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Evidence that dopamine regulates norepinephrine synthesis in the rat superior cervical ganglion during hypoxic stress
Journal of the Autonomic Nervous System, 18 (1987) 185-193
185
Elsevier JAN 00695
Research Papers
Evidence that dopamine regulates norepinephrine synthesis in the rat superior cervical ganglion during hypoxic stress J a m e s J. B r o k a w a n d J o h n T. H a n s e n Department of Cellular and Structural Biology, The University of Texas Health Science Center. San Antonio, TX 78284 (U. S.A.)
(Received 6 March, 1986) (Revised version received and accepted 16 October 1986)
K e y words: H y p o x i c stress; Small intensely fluorescent ( S I F ) cell; S u p e r i o r cervical ganglion;
C a t e c h o l a m i n e turnover; T y r o s i n e h y d r o x y l a s e activity Summary Electrical stimulation of preganglionic nerves is known to increase norepinephrine synthesis in the rat superior cervical ganglion in vitro, an effect which appears to be partially regulated by a non-cholinergic transmitter. In the present study, we sought to determine whether sympathetic stimulation also increases norepinephrine synthesis in the rat ganglion in vivo, and whether dopamine released from ganglionic interneurons might regulate this response. To tackle these questions, rats were pretreated with spiroperidol, a selective dopamine-receptor blocker, and then were sympathetically stimulated by exposure to severe hypoxic stress. Other rats were pretreated with vehicle alone before the hypoxic exposure. Norepinephrine synthesis in ganglia was assessed by measuring endogenous tyrosine hydroxylase activity and norepinephrine turnover. We found that hypoxic stress increased both of these indices of norepinephrine synthesis, but only in rats pretreated with spiroperidol. No such response was detected in rats pretreated with vehicle. These results indicate that sympathetic stimulation increases norepinephrine synthesis in the rat superior cervical ganglion in vivo, and that dopamine released from interneurons might regulate this response.
Introduction T h e s u p e r i o r cervical ganglion ( S C G ) c o n t a i n s a high c o n c e n t r a t i o n of n o r e p i n e p h r i n e d i s t r i b u t e d t h r o u g h o u t the cell b o d i e s a n d processes of the p r i n c i p a l n e u r o n s [11]. A l t h o u g h the precise role of this c a t e c h o l a m i n e in ganglionic function is uncertain, several lines of evidence suggest that s y m p a t h e t i c s t i m u l a t i o n releases n o r e p i n e p h r i n e f r o m nerve t e r m i n a l s within the ganglion to m o d ify neural t r a n s m i s s i o n [33]. If s y m p a t h e t i c stimulation does release n o r e p i n e p h r i n e , then it is likely Correspondence: J.J. Brokaw, Cardiovascular Research Institute, 1327-M, University of California School of Medicine, San Francisco, CA 94143, U.S.A.
that n o r e p i n e p h r i n e levels within the ganglion w o u l d be m a i n t a i n e d b y a c o r r e s p o n d i n g increase in n o r e p i n e p h r i n e synthesis [29,40]. Consistent with this notion, electrical s t i m u l a t i o n of p r e g a n glionic axons has been shown to increase n o r e p i n e p h r i n e synthesis in the rat S C G in vitro [19,38]. F u r t h e r m o r e , this s t i m u l u s - i n d u c e d response app e a r s to be regulated in p a r t b y acetylcholine a n d in p a r t b y a n o n - c h o l i n e r g i c t r a n s m i t t e r [19]. O n e p o s s i b l e c a n d i d a t e for this n o n - c h o l i n e r g i c t r a n s m i t t e r is d o p a m i n e . In m a n y m a m m a l i a n species, d o p a m i n e is the p r i m a r y t r a n s m i t t e r present in a small p o p u l a t i o n of ganglionic cells referred to as small intensely fluorescent ( S I F ) cells [42]. Because S I F cells are often seen s y n a p t i cally i n t e r p o s e d b e t w e e n pre- a n d p o s t g a n g l i o n i c
0165-1838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
186
elements, particularly in the rat SCG [10], it has been suggested that they function as interneurons which regulate ganglionic transmission [42]. According to current hypotheses of SIF cell function [27], sympathetic stimulation releases SIF cell dopamine, which, in turn, acts on principal neurons to produce the slow inhibitory postsynaptic potential (sIPSP) and to augment the slow excitatory postsynaptic potential (sEPSP). However, it is not known if these dopamine-induced alterations in neuronal excitability are accompanied by corresponding alterations in norepinephrine synthesis. Therefore, this study poses the following questions: does sympathetic stimulation increase norepinephrine synthesis in the rat SCG in vivo and, if so, might this response be regulated by SIF cell dopamine? To address these questions, ganglionic norepinephrine synthesis was assessed in rats that were sympathetically stimulated by exposure to severe hypoxic stress [16,39], either with or without prior treatment with a selective dopamine-receptor blocker. To provide a basis of comparison, ganglionic catecholamine synthesis was also systematically characterized under resting conditions.
Materials and Methods
Animals and drugs Male Sprague-Dawley rats (220-310 g) used in this study were suppfied by two sources, Charles River and Harlan Sprague-Dawley. The following drugs were used in this study: a-methyl-p-tyrosine, methyl ester (a-MT, Regis); m-hydroxybenzylhydrazine dihydrochloride (NSD-t015, Sigma); diethyldithiocarbamate, sodium salt (DDC, Regis); pargyline hydrochloride (Sigma); and spiroperidol (a gift from Janssen Pharmaceuticals, Beerse). aMT, NSD-1015, and pargyline were dissolved in normal saline; DDC and spiroperidol were dissolved in dimethylsulfoxide and 0.1 M citric acid, respectively. In all experiments, control rats received injections of the corresponding vehicle. Catecholamine synthesis in the SCG during rest Before we investigated the effects of sympathetic stimulation (hypoxic stress), we first
wanted to characterize catecholamine synthesis in the rat SCG during resting conditions (normoxia). Two indices of catecholamine synthesis were used in this study: catecholamine turnover and tyrosine hydroxylase activity [35]. To determine the turnover of ganglionic norepinephrine and dopamine, rats were treated with a-MT (250 mg/kg, i.p.), an inhibitor of tyrosine hydroxylase [37], and killed at selected timepoints thereafter. After catecholamine measurements, the time-dependent decrease in norepinephrine content was plotted semilogarithmically and a regression line was calculated by the method of least-squares (y = 155.48 - 41.35 x). The fractional rate constant was determined from the relationship: K = 0.693/tl/2 and the norepinephrine turnover rate (pmol/ganglion/h) was calculated by multiplying the rate constant times the initial steady-state concentration of norepinephrine [3]. The decrease in dopamine content was statistically evaluated with oneway analysis of variance (ANOVA) and the Newman-Keuls test. To determine ganglionic tyrosine hydroxylase activity, rats were treated with NSD-1015 (100 mg/kg, i.p.), an inhibitor of L-aromatic amino acid decarboxylase (DOPA decarboxylase) [23], and killed at selected time-points thereafter. After DOPA measurements, the increase in DOPA content, a reflection of tyrosine hydroxylation, was plotted using the function: y = 34.22(1 - e -°165t) + 6.27 The instantaneous rate of DOPA production (pmol/ganglion/min) was calculated from the expression: (d y / d t )t= o = (34.22)(0.165) and the time (min) required for the half-maximum DOPA increase was determined from the relationship:
tl/2
=
0.693/0.165
187 The decrease in dopamine content was statistically evaluated with one-way ANOVA and the Newm a n - K e u l s test. In addition, to determine the relative proportion of ganglionic dopamine serving as norepinephrine precursor, rats were treated with DDC (200 mg/kg, i.p.), an inhibitor of dopamine-/3-hydroxylase [17], and killed at selected time-points thereafter. This procedure also provided a more sensitive measure of norepinephrine turnover [15]. After catecholamine measurements, the increase in dopamine levels and the decrease in norepinephrine levels were statistically evaluated with one-way ANOVA and the Newman-Keuls test. Finally, to determine the contribution of metabolism to the turnover of ganglionic catecholamines, rats were treated with pargyline (100 mg/kg, i.p.), an inhibitor of monoamine oxidase [22], and killed at selected time-points thereafter. After catecholamine measurements, the increases in norepinephrine and dopamine contents were statistically evaluated with one-way ANOVA and the Newman-Keuls test. Catecholamine synthesis in the SCG during sympathetic stimulation To induce physiologic stress, rats were made severely hypoxic by placing them in a chamber (20 liter capacity) that was continuously flushed with a gas mixture consisting of 5% 02 and 95% N 2. The gas concentration was periodically tested with a Fyrite 02 analyzer. We have previously demonstrated that this method is effective in lowering arterial blood gases [4]. To determine the effect of hypoxic stress on ganglionic tyrosine hydroxylase activity, rats were treated with NSD-1015 (100 mg/kg, i.p.) and exposed to either room air (controls) or the hypoxic gas mixture for 20 min. To assess the potential role of dopamine in regulating the response to hypoxia, this same procedure was repeated 30 min after rats had been pretreated with spiroperidol (1 mg/kg, i.p.), a dopamine-receptor antagonist [1,20], or with vehicle. After DOPA measurements, group means were compared with Student's t-test (two-tailed). To determine the effect of hypoxic stress on ganglionic norepinephrine turnover, rats were
treated with DDC (200 mg/kg, i.p.) and exposed to either room air or intermittent hypoxia (20 min hypoxia, 10 min room air, repeated) for 2 h. The same procedure was repeated 30 rain after pretreatment with spiroperidol (1 mg/kg, i.p.) or vehicle. After catecholamine measurements, group means were compared with one-way ANOVA and the Newman-Keuls test. Catecholamine assay After the experimental procedures, rats were killed by decapitation and their ganglia were rapidly removed (5 rain), frozen on dry ice, and stored at - 7 0 ° C until assayed for catecholamines. The concentrations of DOPA, dopamine, and norepinephrine were measured by high performance liquid chromatography with electrochemical detection [28]. Each sample consisted of a single ganglion (one per rat) homogenized in 200 t~l of 0.2 M perchloric acid that contained 16 n g / 2 0 ~1 of dihydroxybenzylamine as an internal standard. Reference standards contained 16 n g / 2 0 ~1 each of DOPA, dopamine, norepinephrine, and dihydroxybenzylamine. All other details of the assay were identical to those we reported previously [4]. Results are expressed as picomols of catecholamine per ganglion.
Results
Catecholamine synthesis in the SCG during rest The steady-state concentrations of ganglionic norepinephrine and dopamine determined from 126 control rats were 175.0 _+ 38.4 and 17.9 _+ 5.2 pmol/ganglion, respectively (mean ___S.D.). Inhibiting tyrosine hydroxylase with a-MT caused a rapid decrease in ganglionic norepinephrine and dopamine (Fig. 1). The norepinephrine content was reduced by 74% from baseline values after 3 h. The half-life and turnover rate of norepinephrine were 1.6 h and 73 pmol/ganglion/h, respectively. Within 1 h after a-MT injection, dopamine content had dropped by 60% from baseline ( P < 0.001) and remained at about this level for 3 h. Inhibiting of DOPA decarboxylase with NSD1015 caused a rapid accumulation of ganglionic
188
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Fig. 1. Decline in the concentrations of norepinephrine and dopamine in the rat superior cervical ganglion (SCG) after the administration of a-methyl-p-tyrosine. Each point represents the mean+ S.E.M. of 8 measurements. *, Significantly different from time-zero controls ( p < 0.001).
D O P A and a corresponding decrease in ganglionic d o p a m i n e (Fig. 2). The increase in D O P A content was h a l f - m a x i m u m in 4.2 min. The rate of D O P A formation was 338 p m o l / g a n g l i o n / h . Since the
d o p a m i n e content was reduced by 55% somewhere between 20 and 40 min, we estimated the half-life to be approximately 30 min. The greatest reduction in d o p a m i n e occurred within the first 5 min. Inhibiting dopamine-C/-hydroxylase with D D C caused a small decrease in ganglionic norepinephrine that was accompanied by a larger but transient increase in ganglionic d o p a m i n e (Fig. 3). The norepinephrine content was decreased by an average of 33% from baseline at all time-points ( p < 0.001). The d o p a m i n e content was increased by 202% above baseline after 1 h ( p < 0.00t), by 95% after 2 h ( p < 0.01), and by 42% after 3 h (not significant). In addition, the a m o u n t of d o p a m i n e present at 1 h was significantly different from that present at 2 h ( P < 0.005) and 3 h ( P < 0.001). Inhibiting m o n o a m i n e oxidase with pargyline caused a small increase in ganglionic norepinephrine, but no change in ganglionic d o p a m i n e (Table I). The norepinephrine content was increased by 23% above baseline after 1 h (not significant) and b y 42% after 2 h ( P < 0.025). The d o p a m i n e content was not significantly altered at either timepoint.
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Fig. 2. Increase in the concentration of DOPA and decline in the concentration of dopamine in the rat superior cervical ganglion (SCG) after the administration of NSD-1015. Each point represents the mean+S.E.M, of 8 measurements. *, Significantly different from time-zero controls (P < 0.001).
on the concentrations of norepinephrine (NE) and dopamine (DA) in the rat superior cervical ganglion (SCG). Each bar represents the mean+ S.E.M: of 8 measurements. Probability values refer to comparisons between the control levels of NE and DA at time-zero, and their respective levels of subsequent time-points; in addition, the amount of DA present at 1 h is significantly different from that present at 2 h ( P < 0.005) and 3 h (P < 0.001).
189 VEH/CLE PRETREATMENT
TABLE I
Effects of pargvline on catecholamine leuels in the superior cercical ganglion of rats
160 -
Data are mean + S.E.M. for 10 rats. 80-
Time after drug (t7)
Catecholamines (pmol/ganglion) N orepinephrine
Dopamine
0 1 2
171.1 + 10.3 210.8 + 16.0 243.6_+23.3 *
18.1 +_0.9 17.6 _+1.4 17.5+3.0
* Significantly different from time-zero controls ( P < 0.025).
Catecholamine synthesis in the S C G during sympathetic stimulation After tyrosine hydroxylase was inhibited, the amount of ganglionic DOPA that accumulated during 20 min of hypoxic stress was dependent on the pretreatment (Table II). In rats pretreated with spiroperidol, hypoxia elicited a 93% greater increase in DOPA content than was found in the normoxic group ( p < 0.05). In rats pretreated with vehicle, there was no significant difference in DOPA content between the hypoxic and normoxic groups. After dopamine-/3-hydroxylase was inhibited, the degree by which ganglionic catecholamine levels were altered during 2 h of hypoxic stress was also dependent on the pretreatment (Fig. 4).
TABLE
II
Effects of t~vpoxie stress and spiroperidol on (vrosine hvdro:(vlase actwi~ in the superior cervical ganglion of rats Spiroperidol or its vehicle was injected into rats 30 min before the experiment. Tyrosine hydroxylase activity was assessed by measuring the amount of ganglionic DOPA accumulated 20 min after injection of NSD-1015 into rats breathing either room air or 5% 02 in N 2. Data are expressed as m e a n + S.E.M. for 7 or 8 rats.
Pretreatment
Vehicle Spiroperidol
DOPA (pmol/ganglion) Normoxic
Hypoxic
32.7_+ 2.4 28.6 + 2.4
38.0_+ 10.5 55.3 _+ 9.9 *
* Significantly different from the corresponding normoxic group ( P < 0.05).
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Fig. 4. Effects of spiroperidol pretreatment on the concentrations of norepinephrine (NE) and dopamine (DA) in the superior cervical ganglion (SCG) of control rats and rats exposed to either normoxic or hypoxic conditions after the administration of diethyldithiocarbamate (DDC). Each bar represents the mean_+ S.E.M. of 8 measurements. Probability values refer to comparisons between the control levels of NE and DA, and their respective levels in the normoxic and hypoxic groups. Also, in the lower panel, there are significant differences in the levels of both NE (P < 0.005) and DA ( P < 0.001) in the hypoxic group when compared to their respective levels in the normoxic group: no corresponding differences are present in the upper panel.
In rats pretreated with spiroperidol, hypoxia elicited a much greater change in both the amount of dopamine accumulated ( P < 0 . 0 0 1 ) and the amount of norepinephrine depleted (P < 0.005) than in the normoxic group. In rats pretreated with vehicle, there were no significant differences in catecholamine levels between the hypoxic and normoxic groups.
Discussion The results of this study indicate that sympathetic stimulation increases norepinephrine synthesis in the rat SCG in vivo, and that SIF cell dopamine might regulate this response. In addition, we find that ganglionic norepinephrine ex-
190
hibits a rapid turnover and synthesis during resting conditions, and speculate that ganglionic dopamine is segregated into two pools with different rates of turnover. Catecholamine synthesis in the SCG during rest Similar to the findings of others [3,9,14], our values for the half-life (1.6 h) and turnover rate (73 p m o l / g a n g l i o n / h ) of norepinep'hrine indicate that this catecholamine turns over rapidly in the rat SCG. However, the relatively modest increase in norepinephrine content that we observed 2 h after injecting pargyline suggests that degradation by monoamine oxidase is not a major contributor to this rapid turnover. Perhaps other processes, such as axonal transport [2] or synaptic release and overflow [31], contribute more significantly to the elimination of ganglionic norepinephrine. In agreement with the turnover data, our measure of tyrosine hydroxylase activity, 338 pmol D O P A / g a n g l i o n / h , indicates a rapid synthesis of ganglionic norepinephrine. However, the rate of DOPA formation is considerably higher than the rate we determined for norepinephrine turnover. This disparity might be explained by assuming that a substantial portion of the dopamine measured in the ganglion is present in the cytoplasm of principal neurons as norepinephrine precursor and is thus able to limit tyrosine hydroxylase activity by end-product inhibition. If this assumption is correct, then the rapid loss of dopamine that occurred within 5 rain after injection of NSD-1015 might have permitted an exaggerated rate of DOPA formation. In support of this possibility, Ip et al. [19] reported rates as high as 250 pmol D O P A / g a n g l i o n / h for the rat SCG in Vitro. Like Karoum et al. [21], we estimated the halflife of ganglionic dopamine to be considerably less than 1 h, which indicates a very high rate of turnover. Although measures of total dopamine turnover cannot distinguish between the rates of turnover associated with SIF cells and principal neurons, some clues to the relative contributions made by these separate dopamine sources can be deduced from the effects of inhibiting synthesis. For example, within 1 h after a - M T injection, and 40 min after NSD-1015 injection, the ganglionic dopamine content had dropped by about 60%,
leaving some 7 pmol of dopamine that was resistant to further significant depletion for at least 3 h. According to Koslow [26], approximately 40% of the dopamine in the rat SCG is stored in SIF cells. Applying this figure to our measure of ganglionic dopamine (18 pmol/ganglion) would mean that about 7 pmol of dopamine is contained in SIF cells. Therefore, we speculate that the 7 pmol of dopamine remaining 1 h after inhibiting synthesis represents SIF cell dopamine that is slowly turning over, whereas the 60% that is rapidly depleted represents precursor dopamine in principal neurons. This notion is consistent with reports which have shown that SIF cell catecholamines have a very slow turnover in the rat SCG [32,41]. We obtained further evidence for this segregation of dopamine by blocking the conversion of dopamine to norepinephrine and measuring subsequent changes in ganglionic dopamine levelsl Because dopamine content increased rapidly during the first hour after injection of DDC, it is likely that most of the ganglionic dopamine is serving as a substrate for norepinephrine synthesis. The gradual decline in dopamine content thereafter might indicate an increased rate of degradation or release, and could explain why, in an earlier study by Koslow [26], no change in dopamine content was detected 2 h after injection of DDC. Moreover, the absence of any significant alteration in dopamine levels 2 h after injection of pargyline is also consistent with the segregation of ganglionic dopamine into two pools with different rates of turnover. If SIF cell dopamine has a slow turnover, then a correspondingly slow rate of dopamine accumulation would be expected after inhibition of monoamine oxidase [35]. By similar reasoning, the rapid conversion of precursor dopamine to norepinephrine in the storage granules of principal neurons would leave little dopamine available for degradation by monoamine oxidase located in the mitochondrial membrane [36]. Catecholamine synthesis in the SCG during sympathetic stimulation Consistent with previous in vitro findings [19,38], we found that sympathetic stimulation
191
(hypoxic stress) increased endogenous tyrosine hydroxylase activity and norepinephrine turnover in the rat SCG in vivo. However, this stimulus-induced increase in norepinephrine synthesis was demonstrable only in rats that were pretreated with spiroperidol. No such response was seen in rats that were pretreated with vehicle. Because spiroperidol is a highly selective dopamine-receptor antagonist [20], with no significant adrenoceptor activity at the dose used [1], these results suggest that dopamine is involved in the regulation of norepinephrine synthesis in principal neurons during stress. Since spiroperidol was administered systemically, we cannot exclude the possibility that the effects we observed may have been due to a centrally mediated potentiation of sympathetic outflow, unrelated to intrinsic alterations in ganglionic function. It is also possible that norepinephrine synthesis was increased by the actions of adrenal catecholamines reaching the ganglion via the circulation [39]. Nevertheless, given the abundance of dopamine-containing SIF cells in the rat SCG [25,26,34], and the reported effects of dopamine on ganglionic transmission [24,27], a reasonable explanation for our data would be that spiroperidol blocked the postsynaptic actions of SIF cell dopamine on principal neurons. If this interpretation is correct, it suggests that dopamine released from SIF cells inhibits the ability of principal neurons to increase their rates of norepinephrine synthesis in response to sympathetic stimulation. Alternatively, SIF cell dopamine may act presynaptically to inhibit the release of acetylcholine from preganglionic axon terminals [30]. In either case, norepinephrine synthesis would increase during sympathetic stimulation when the inhibitory influence of dopamine is blocked with a selective dopamine-receptor antagonist. In contrast, since spiroperidol did not increase tyrosine hydroxylase activity in the absence of sympathetic stimulation, it might therefore be assumed that SIF cell dopamine does little to modify resting norepinephrine synthesis. This observation is consistent with the reported inability of exogenous dopamine to alter tyrosine hydroxylase activity in the rat SCG in vitro [18]. At present, the only documented role for SIF
cell dopamine attributed to a specific interaction with a classical dopamine-receptor is the modulation of the sEPSP response in rabbit principal neurons via activation of dopamine-sensitive adenylate cyclase [24,27]. In this context, it is of interest that neither the electrophysiologic responses (sIPSP/sEPSP) [5,7] nor the production of cAMP [6,8] appears to be mediated by dopamine-receptors in the rat SCG. From our data, however, it appears that SIF dopamine may exert a unique effect on norepinephrine synthesis in the rat SCG, presumably by interacting with specific dopamine-receptors on principal neurons or preganglionic terminals. Such a mechanism could provide an inhibitory feed-back system to regulate norepinephrine synthesis in the SCG during sympathetic stimulation. A similar mechanism has been proposed for the role of dopamine in sympathetically innervated tissues [12,13].
Acknowledgements We thank Dr. Vojtech Licko for his assistance in evaluating our data.The research was supported by N I H Grant HL-25508 and by AHA Grant 83-733 to J . T . H . J . T . H . is the recipient of N I H Research Career Development Award K04 HL00680.
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192 adrenoceptors in rat sympathetic ganglia, Brit. J. PharmacoL, 65 (1979) 435-445. 6 Brown, D.A., Caulfield, M.P. and Kirby, P.J., Relation between catecholamine-induced cyclic AMP changes and hyperpolarization in isolated rat sympathetic ganglia, J. Physiol. (L,ondon), 290 (1979) 441-451. 7 Brown, D.A. and Dunn, P.M., Depolarization of rat isolated superior cervical ganglia mediated by/32-adrenoce ptors, Brit. J. Pharmacol., 79 (1983) 429-439. 8 Brown, D.A. and Dunn, P.M., Cyclic adenosine 3',5'monophosphate and r-effects in rat isolated superior cervical ganglia, Brit. J. Pharmacol., 79 (1983) 441-449. 9 Brown, J.H., Nelson, D.L. and Molinoff, P.B., Organ culture of rat superior cervical ganglion, J. Pharmacol. Exp. Ther., 201 (1977) 298-311. 10 Case, C.P. and Matthews, M.R., A quantitative study of structural features, synapses and nearest-neighbor relationships of small, granule-containing cells in the rat superior cervical sympathetic ganglion at various adult stages, Neuroscience, 15 (1985) 237-282. 11 Dail, W.G. and Barton, S., Structure and organization of mammalian sympathetic ganglia. In L.-G. Elfvin (Ed.), Autonomic" Ganglia, John Wiley, Chichester, 1983, pp. 3-25. 12 Dubocovich, M.L. and Langer, S.Z., Dopamine and alpha adrenoceptor agohists inhibit neurotransmission in the cat spleen through different presynaptic receptors, J. Pharmacol. Exp. Ther, 212 (1980) 144-152. 13 Enero, M.A. and Langer, S.Z., Inhibition by dopamine of 3H-noradrenaline release elicited by nerve stimulation in the isolated cat's nictitating membrane, Naunyn-Schmiedeberg's Arch. Pharmacol., 289 (1975) 179-303. 14 Fischer, J.E. and Snyder, S., Disposition of 3H-norepinephrine in sympathetic ganglia, J. Pharmacol. Exp. Ther., 150 (1965) 190-195. 15 Fuller, R.W., Snoddy, H.D. and Perry, K.W, Dopamine accumulation after dopamine-fl-hydroxylase inhibition in rat heart as an index of norepinephrine turnover, Life Sci., 31 (1982) 563-570. 16 Goldman, R.H. and Harrison, D.C., The effects of hypoxia and hypercarbia on myocardial catecholamines, J. Pharmacol. Exp. Ther., 174 (1970)307-314. 17 Goldstein, M., Inhibition of norepinephrine biosynthesis at the dopamine-fl-hydroxylation stage, PharmacoL Re~,., 18 (1966) 77-82. 18 Ip, N.Y., Baldwin, C. and Zigmond, R.E., Regulation of the concentration of adenosine 3',5'-cyclic monophosphate and the activity of tyrosine hydroxylase in the rat superior cervical ganglion by three neuropeptides of the secretin family, J. Neurosci., 5 (1985) 1947-1954. 19 Ip, N.Y., Perlman, R.L. and Zigmond, R.E., Acute transsynaptic regulation of tyrosine 3-mono-oxygenase in the rat superior cervical ganglion: evidence for both cholinergic and non-cholinergic mechanisms, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 2081-2085. 20 Iversen, L.L., Horn, A.S. and Miller, R.J., Pre- and postsynaptic receptors. In E. Usdin and W.E. Bunney, Jr. (Eds.), Modern Pharmacology-Toxicology, Vol. 3, MarcelDekker, New York, 1975. pp. 207-243.
21 Karoum, F., Garrison, C.K., Neff, N. and Wyatt, R.J., Trans-synaptic modulation ol dopamine metabolism in the rat superior cervical ganglion, J. Pharmacol. Exp. Ther., 201 (1977) 654-661. 22 Kehr, W., 3-methoxytyramine as an indicator of impulseinduced dopamine release in rat brain in vivo, NaunvnSchmiedeberg's Arch, PharmacoL, 293 (1976) 209-215. 23 Kehr, W., Carlsson, A., Lindqvist, M., Magnusson, T. and Atack, C., Evidence for a receptor-mediated feedback control of striatal tyrosine hydroxylase activity, J. Pharm. Pharmacol., 24 (1972) 744-747. 24 Kobayashi, H. and Tosaka, T., Slow synaptic actions in mammalian sympathetic ganglia, with special reference to the possible roles played by cyclic nucleotides. In L.-G. Elfvin (Ed.), Autonomic" Ganglia, John Wiley, Chichester, 1983, pp. 281-307. 25 Ktnig, R. and Heym, C., lmmunofluorescent localization of dopamine-fl-hydroxylase in the small intensely fluorescent cells of the rat superior cervical ganglion, Neurosci. Lett., 10 (1978) 187-191. 26 Koslow, S.H., Mass fragmentographic analysis of SIF cell catecholamines of normal and experimental rat sympathetic ganglia. In O. Erank6 (Ed.), SIF Cells: Structure and Function of the Small, Intensely Fluorescent Sympathetic Cells, U.S. Government Printing Office, Washington, DC, 1976, pp. 82-88. 27 Libet, B., Dopaminergic synaptic processes in the superior cervical ganglion: models for synaptic actions. In A.S. Horn, J. Korf and B.H.C. Westerink (Eds.), The Neurobiology of Dopamine, Academic, New York, 1979, pp. 453-474. 28 Mefford, I.N., Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain, J. Neurosci. Meth., 3 (1981) 207-224. 29 Murrin, L.C. and Roth, R.H., Dopaminergic neurons: effects of electrical stimulation on dopamine biosynthesis, Molec. PharmacoL, 12 (1976) 463-475. 30 Nishi, S., The catecholarnine-mediated inhibition in ganglionic transmission. In C.M. Brooks, K. Koisumi and A. Sato (Eds.), Integrative Functions of the Autonomic Nervous System, University of Tokyo Press and Elsevier/NorthHolland Biomedical Press, Amsterdam, 1979, pp. 223-233. 31 Noon, J.P., McAfee, D.A. and Roth R.H., Norepinephrine release from nerve terminals within the rabbit superior cervical ganglion, Naunyn- Schmiedeberg's Archl Pharmacol., 291 (1975) 139-162. 32 Norberg, K.-A., Ritztn, M. and Ungerstedt, U., Histochemical studies on a special catechotamine-containing cell type in sympathetic ganglia, Acta PhysioL Scand., 67 (1966) 260-270. 33 Norberg, K.-A. and SfiSqvist, F., New possibilities for adrenergic modulation of ganglionic transmission, PharmacoL Rev., 18 (1966) 743-751. 34 Rybarczyk, K.E., Baker, H.A., Burke, J.P., Hartman, B,K. and Van Orden, L.S. III, Histochemical and immunocytochemical identification of catecholamines, dopamine-/3-
193
35
36
37
38
hydroxylase and phenylethanolamine-N-methyltransferase. In O. Erank~5 (Ed.), SIF Cells: Structure and Function of the Small Intensely Fluorescent Sympathetic Cells, U.S. Government Printing Office, Washington, DC, 1976, pp. 68-81. Sharman, D.F., The turnover of catecholamines. In C.J. Pycock and P.V. Taberner (Eds.), Central Neurotransmitter Turnover, University Park Press, Baltimore, 1981, pp. 20-58. Snider, S.R., Almgren, O. and Carlsson, A., The occurrence and functional significance of dopamine in some peripheral adrenergic nerves of the rat, Naunyn-Schmiedeberg's Arch. PharmacoL, 278 (1973) 1-12. Spector, S., Sjoerdsma, A. and Undenfriend, S., Blockade of endogenous norepinephrine synthesis by a-methyl-tyrosine, an inhibitor of tyrosine hydroxylase, J. Pharmacol. Exp. Ther., 147 (1965) 86-95. Steinberg, M.I. and Keller, C.E., Enhanced catecholamine synthesis in isolated rat superior cervical ganglia caused by
39
40
41
42
nerve stimulation: dissociation between ganglionic transmission and catecholamine synthesis, J. PharmacoL Exp. Ther., 204 (1978) 384-399. Steinsland, O.S., Passo, S.S. and Nahas, G.G., Biphasic effect of hypoxia on adrenal catecholamine content, Am. J. Pt~vsioL, 218 (1970) 995-998. Thoa, N.B., Johnson, D.J., Kopin, I.J. and Weiner, N., Acceleration of catecholamine formation in the guinea-pig vas deferens after hypogastric nerve stimulation: roles of tyrosine hydroxylase and new protein synthesis, J. Pharmacol. Exp. Ther., 178 (1971) 442-449. Van Orden, L.S. III, Burke, J.P., Geyer, M. and Lodoen, F.V., Localization of depletion-sensitive and depletion-resistant norepinephrine storage sites in autonomic ganglia, J. PharmacoZ Exp. Ther., 174 (1970) 56-71. Williams, T. and Jew, J., Monoamine connections in sympathetic ganglia. In L-G. Elfvin (Ed.), Autonomic Ganglia, John Wiley, Chichester, 1983, pp. 235-264.
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Elsevier JAN 00695
Research Papers
Evidence that dopamine regulates norepinephrine synthesis in the rat superior cervical ganglion during hypoxic stress J a m e s J. B r o k a w a n d J o h n T. H a n s e n Department of Cellular and Structural Biology, The University of Texas Health Science Center. San Antonio, TX 78284 (U. S.A.)
(Received 6 March, 1986) (Revised version received and accepted 16 October 1986)
K e y words: H y p o x i c stress; Small intensely fluorescent ( S I F ) cell; S u p e r i o r cervical ganglion;
C a t e c h o l a m i n e turnover; T y r o s i n e h y d r o x y l a s e activity Summary Electrical stimulation of preganglionic nerves is known to increase norepinephrine synthesis in the rat superior cervical ganglion in vitro, an effect which appears to be partially regulated by a non-cholinergic transmitter. In the present study, we sought to determine whether sympathetic stimulation also increases norepinephrine synthesis in the rat ganglion in vivo, and whether dopamine released from ganglionic interneurons might regulate this response. To tackle these questions, rats were pretreated with spiroperidol, a selective dopamine-receptor blocker, and then were sympathetically stimulated by exposure to severe hypoxic stress. Other rats were pretreated with vehicle alone before the hypoxic exposure. Norepinephrine synthesis in ganglia was assessed by measuring endogenous tyrosine hydroxylase activity and norepinephrine turnover. We found that hypoxic stress increased both of these indices of norepinephrine synthesis, but only in rats pretreated with spiroperidol. No such response was detected in rats pretreated with vehicle. These results indicate that sympathetic stimulation increases norepinephrine synthesis in the rat superior cervical ganglion in vivo, and that dopamine released from interneurons might regulate this response.
Introduction T h e s u p e r i o r cervical ganglion ( S C G ) c o n t a i n s a high c o n c e n t r a t i o n of n o r e p i n e p h r i n e d i s t r i b u t e d t h r o u g h o u t the cell b o d i e s a n d processes of the p r i n c i p a l n e u r o n s [11]. A l t h o u g h the precise role of this c a t e c h o l a m i n e in ganglionic function is uncertain, several lines of evidence suggest that s y m p a t h e t i c s t i m u l a t i o n releases n o r e p i n e p h r i n e f r o m nerve t e r m i n a l s within the ganglion to m o d ify neural t r a n s m i s s i o n [33]. If s y m p a t h e t i c stimulation does release n o r e p i n e p h r i n e , then it is likely Correspondence: J.J. Brokaw, Cardiovascular Research Institute, 1327-M, University of California School of Medicine, San Francisco, CA 94143, U.S.A.
that n o r e p i n e p h r i n e levels within the ganglion w o u l d be m a i n t a i n e d b y a c o r r e s p o n d i n g increase in n o r e p i n e p h r i n e synthesis [29,40]. Consistent with this notion, electrical s t i m u l a t i o n of p r e g a n glionic axons has been shown to increase n o r e p i n e p h r i n e synthesis in the rat S C G in vitro [19,38]. F u r t h e r m o r e , this s t i m u l u s - i n d u c e d response app e a r s to be regulated in p a r t b y acetylcholine a n d in p a r t b y a n o n - c h o l i n e r g i c t r a n s m i t t e r [19]. O n e p o s s i b l e c a n d i d a t e for this n o n - c h o l i n e r g i c t r a n s m i t t e r is d o p a m i n e . In m a n y m a m m a l i a n species, d o p a m i n e is the p r i m a r y t r a n s m i t t e r present in a small p o p u l a t i o n of ganglionic cells referred to as small intensely fluorescent ( S I F ) cells [42]. Because S I F cells are often seen s y n a p t i cally i n t e r p o s e d b e t w e e n pre- a n d p o s t g a n g l i o n i c
0165-1838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
186
elements, particularly in the rat SCG [10], it has been suggested that they function as interneurons which regulate ganglionic transmission [42]. According to current hypotheses of SIF cell function [27], sympathetic stimulation releases SIF cell dopamine, which, in turn, acts on principal neurons to produce the slow inhibitory postsynaptic potential (sIPSP) and to augment the slow excitatory postsynaptic potential (sEPSP). However, it is not known if these dopamine-induced alterations in neuronal excitability are accompanied by corresponding alterations in norepinephrine synthesis. Therefore, this study poses the following questions: does sympathetic stimulation increase norepinephrine synthesis in the rat SCG in vivo and, if so, might this response be regulated by SIF cell dopamine? To address these questions, ganglionic norepinephrine synthesis was assessed in rats that were sympathetically stimulated by exposure to severe hypoxic stress [16,39], either with or without prior treatment with a selective dopamine-receptor blocker. To provide a basis of comparison, ganglionic catecholamine synthesis was also systematically characterized under resting conditions.
Materials and Methods
Animals and drugs Male Sprague-Dawley rats (220-310 g) used in this study were suppfied by two sources, Charles River and Harlan Sprague-Dawley. The following drugs were used in this study: a-methyl-p-tyrosine, methyl ester (a-MT, Regis); m-hydroxybenzylhydrazine dihydrochloride (NSD-t015, Sigma); diethyldithiocarbamate, sodium salt (DDC, Regis); pargyline hydrochloride (Sigma); and spiroperidol (a gift from Janssen Pharmaceuticals, Beerse). aMT, NSD-1015, and pargyline were dissolved in normal saline; DDC and spiroperidol were dissolved in dimethylsulfoxide and 0.1 M citric acid, respectively. In all experiments, control rats received injections of the corresponding vehicle. Catecholamine synthesis in the SCG during rest Before we investigated the effects of sympathetic stimulation (hypoxic stress), we first
wanted to characterize catecholamine synthesis in the rat SCG during resting conditions (normoxia). Two indices of catecholamine synthesis were used in this study: catecholamine turnover and tyrosine hydroxylase activity [35]. To determine the turnover of ganglionic norepinephrine and dopamine, rats were treated with a-MT (250 mg/kg, i.p.), an inhibitor of tyrosine hydroxylase [37], and killed at selected timepoints thereafter. After catecholamine measurements, the time-dependent decrease in norepinephrine content was plotted semilogarithmically and a regression line was calculated by the method of least-squares (y = 155.48 - 41.35 x). The fractional rate constant was determined from the relationship: K = 0.693/tl/2 and the norepinephrine turnover rate (pmol/ganglion/h) was calculated by multiplying the rate constant times the initial steady-state concentration of norepinephrine [3]. The decrease in dopamine content was statistically evaluated with oneway analysis of variance (ANOVA) and the Newman-Keuls test. To determine ganglionic tyrosine hydroxylase activity, rats were treated with NSD-1015 (100 mg/kg, i.p.), an inhibitor of L-aromatic amino acid decarboxylase (DOPA decarboxylase) [23], and killed at selected time-points thereafter. After DOPA measurements, the increase in DOPA content, a reflection of tyrosine hydroxylation, was plotted using the function: y = 34.22(1 - e -°165t) + 6.27 The instantaneous rate of DOPA production (pmol/ganglion/min) was calculated from the expression: (d y / d t )t= o = (34.22)(0.165) and the time (min) required for the half-maximum DOPA increase was determined from the relationship:
tl/2
=
0.693/0.165
187 The decrease in dopamine content was statistically evaluated with one-way ANOVA and the Newm a n - K e u l s test. In addition, to determine the relative proportion of ganglionic dopamine serving as norepinephrine precursor, rats were treated with DDC (200 mg/kg, i.p.), an inhibitor of dopamine-/3-hydroxylase [17], and killed at selected time-points thereafter. This procedure also provided a more sensitive measure of norepinephrine turnover [15]. After catecholamine measurements, the increase in dopamine levels and the decrease in norepinephrine levels were statistically evaluated with one-way ANOVA and the Newman-Keuls test. Finally, to determine the contribution of metabolism to the turnover of ganglionic catecholamines, rats were treated with pargyline (100 mg/kg, i.p.), an inhibitor of monoamine oxidase [22], and killed at selected time-points thereafter. After catecholamine measurements, the increases in norepinephrine and dopamine contents were statistically evaluated with one-way ANOVA and the Newman-Keuls test. Catecholamine synthesis in the SCG during sympathetic stimulation To induce physiologic stress, rats were made severely hypoxic by placing them in a chamber (20 liter capacity) that was continuously flushed with a gas mixture consisting of 5% 02 and 95% N 2. The gas concentration was periodically tested with a Fyrite 02 analyzer. We have previously demonstrated that this method is effective in lowering arterial blood gases [4]. To determine the effect of hypoxic stress on ganglionic tyrosine hydroxylase activity, rats were treated with NSD-1015 (100 mg/kg, i.p.) and exposed to either room air (controls) or the hypoxic gas mixture for 20 min. To assess the potential role of dopamine in regulating the response to hypoxia, this same procedure was repeated 30 min after rats had been pretreated with spiroperidol (1 mg/kg, i.p.), a dopamine-receptor antagonist [1,20], or with vehicle. After DOPA measurements, group means were compared with Student's t-test (two-tailed). To determine the effect of hypoxic stress on ganglionic norepinephrine turnover, rats were
treated with DDC (200 mg/kg, i.p.) and exposed to either room air or intermittent hypoxia (20 min hypoxia, 10 min room air, repeated) for 2 h. The same procedure was repeated 30 rain after pretreatment with spiroperidol (1 mg/kg, i.p.) or vehicle. After catecholamine measurements, group means were compared with one-way ANOVA and the Newman-Keuls test. Catecholamine assay After the experimental procedures, rats were killed by decapitation and their ganglia were rapidly removed (5 rain), frozen on dry ice, and stored at - 7 0 ° C until assayed for catecholamines. The concentrations of DOPA, dopamine, and norepinephrine were measured by high performance liquid chromatography with electrochemical detection [28]. Each sample consisted of a single ganglion (one per rat) homogenized in 200 t~l of 0.2 M perchloric acid that contained 16 n g / 2 0 ~1 of dihydroxybenzylamine as an internal standard. Reference standards contained 16 n g / 2 0 ~1 each of DOPA, dopamine, norepinephrine, and dihydroxybenzylamine. All other details of the assay were identical to those we reported previously [4]. Results are expressed as picomols of catecholamine per ganglion.
Results
Catecholamine synthesis in the SCG during rest The steady-state concentrations of ganglionic norepinephrine and dopamine determined from 126 control rats were 175.0 _+ 38.4 and 17.9 _+ 5.2 pmol/ganglion, respectively (mean ___S.D.). Inhibiting tyrosine hydroxylase with a-MT caused a rapid decrease in ganglionic norepinephrine and dopamine (Fig. 1). The norepinephrine content was reduced by 74% from baseline values after 3 h. The half-life and turnover rate of norepinephrine were 1.6 h and 73 pmol/ganglion/h, respectively. Within 1 h after a-MT injection, dopamine content had dropped by 60% from baseline ( P < 0.001) and remained at about this level for 3 h. Inhibiting of DOPA decarboxylase with NSD1015 caused a rapid accumulation of ganglionic
188
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I
I
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I 3
20
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[ 1 Time [h)
Fig. 1. Decline in the concentrations of norepinephrine and dopamine in the rat superior cervical ganglion (SCG) after the administration of a-methyl-p-tyrosine. Each point represents the mean+ S.E.M. of 8 measurements. *, Significantly different from time-zero controls ( p < 0.001).
D O P A and a corresponding decrease in ganglionic d o p a m i n e (Fig. 2). The increase in D O P A content was h a l f - m a x i m u m in 4.2 min. The rate of D O P A formation was 338 p m o l / g a n g l i o n / h . Since the
d o p a m i n e content was reduced by 55% somewhere between 20 and 40 min, we estimated the half-life to be approximately 30 min. The greatest reduction in d o p a m i n e occurred within the first 5 min. Inhibiting dopamine-C/-hydroxylase with D D C caused a small decrease in ganglionic norepinephrine that was accompanied by a larger but transient increase in ganglionic d o p a m i n e (Fig. 3). The norepinephrine content was decreased by an average of 33% from baseline at all time-points ( p < 0.001). The d o p a m i n e content was increased by 202% above baseline after 1 h ( p < 0.00t), by 95% after 2 h ( p < 0.01), and by 42% after 3 h (not significant). In addition, the a m o u n t of d o p a m i n e present at 1 h was significantly different from that present at 2 h ( P < 0.005) and 3 h ( P < 0.001). Inhibiting m o n o a m i n e oxidase with pargyline caused a small increase in ganglionic norepinephrine, but no change in ganglionic d o p a m i n e (Table I). The norepinephrine content was increased by 23% above baseline after 1 h (not significant) and b y 42% after 2 h ( P < 0.025). The d o p a m i n e content was not significantly altered at either timepoint.
4o
[] NE 200-
2 20
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Fig. 3. Effects of the a d m i n i s t r a t i o n of d i e t h y l d i t h i o c a r b a m a t e 4O Time [mini
Fig. 2. Increase in the concentration of DOPA and decline in the concentration of dopamine in the rat superior cervical ganglion (SCG) after the administration of NSD-1015. Each point represents the mean+S.E.M, of 8 measurements. *, Significantly different from time-zero controls (P < 0.001).
on the concentrations of norepinephrine (NE) and dopamine (DA) in the rat superior cervical ganglion (SCG). Each bar represents the mean+ S.E.M: of 8 measurements. Probability values refer to comparisons between the control levels of NE and DA at time-zero, and their respective levels of subsequent time-points; in addition, the amount of DA present at 1 h is significantly different from that present at 2 h ( P < 0.005) and 3 h (P < 0.001).
189 VEH/CLE PRETREATMENT
TABLE I
Effects of pargvline on catecholamine leuels in the superior cercical ganglion of rats
160 -
Data are mean + S.E.M. for 10 rats. 80-
Time after drug (t7)
Catecholamines (pmol/ganglion) N orepinephrine
Dopamine
0 1 2
171.1 + 10.3 210.8 + 16.0 243.6_+23.3 *
18.1 +_0.9 17.6 _+1.4 17.5+3.0
* Significantly different from time-zero controls ( P < 0.025).
Catecholamine synthesis in the S C G during sympathetic stimulation After tyrosine hydroxylase was inhibited, the amount of ganglionic DOPA that accumulated during 20 min of hypoxic stress was dependent on the pretreatment (Table II). In rats pretreated with spiroperidol, hypoxia elicited a 93% greater increase in DOPA content than was found in the normoxic group ( p < 0.05). In rats pretreated with vehicle, there was no significant difference in DOPA content between the hypoxic and normoxic groups. After dopamine-/3-hydroxylase was inhibited, the degree by which ganglionic catecholamine levels were altered during 2 h of hypoxic stress was also dependent on the pretreatment (Fig. 4).
TABLE
II
Effects of t~vpoxie stress and spiroperidol on (vrosine hvdro:(vlase actwi~ in the superior cervical ganglion of rats Spiroperidol or its vehicle was injected into rats 30 min before the experiment. Tyrosine hydroxylase activity was assessed by measuring the amount of ganglionic DOPA accumulated 20 min after injection of NSD-1015 into rats breathing either room air or 5% 02 in N 2. Data are expressed as m e a n + S.E.M. for 7 or 8 rats.
Pretreatment
Vehicle Spiroperidol
DOPA (pmol/ganglion) Normoxic
Hypoxic
32.7_+ 2.4 28.6 + 2.4
38.0_+ 10.5 55.3 _+ 9.9 *
* Significantly different from the corresponding normoxic group ( P < 0.05).
(.9
~ g
0-
~ 2400
$PlROPERIDOL PRETREATMENT
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I~D,d ***p
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80-
O-
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DDC, 2 h NormoxlC
DDC, 2 h Hypoxic
Fig. 4. Effects of spiroperidol pretreatment on the concentrations of norepinephrine (NE) and dopamine (DA) in the superior cervical ganglion (SCG) of control rats and rats exposed to either normoxic or hypoxic conditions after the administration of diethyldithiocarbamate (DDC). Each bar represents the mean_+ S.E.M. of 8 measurements. Probability values refer to comparisons between the control levels of NE and DA, and their respective levels in the normoxic and hypoxic groups. Also, in the lower panel, there are significant differences in the levels of both NE (P < 0.005) and DA ( P < 0.001) in the hypoxic group when compared to their respective levels in the normoxic group: no corresponding differences are present in the upper panel.
In rats pretreated with spiroperidol, hypoxia elicited a much greater change in both the amount of dopamine accumulated ( P < 0 . 0 0 1 ) and the amount of norepinephrine depleted (P < 0.005) than in the normoxic group. In rats pretreated with vehicle, there were no significant differences in catecholamine levels between the hypoxic and normoxic groups.
Discussion The results of this study indicate that sympathetic stimulation increases norepinephrine synthesis in the rat SCG in vivo, and that SIF cell dopamine might regulate this response. In addition, we find that ganglionic norepinephrine ex-
190
hibits a rapid turnover and synthesis during resting conditions, and speculate that ganglionic dopamine is segregated into two pools with different rates of turnover. Catecholamine synthesis in the SCG during rest Similar to the findings of others [3,9,14], our values for the half-life (1.6 h) and turnover rate (73 p m o l / g a n g l i o n / h ) of norepinep'hrine indicate that this catecholamine turns over rapidly in the rat SCG. However, the relatively modest increase in norepinephrine content that we observed 2 h after injecting pargyline suggests that degradation by monoamine oxidase is not a major contributor to this rapid turnover. Perhaps other processes, such as axonal transport [2] or synaptic release and overflow [31], contribute more significantly to the elimination of ganglionic norepinephrine. In agreement with the turnover data, our measure of tyrosine hydroxylase activity, 338 pmol D O P A / g a n g l i o n / h , indicates a rapid synthesis of ganglionic norepinephrine. However, the rate of DOPA formation is considerably higher than the rate we determined for norepinephrine turnover. This disparity might be explained by assuming that a substantial portion of the dopamine measured in the ganglion is present in the cytoplasm of principal neurons as norepinephrine precursor and is thus able to limit tyrosine hydroxylase activity by end-product inhibition. If this assumption is correct, then the rapid loss of dopamine that occurred within 5 rain after injection of NSD-1015 might have permitted an exaggerated rate of DOPA formation. In support of this possibility, Ip et al. [19] reported rates as high as 250 pmol D O P A / g a n g l i o n / h for the rat SCG in Vitro. Like Karoum et al. [21], we estimated the halflife of ganglionic dopamine to be considerably less than 1 h, which indicates a very high rate of turnover. Although measures of total dopamine turnover cannot distinguish between the rates of turnover associated with SIF cells and principal neurons, some clues to the relative contributions made by these separate dopamine sources can be deduced from the effects of inhibiting synthesis. For example, within 1 h after a - M T injection, and 40 min after NSD-1015 injection, the ganglionic dopamine content had dropped by about 60%,
leaving some 7 pmol of dopamine that was resistant to further significant depletion for at least 3 h. According to Koslow [26], approximately 40% of the dopamine in the rat SCG is stored in SIF cells. Applying this figure to our measure of ganglionic dopamine (18 pmol/ganglion) would mean that about 7 pmol of dopamine is contained in SIF cells. Therefore, we speculate that the 7 pmol of dopamine remaining 1 h after inhibiting synthesis represents SIF cell dopamine that is slowly turning over, whereas the 60% that is rapidly depleted represents precursor dopamine in principal neurons. This notion is consistent with reports which have shown that SIF cell catecholamines have a very slow turnover in the rat SCG [32,41]. We obtained further evidence for this segregation of dopamine by blocking the conversion of dopamine to norepinephrine and measuring subsequent changes in ganglionic dopamine levelsl Because dopamine content increased rapidly during the first hour after injection of DDC, it is likely that most of the ganglionic dopamine is serving as a substrate for norepinephrine synthesis. The gradual decline in dopamine content thereafter might indicate an increased rate of degradation or release, and could explain why, in an earlier study by Koslow [26], no change in dopamine content was detected 2 h after injection of DDC. Moreover, the absence of any significant alteration in dopamine levels 2 h after injection of pargyline is also consistent with the segregation of ganglionic dopamine into two pools with different rates of turnover. If SIF cell dopamine has a slow turnover, then a correspondingly slow rate of dopamine accumulation would be expected after inhibition of monoamine oxidase [35]. By similar reasoning, the rapid conversion of precursor dopamine to norepinephrine in the storage granules of principal neurons would leave little dopamine available for degradation by monoamine oxidase located in the mitochondrial membrane [36]. Catecholamine synthesis in the SCG during sympathetic stimulation Consistent with previous in vitro findings [19,38], we found that sympathetic stimulation
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(hypoxic stress) increased endogenous tyrosine hydroxylase activity and norepinephrine turnover in the rat SCG in vivo. However, this stimulus-induced increase in norepinephrine synthesis was demonstrable only in rats that were pretreated with spiroperidol. No such response was seen in rats that were pretreated with vehicle. Because spiroperidol is a highly selective dopamine-receptor antagonist [20], with no significant adrenoceptor activity at the dose used [1], these results suggest that dopamine is involved in the regulation of norepinephrine synthesis in principal neurons during stress. Since spiroperidol was administered systemically, we cannot exclude the possibility that the effects we observed may have been due to a centrally mediated potentiation of sympathetic outflow, unrelated to intrinsic alterations in ganglionic function. It is also possible that norepinephrine synthesis was increased by the actions of adrenal catecholamines reaching the ganglion via the circulation [39]. Nevertheless, given the abundance of dopamine-containing SIF cells in the rat SCG [25,26,34], and the reported effects of dopamine on ganglionic transmission [24,27], a reasonable explanation for our data would be that spiroperidol blocked the postsynaptic actions of SIF cell dopamine on principal neurons. If this interpretation is correct, it suggests that dopamine released from SIF cells inhibits the ability of principal neurons to increase their rates of norepinephrine synthesis in response to sympathetic stimulation. Alternatively, SIF cell dopamine may act presynaptically to inhibit the release of acetylcholine from preganglionic axon terminals [30]. In either case, norepinephrine synthesis would increase during sympathetic stimulation when the inhibitory influence of dopamine is blocked with a selective dopamine-receptor antagonist. In contrast, since spiroperidol did not increase tyrosine hydroxylase activity in the absence of sympathetic stimulation, it might therefore be assumed that SIF cell dopamine does little to modify resting norepinephrine synthesis. This observation is consistent with the reported inability of exogenous dopamine to alter tyrosine hydroxylase activity in the rat SCG in vitro [18]. At present, the only documented role for SIF
cell dopamine attributed to a specific interaction with a classical dopamine-receptor is the modulation of the sEPSP response in rabbit principal neurons via activation of dopamine-sensitive adenylate cyclase [24,27]. In this context, it is of interest that neither the electrophysiologic responses (sIPSP/sEPSP) [5,7] nor the production of cAMP [6,8] appears to be mediated by dopamine-receptors in the rat SCG. From our data, however, it appears that SIF dopamine may exert a unique effect on norepinephrine synthesis in the rat SCG, presumably by interacting with specific dopamine-receptors on principal neurons or preganglionic terminals. Such a mechanism could provide an inhibitory feed-back system to regulate norepinephrine synthesis in the SCG during sympathetic stimulation. A similar mechanism has been proposed for the role of dopamine in sympathetically innervated tissues [12,13].
Acknowledgements We thank Dr. Vojtech Licko for his assistance in evaluating our data.The research was supported by N I H Grant HL-25508 and by AHA Grant 83-733 to J . T . H . J . T . H . is the recipient of N I H Research Career Development Award K04 HL00680.
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