at 25°C. AChE Acid P'tase 90 & 9 77 k 5 Deprenyl was used at 100 ng/ml without preincubation in CAT 8.5 & 2.1 9.6 f 1.5 an experiment testing the additivity of clorgyline and deprenyl inhibition. The extent of inhibition by deprenyl Accumulations are expressed in terms of absolute acat 100ng/ml was the same with and without 15 min pretivity accumulating at a tie after 12 h t n vim. M A 0 was incubation. assayed at 2 tryptamine concentrations, I pM and 20pM. The amount of axoplasmically transported M A 0 ac- Number of nerves is the same as in Table I with the excep cumulating at a ligature was calculated by subtracting tion of CAT accumulation in sympathectomized nerves 2 mm worth of intrinsic segment activity (corrected for a where n was I. 10% increase in amount of enzyme at the ligature induced * P < 0.01. & MCDOUGAL, t P < 0.05. by the mechanical act of ligation, SCHMIDT
+ +
+
+
+
*
Effects of sympathectomy on axonal transport
539
TABLE 3. ADDITIVITY OF CLORGYLINE AND DEPRENYL INHIBI-
3 1000 K
TION OF
M A 0 ACTIVITY
I-
z
s
Clorgyline Deprenyl Clorgyline and deprenyl (percentage inhibition)
& Unligated
50-
a
->
INTRINSIC
P W
\
.
k
1
!-
:: 0 -
TRANSPORTED \
-10- A @ o io-'O 1 0 ' ~ I O - ~ lo-'
IO-~
CLORGYLINE CONCENTRATION
I-
2 0
L,, 0 1
(M)
#
50
DEPRENYL ( n g / m l )
I+
100 1000
FIG.1. Effect of clorgyline and deprenyl on intrinsic and accumulated M A 0 activity in control sciatic nerve. M A 0 of the intrinsic (-) and pre-tie (. . . . .) segments and transported M A 0 activity (-----) of the rat sciatic nerves 2 days after tying, calculated as described in Methods, was examined in the presence of increasing concentrations of (A) clorgyline and (B) deprenyl. See Materials and Methods for details. All M A 0 activities are expressed as a percentage of the uninhibited enzyme activity. Uninhibited M A 0 activities of pre-tie and intrinsic segments and transported M A 0 activity were 4099 f 75, 1768 f 47, and 2134 & 94 pmol/h/2 mm nerve respectively in the clorgyline experiment and 4320 f 121, 1802 f 127, and 2318 f 118 pmol/h/2 mm nerve in the deprenyl experiment. Each point represents the mean f S.E. of 6 nerves. However, there were very appreciable changes in enzyme accumulation at a ligature (Table 2). The amount of M A 0 activity accumulating at a tie was decreased 65% or more by sympathectomy. Accumulation of hexokinase, another mitochondria1 enzyme, was reduced 26%, while that of still a third mitochondrial enzyme, GDH, was unchanged. The accumulation of AChE activity was decreased 40% by sympathectomy. Accumulations of the lysosoma1 enzyme acid P'tase and the soluble CAT were not altered significantly. The profound decrease in transported M A 0 activity with a minimal change in M A 0 activity of intrinsic nerve segments and the somewhat different ratios of accumulated and intrinsic M A 0 activities with different substrate concentrations suggested the possibility of several forms of M A 0 in this system. The apparent K , for tryptamine for M A 0 in sciatic nerve homogenate was about 7 PM. Both transported and intrinsic M A 0 activity are sensitive to the type
N.C. 30/3-C
. 54
nerve
0
Pre-tie Intrinsic
79 f 2 57 f 1
41 f 2
91 f 1
31 f 2 46 f 3
91 + 2 89 2
+
M A 0 activity of unligated nerve and pre-tie and intrinsic nerve segments of 2 day in uiuo ligated nerve was assayed without inhibitors, or in the presence of l o T 8 M-clorgyline, 100 ng/ml deprenyl, or 10- * M-ClOrgyline + 100 ng/ml deprenyl. Activity is expressed as percentage of inhibition compared to uninhibited control MA0 activity. Absolute M A 0 activities of unligated, pre-tie and intrinsic nerve segments were 1993 _+ 139, 3666 _+ 354, and 1444 _+ 112 pmol/h/2 m m nerve respectively. Each value represents the mean f S.E. of 6 nerves.
A M A 0 inhibitor clorgyline at very low concentrations (Fig. 1A). At lo-' M, clorgyline inhibited transported M A 0 activity completely but intrinsic segment M A 0 activity was only inhibited 60%. Inhibition by the type B M A 0 inhibitor deprenyl reached an approximate plateau of inhibition between 10 and 100ng/ml of inhibitor (Fig. 1B). Intrinsic M A 0 activity was inhibited 40% over this range and transported M A 0 only 10%. The additivity of inhibition of M A 0 activity with clorgyline and deprenyl was investigated with enzyme from unligated nerve and intrinsic and pre-tie segment M A 0 from two day ligated nerves (Table 3). Approximate additivity of inhibition occurs with enzyme from all three sources; however, some overlap of inhibition probably occurs, so that the inhibition produced by the two inhibitors together was somewhat less than the sum of inhibitions produced by each one separately. DISCUSSION The fact that there was no change in weight per unit length of nerve produced by sympathectomy (Table 1) suggests that the mass of fibers removed by sympathectomy was relatively small; however, a decrease of less than 1045% would not have been discernible. Therefore, these fibers must be relativel; rich in transported M A 0 activity, since approx 213 of the M A 0 accumulating at a ligature would be contained in less, perhaps much less, than 1/5 the mass of axons. In contrast to the transported enzyme activity, the intrinsic MAO activity associated with the sympathetic fibers did not appear particularly high. The accumulation of some M A 0 activity in sympathectomized nerves is not necessarily evidence for the incomplete removal of sympathetic fibers. The littermates of these animals were shown to have essentially no surviving peripheral sympathetic neurons (DOUGLAS et al., 1979, and M A 0 is known not to be restricted to sympathetic neurons (BLQOMet al.,
540
R. E. SCHMIDT, C. D. Ross and D. B. MCDOUGAL. JR.
1972; THOENEN, 1972). Some studies, however, three accumulating mitochondria1 enzymes provides reported the absence of M A 0 in cholinergic axons additional evidence that mitochondria in the nervous (CONSOLO et al., 1968) and the relative absence of system are not homogeneous (MCDOUGALet al., M A 0 in sensory and cholinergic cells compared to 1964; NEIDLEet al., 1969). In rat brain, M A 0 sedisympathetic ganglion cells (KOELLE& VALK, 1954). ments in a different fraction than GDH (NEIDLEet DAHLSTROM et al. (1 969) also measured the accumu- al., 1969), and in the same system mitochondria were lation of M A 0 at a ligature in sciatic nerves of con- separated into several subclasses with different relatrol and (surgically) sympathectomized rats. They tive activities against various substrates for M A 0 found no accumulation in control animals until 3 (KROON& VELDSTRA,1972). In the present instance, days after ligation, and in sympathectomized animals, since GDH, HK and M A 0 have been shown to acnone until the seventh day. The amount of accumu- cumulate as a result of axoplasmic transport (PART1978), it lation we found in normal animals at 7 days (SCHMIDT LOW et a/., 1972; SCHMiDT & MCDOUGAL, & MCDOUGAL, 1978) was about the same as that now appears that different neurons can have mitoet al. (1969), but chondria with different enzyme compositions. found in controls by DAHLSTROM we detected no measurable delay between ligation The 40% decrease in AChE transport suggests that and the onset of accumulation of MA0 activity. Some sympathetic fibers are relatively rich in transported of the discrepancy may be explainable by the diluting A G E , while the substantial decrease in intrinsic effect of the use of larger nerve segments (5mm) by A G E activity suggests relatively large amounts of et al., since the accumulation is mostly AChE in their axolemmata. It has been shown in rat DAHLSTROM confined to the first 2 m m adjacent to the ligature. superior cervical ganglion that adrenergic neurons Transport to MA0 has been observed to decrease (identified by catecholamine fluorescence) contain hisin amphibians subjected to operative sympathectomy tochemically demonstrable AChE in their cell bodies (H~KONEN , 1963). Since guanethidine has been (MCLEAN & BURNSTOCK,1972). Use of kinetic studies, selective inhibitors, immuno- shown to be specific for adrenergic neurons (JENSENet al., 1971 ; ANGEprecipitation, substrate specificity, thermal denatur- HOLM& JUUL, 1971; BURNSTOCK ation, and electrophoretic mobility has revealed the LETTI & LEVI-MONTALCINI, 1972), one would expect existence of two isozymes or isozymic classes of M A 0 that guanethidine treatment would leave cholinergic 1968; GORIDIS & NEE, 1971 ; SHm & EIDU- sympathetic axons untouched. Thus the decrease in (JOHNSON, SON, 1971; YANG et al., 1972; YANG & NEFF, 1973, AChE transport cannot be explained by destruction 1974; MCCAULEY & RACER, 1973). One form of of these neurons. AChE staining of rat sciatic nerve MAO, designated type A, is more active against the suggests that sympathetic fibers are rich in axolemmal neurotransmitters norepinephrine and serotonin, is AChE (SCHLAEPFER & TORACK, 1966). Our observaexquisitely sensitive to clorgyline, harmine, and har- tions are consistent with the notion that CAT is not maline, but is resistant to inhibition by deprenyl. concentrated in adrenergic axons; however, it should Type B enzyme is more active against phenylethyl- be stressed that even appreciable changes in CAT amine and benzylamine, is insensitive to clorgyline transport might have been overlooked in this study and sensitive to deprenyl and pargyline. Both types since the time allowed for accumulation was short of enzyme are active against tyramine and tryptamine. relative to the apparent rate of transport of this In certain experimental systems, such as the innerva- enzyme. The lack of change in accumulation of acid Ptase tion of the pineal gland by the cervical sympathetic ganglion, type A MA0 seems to be concentrated in activity in response to sympathectomy does not mean the neurons of the ganglion, including the nerves in that adrenergic axons lack fysosomes, but only that the gland (YANG et al., 1972). Type B appears to be lysosomes containing acid Ptase are not concentrated localized in the (non-neuronal) parenchymal cells of in these axons. Indeed, electron microscopic studies the pineal gland. However, the type A enzyme is of rat sympathetic axons reveal dense bodies accumu8z &ISMAN, 1972). probably not exclusively neuronal. Our experiments lating at a ligature (MATTHEWS are consistent with the neuronal localization of type A enzyme, since only intraneuronal materials would Acknowledgements-We thank Ms. MARGARETYv for be expected to accumulate by axonal transport at a skillful technical assistance. Supported by NIH grants ligature under the conditions of our experiments. GM-02016, NS-11941 and NS-06800. Transported enzyme is almost entirely type A; unligated or intrinsic segment activity seems to be composed of 213 type A enzyme and 1/3 type B enzyme. REFERENCES Intrinsic activity may be localized to Schwann cells, A N G F L E P. ~ U. & LEVI-MONTALCINI R. (1972) Proc. nutn. connective tissue, vascular tissue or non-sympathetic Acud. SCL, U.S.A. 69, 86-88. axons. Preliminary experiments with M A 0 activity BLOOMF. E., SIMSK. L., WEITSENH.A,, DAVISG. A. accumulating distal to a tie revealed it also to be & HANKER J. S. (1972) Adv. Biochem. Pharmuc. 5, mostly type A enzyme, i.e. it was inhibited 90% by 243-262. 10- 8w-clorgyline. BURNSTOCKG., EVANS B., GANNON B. J., HEATHJ. W. 8c JAMES V. (1971) Br. J . Pharmuc. 43, 295-301. The differential response to sympathectomy of the
Effects of sympathectomy on axonal transport S., GIACOBINI E. & KARJALAINEN K. (1968) Acta. CONSOLO physiol. scand. 74, 513-520. DAHLSTR~M A., JONASON J., & NORBERG K. A. (1969) Eur. J. Pharmac. 6, 248-254. DOUGLAS J. R., JR., JOHNSONE. M., JR., MARSHAL G. R., B. K. & NEEDLEMAN P. (1975) CirHEISTJ., HARTMANN culation Res. 36 and 37, Suppl. 1, 171-178. GARCIA-BUNUEL L., MCDOUGAL D. B., JR., BURCHH. B., JONESE. M. & TOUHILLE. (1962) J. Neurochem. 9, 589-594. GORIDISC. & NEFFN. H. (1971) Neuropharmacology 10, 557-564. G u m L., ALBERS R. W. & BROWNW. C. (1964) Exp. Neurol. 10, 236-250. HARKONEN M. (1964) Acta physrol. scand. 63, Suppl. 237, 3-94. JENSEN-HOLM J. & JUULP. (1971) Acta Pharmac. Toxicol. 30, 308-320. JOHNSON J. P. (1968) Biochem. Pharmac. 17, 1285-1297. KOELLEG . B. & VALK A. de T. (1954) J. Physiol., Lond. 126, 434447. KROONM. C. & VELDSTRA H. (1972) FEBS Lett. 24, 173-176. LOWRY0. H. & PASSONNEAU J. V. (1972) A Flexible Sysrem of Enzymatic Analysis. Academic Press, New York. LOWRY0. H., ROBERTS N. R., SCHULZD. W., CLOW J E. & CLARKJ. R. (1961) J . biol. Chem. 236, 28 13-2820. LOWRY0. H., ROBERTSN. R., Wu M.-L., HIXONW. S. & CRAWFORD E. J. (1954) J. biol. Chem. 207, 19-37.
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MATTHEWS M. R. & RAISMANG. (1972)Proc. R. SOC.,Lond. B. 181, 43-79. MCCAULEY R. & RACKERE. (1973) Mol. Cell Biochem. 1, 73-81. MCDOUGAL D. B., JR., SCHIMKE R. T., JONESE. M., TouHILL E. (1964) J. gen Physiol. 47, 419-432. G . (1972) Z . Zellforsch. 124, MCLEANJ. R. & BURNSTOCK 44-56. NEIDLEA,, VAN DEN BERGC. 0. & GRYNBAUM A. (1969) J . Neurochem. 16, 225-234. PARTLOW L. M., Ross C. D., MOTWANI R. & MCDOUGAL D. B., JR. (1972) J. gen. Physiol. 60, 388405. Ross C. D. & MCDOUGAL D. B., JR. (1976) J. Neurochem. 26, 521-526. SCHLAEPFER W. W. & TORACKR. M. (1966) J. Histochem. Cytochem. 14, 369-378. SCHMIDT R. E. & MCDOUGAL D. B., JR. (1978) J . Neurochem. 30, 527-535. SHIH J.-H. C . & EIDUSONS. (1971) J. Neurochem. 18, 1221-1 227. THOENEN H. (1972) Handbook of Experimental Pharmacology (BLASCHKO H. & MUSCHOLL E., eds) Vol. XXXIII, pp. 813-844, Springer, Berlin. WURTMAN R. J. & AXELRODJ. (1963) Biochem. Pharmac. 12, 143S1441. YANG H.-Y. T., GORIDIS C. & NEFFN. H. (1972) J. Neurochem. 19, 1241-1250. YANC H.-Y. T. & NEFFN. H. (1973) J . Pharmac. exp. Ther. 187, 365-371. YANG H.-Y. T. & NEFFN. H. (1974) J. Pharmac. exp. Ther. 189, 733-740.
