ABSTRACT. Electrical stimulation of the superior cervical ganglion of the rat increased the phosphorylation of tyrosine hydroxylase (tyrosine 3-monooxygenase, ...
Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7243-7247, November 1984 Neurobiology
Electrical stimulation increases phosphorylation of tyrosine hydroxylase in superior cervical ganglion of rat (catecholamine synthesis/sympathetic ganglion/protein kinases)
ANNE L. CAHILL AND ROBERT L. PERLMAN* Department of Physiology and Biophysics, University of Illinois College of Medicine, P.O. Box 6998, Chicago, IL 60680
Communicated by Ira Pastan, July 16, 1984
ABSTRACT Electrical stimulation of the superior cervical ganglion of the rat increased the phosphorylation of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) in this tissue. Ganglia were incubated with [32pJp1 for 90 min and were then electrically stimulated via the preganglionic nerve. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. 32P-labeled tyrosine hydroxylase was visualized by radioautography, and the incorporation of 32p into the enzyme was quantitated by densitometry of the radioautograms. Stimulation of ganglia at 20 Hz for 5 min increased the incorporation of 32p into tyrosine hydroxylase to a level 5-fold that found in unstimulated control ganglia. The increase in phosphorylation of tyrosine hydroxylase was dependent on the duration and frequency of stimulation. Preganglionic stimulation did not increase the phosphorylation of tyrosine hydroxylase in a medium that contained low Ca2+ and high Mg2 . Increases in phosphorylation were reversible; within 30 min after the cessation of stimulation, the incorporation of 32p into tyrosine hydroxylase decreased to the level found in unstimulated ganglia. The nicotinic antagonist hexamethonium reduced the increase in 32p incorporation into tyrosine hydroxylase by about 50%, while the muscarinic antagonist atropine had no effect. Thus, preganglionic stimulation appeared to increase the phosphorylation of tyrosine hydroxylase in part by a nicotinic mechanism and in part by a noncholinergic mechanism. Antidromic stimulation of ganglia also increased the phosphorylation of tyrosine hydroxylase. Two-dimensional gel electrophoresis revealed that electrical stimulation also increased the incorporation of 32p into at least six other phosphoproteins in the ganglion.
Electrical stimulation of sympathetic nerves increases the rate of catecholamine biosynthesis in these nerves (1). This acceleration of catecholamine synthesis was initially thought to be due to the release of catecholamines from the nerve terminals, resulting in a decrease in intraneuronal catecholamine levels and a relief of the feedback inhibition of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) by catecholamines (2). It is now clear, however, that stimulation of both peripheral and central adrenergic nerves causes a stable activation of tyrosine hydroxylase and a change in the kinetic properties of the enzyme (most commonly, a decrease in the apparent Km for pteridine cofactor and an increase in the Ki for catecholamines) (3-6). There is evidence that the activation of tyrosine hydroxylase may be mediated by phosphorylation of the enzyme. Tyrosine hydroxylase is a phosphoprotein (7) and can be activated in vitro by phosphorylation (8-12). Moreover, phosphorylation of tyrosine hydroxylase is increased in situ in adrenal chromaffin cells by stimuli that increase the rate of catecholamine synthesis in these cells (13, 14). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Preganglionic stimulation increases tyrosine hydroxylase activity in the superior cervical ganglion (SCG) of the rat (15, 16). This increase in tyrosine hydroxylase activity cannot be due to the relief of feedback inhibition, since preganglionic stimulation does not evoke significant catecholamine release in the ganglion (15, 17). The effect of preganglionic stimulation on tyrosine hydroxylase activity appears to be mediated in part by a nicotinic mechanism and in part by a noncholinergic mechanism (16). Nicotinic agonists, muscarinic agonists, and agents that raise ganglionic levels of cAMP all increase tyrosine hydroxylase activity in the ganglion (18-20). We have recently found that all of these agents also increase the phosphorylation of tyrosine hydroxylase in the SCG (21). We now report the effects of preganglionic nerve stimulation on the phosphorylation of tyrosine hydroxylase in the ganglion.
MATERIALS AND METHODS Materials. [32p]p, (carrier-free) was purchased from New England Nuclear. Hexamethonium chloride [1,6-bis(trimethylammonium)hexane dichloride] was purchased from Mann Research Laboratories (New York). Atropine sulfate was purchased from Sigma. Inactivated Staphylococcus aureus (Pansorbin) was purchased from Calbiochem-Behring. Chemicals for electrophoresis were purchased from BioRad. Rabbit anti-tyrosine hydroxylase serum was a gift from A. W. Tank and N. Weiner (22). A superfusion chamber equipped with suction bipolar electrodes was built and donated by D. A. McAfee (23). Superfusion medium contained 116 mM NaCl, 5.3 mM KCl, 1.8 mM CaCI2, 0.8 mM MgSO4, 5 mM glucose, 0.1 mM EDTA, and 25 mM Hepes adjusted to pH 7.4 with NaOH. In the low Ca2+/high Mg2+ medium, CaCI2 was reduced to 0.1 mM and MgSO4 was increased to 10 mM. Animals. Male Sprague-Dawley rats (175-200 g) were purchased from King Animal Laboratories (Oregon, WI) and were maintained in our facility for at least a week before use. Rats were killed by cervical dislocation and their SCG were removed and desheathed in oxygenated superfusion medium. Labeling of SCG with 32p. Ganglia were incubated in pairs for 90 min at 37°C in 0.3 ml of oxygenated superfusion medium containing 225 ,uCi of [32p]p, per ml (1 ,uCi = 37 kBq). [32p]p; was the sole source of phosphate in the medium. After incubation with the isotope, ganglia were rinsed three times in 2 ml of superfusion medium and then transferred to the superfusion chamber. Electrical Stimulation. One of the pair of 32P-labeled ganglia was inserted into the suction bipolar electrodes as described by McAfee (23). The other, control ganglion was placed in the same superfusion chamber but was not inserted into the electrodes. Ganglia were superfused at 1.1 ml/min Abbreviation: SCG, superior cervical ganglion. *To whom reprints requests should be addressed.
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with oxygenated medium at 340C. Hexamethonium and atropine were included in the medium as indicated. Ganglia were supramaximally stimulated at 20 Hz (0.5-msec pulse duration) through the preganglionic nerve. The compound action potential was differentially recorded from the postganglionic (internal carotid) nerve. The stimulation voltage was adjusted to twice that necessary to give the maximal postganglionic response. A compound action potential of 1.5-3 mV was obtained under these conditions. Immediately after the stimulation period, each ganglion was homogenized in 100 A.l of an ice-cold phosphatase-inhibiting buffer suggested by Yamauchi and Fujisawa (24) and Meligeni et al. (11): 30 mM potassium phosphate, pH 7.5/5 mM NaF/1 mM EDTA/0.5 mM phenylmethylsulfonyl fluoride/0.5% Nonidet P-40. Homogenates were centrifuged for 45 sec in a Beckman Microfuge, and 80 A.l of each supernatant was taken for immunoprecipitation of 32P-labeled tyrosine hydroxylase. In experiments where total 32P-labeled proteins were examined, ganglia were homogenized in 100 Al of ice-cold 3% (wt/vol) trichloroacetic acid. The precipitate was collected by centrifugation in a Microfuge for 4 min at 40C. The precipitate was washed twice with 1 ml of 2% trichloroacetic acid/5 mM KH2PO4 and once with 1 ml of icecold acetone. Each time the precipitate was resuspended by sonication and collected by centrifugation. The washed precipitate was suspended in 50 ,l of the isoelectric focusing sample buffer described by O'Farrell (25). Two-dimensional gel electrophoresis was carried out according to O'Farrell
(25). Immunoprecipitation and NaDodSO4/PAGE. 32P-labeled tyrosine hydroxylase was isolated from homogenates of the SCG by immunoprecipitation followed by NaDodSO4/
PAGE. Immunoprecipitation was carried out according to Jones (26) with modifications (21). Slab gel electrophoresis was carried out in 11% acrylamide gels according to the method of Laemmli (27). Radioautography and Densitometry. Dried slab gels were exposed overnight at -70°C to Kodak XAR film backed with a DuPont Cronex Xtra Life intensifying screen. The developed film was scanned with an EC910 densitometer equipped with a Hewlett-Packard 3390A digital integrator. Exposure time and sensitivity of the densitometer were adjusted so that the integrated area under the peak was proportional to radioactivity. Incorporation of 32p into tyrosine hydroxylase was expressed in arbitrary units; one unit is equal to one thousand area units on the integrator. 32p incorporation into stimulated ganglia was calculated relative to that in control, unstimulated ganglia analyzed on the same
gel.
RESULTS When ganglia are incubated in medium containing 32p, they incorporate a detectable amount of this isotope into tyrosine hydroxylase. If the labeled ganglia are incubated under control conditions (i.e., without stimulation), the amount of radioactivity associated with tyrosine hydroxylase remains constant for at least 20 min (21). Electrical stimulation of the ganglia increased the incorporation of 32p into tyrosine hydroxylase (Fig. 1). The incorporation of 32p was increased
more than 3-fold after stimulation for 30 sec at 20 Hz and continued to increase with up to 5 min of stimulation at this frequency; after 5 min of stimulation, the phosphorylation of tyrosine hydroxylase was increased 5-fold (Fig. 2). The phosphorylation of tyrosine hydroxylase was also dependent upon the frequency of stimulation (Fig. 3). Stimulation for 5 min at 10 Hz increased 32p incorporation to the same extent as did 20-Hz stimulation, but stimulation at 5 and 2 Hz increased 32p incorporation into tyrosine hydroxylase to lesser degrees. When ganglia were stimulated in a low Ca2+/high
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FIG. 1. Electrical stimulation increases 32P incorporation into tyrosine hydroxylase. A pair of ganglia were labeled with [32p]Pi as described in Materials and Methods. One of the ganglia was then inserted into suction electrodes and stimulated at 20 Hz for 5 min. The stimulation voltage was 8 V. A 2.5-mV compound action potential was recorded from the postganglionic internal carotid nerve. The contralateral ganglion was placed in the same superfusion chamber during stimulation but was not stimulated. Immediately after electrical stimulation, the ganglia were homogenized individually and 32p_ labeled tyrosine hydroxylase was isolated by immunoprecipitation with 5 pul of anti-tyrosine hydroxylase serum. The immunoprecipitates were recovered with Pansorbin, and the precipitated proteins were separated by NaDodSO4/PAGE. The 32P-labeled tyrosine hydroxylase band in the gel (arrow) was visualized by radioautography and quantitated by densitometry. Lane 1, the stimulated ganglion; lane 2, the contralateral unstimulated ganglion. Positions of molecular mass marker proteins (not shown) are indicated on the left.
Mg2+ medium, no compound action potential could be recorded from the postganglionic nerve and the phosphorylation of tyrosine hydroxylase was not increased (Fig. 3). Increases in phosphorylation caused by electrical stimulation were completely reversible. When ganglia were stimulated at 20 Hz for 5 min and then incubated in superfusion medium for 30 min, the incorporation of 32p into tyrosine hydroxylase was 110 ± 8% (range, n = 2) the level found in unstimulated ganglia. Antidromic stimulation (20 Hz, 5 min) increased phosphorylation of tyrosine hydroxylase to 439 + 24% (SEM, n = 3) of the level found in unstimulated ganglia. Electrical stimulation did not increase the incorporation of 32p into acid-precipitable material. After stimulation for 5 min at 20 Hz, the acid-precipitable radioactivity in stimulated ganglia was 95 ± 8% (SEM, n = 8) of that in unstimulated ganglia. The nicotinic antagonist hexamethonium partially blocked the increase in phosphorylation of tyrosine hydroxylase due to electrical stimulation (Table 1). At a concentration that completely blocked ganglionic transmission (3 mM), hexamethonium reduced the incorporation of 32p into tyrosine hydroxylase by -50%. The muscarinic antagonist atropine (3 ,uM) had no effect on 32p incorporation in these experiments. When hexamethonium and atropine were both present in the superfusion medium, preganglionic stimulation still caused a 2-fold increase in tyrosine hydroxylase phosphorylation (Table 1). When ganglionic phosphoproteins were separated by two-
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Proc. Natl. Acad Sci. USA 81 (1984)
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FIG. 2. Phosphorylation of tyrosine hydroxylase in the SCG inwith the duration of electrical stimulation of the SCG at 20 Hz. 32P-labeled ganglia were stimulated through the preganglionic nerve at 20 Hz for the times indicated. Voltage was adjusted to twice that necessary to elicit the maximal compound action potential recorded from the postganglionic internal carotid nerve and was generally 6-8 V. Immediately after stimulation, ganglia were homogenized and 32P-labeled tyrosine hydroxylase was isolated and quarititated as described in the legend to Fig. 1. The incorporation of 32P into tyrosine hydroxylase in each stimulated ganglion is expressed as a percent of the 32p incorporation into unstimulated ganglia. Each value is the mean ± SEM for 4 or 5 stimulated ganglia.
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DISCUSSION Electrical stimulation of the SCG has been shown to activate tyrosine hydroxylase and to increase the rate of catecholamine synthesis in the ganglion (15, 16). We have shown that electrical stimulation increases the incorporation of 32p into tyrosine hydroxylase in the ganglion. The reversibility of this effect is evidence that electrical stimulation actually increases the phosphate content of tyrosine hydroxylase and does not merely increase the specific activity of the enzymebound phosphate (28). Other workers have shown that phosphorylation of tyrosine hydroxylase in vitro results in activation of the enzyme (8-10, 12, 13). It is likely, therefore, that the activation of tyrosine hydroxylase produced by nerve stimulation is mediated by phosphorylation of the enzyme. The activity of tyrosine hydroxylase in the SCG is increased by nicotinic and muscarinic agonists and by agents that raise cAMP levels in the ganglion (16, 20). We have recently found that all of these agents also increase the phosphorylation of tyrosine hydroxylase in this tissue (21). We believe that phosphorylation plays a role in the activation of tyrosine hydroxylase produced by all of these agents. It is difficult to evaluate the contributions of these various mechanisms to the activation and phosphorylation of tyrosine hydroxylase produced by nerve stimulation. Ip et al. (16) reported that the increase in tyrosine hydroxylase activity produced by preganglionic nerve stimulation (10 Hz, 30 min)
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dimensional gel electrophoresis, -40 distinct phosphoprotein spots could be visualized (Fig. 4). Electrical stimulation increased the phosphorylation of several proteins in addition to tyrosine hydroxylase. The apparent molecular weights and isoelectric points (in parentheses) of these other proteins are 67,000 (5.1), 59,000 (5.7), 21,000 (5.6), 21,000 (5.8), 19,000 (5.0), and 19,000 (5.1). The identity of these phosphoproteins is not known.
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FIG. 3. Phosphorylation of tyrosine hydrokylase in the SCG infrequency. 3Plbedgniaweesiu lated through the preganglionic nerve for 5 'Min at the indicated frequencies (o). Voltage was adjusted to twice that necessary to elicit 0~~~~~~~~~ the maximal compound action potential recorded from the postgan32P-labeled ganglionic internal carotid nerve. In some experiments, yhxmtoi bynresiuainwsprial were stimulated for 5 min at 20 Hzniie after the normal glia sup'erfusion medium was replaced by low Ca"+/high Mg2+ medium (0). 32P-labeled tyrosine hydroxylase was isolated and quantitated -as described in the legend to Fig. 1. Incorporation Of 32p into tyrosine hydroxylase in each stimulated ganglion is expressed as a percent of 3pincorporation in unstimulated ganglia from the same experiment. Each value is the mean -+- SEM for 3-5 stimulated ganglia. creases with stimulation
was inhibited -50% by the nicotinic antagonist hexamethonium and was not inhibited by the muscarinic antagonist atropine. This suggested that the activation of tyrosine hydroxylase by nerve stimulation was mediated in part by a nicotinic mechanism and in part by a noncholinergic mechanism. Our results are consistent with this proposal. Thus, the increase intut tyrosine h'wsaayn dtoxylase phosphorylation produced idyCeo bnv
Table 1. Effect of cholinergic antagonists on phosphorylation of tyrosine hydroxylase in electrically stimulated ganglia 32P incorporation, % control Experiment 2 Experiment 1 Superfusion medium 383 568 No additions 254 224 Hexamethonium (3 mM) 662 359 Atropine (3 ,uM) Hexamethonium (3 mM) + atropine (3 AtM) 255 231 Paired 32P-labeled ganglia were placed in a superfusion chamber and one of the pair of ganglia was inserted into suction electrodes and electrically stimulated at 20 Hz for 5 min. When hexamethonium was used, a single test stimulus (0.5-ms duration) was given to check proper insertion of the ganglion into the electrodes before superfusion with hexamethonium was started. Ganglia were then superfused with hexamethonium for 8 min before repetitive stimulation was started. (After 5 min of superfusion, no compound action potential could be recorded from the postganglionic internal carotid nerve.) When atropine was used, it was present throughout the 90 min labeling period as well as during stimulation. At the end of the electrical stimulation period, ganglia were homogenized and 32P-labeled tyrosine hydroxylase was isolated and quantitated as described in the legend to Fig. 1. Typical results from two experiments are shown.
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FIG. 4. Patterns of protein phosphorylation in control and electrically stimulated ganglia. A pair of ganglia were labeled with [32P]Pi, and then one of the ganglia was stimulated for 5 min at 20 Hz. Immediately after stimulation, the ganglia were homogenized in 3%c trichloroacetic acid and the precipitated material was subjected to two-dimensional gel electrophoresis (25). Radioautograms of 32p labeled proteins in the control ganglion (A) and the stimulated ganglion (B). As indicate spots increased in 32p. Tyrosine hydroxylase is indicated by an arrow.
and was not affected by atropine. It appears, therefore, that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a nicotinic and a noncholinergic mechanism. The phosphorylation of tyrosine hydroxylase produced by nicotinic agonists is dependent upon extracellular Ca2+ and is presumably mediated by a Ca2'-dependent protein kinase (21). We cannot test whether the phosphorylation of tyrosine hydroxylase by nerve stimulation requires extracellular Ca2 since Ca + is required for the release of neurotransmitters from the preganglionic nerve terminals. We presume, however, that the nicotinic component of the action of preganglionic stimulation is due to the activation of a Ca2+-dependent protein kinase in the ganglion. A variety of indirect evidence suggests that the noncholinergic component of nerve stimulation-induced protein phosphorylation may be due to the activation of a cAMP-dependent protein kinase. Preganglionic nerve stimulation can increase the content of cAMP in the SCG by a noncholinergic mechanism (29, 30), and agents that raise cAMP levels in the ganglion can cause activation and phosphorylation of tyrosine hydroxylase (20, 21). Several peptides, including vasoactive intestinal peptide and secretin, also increase the content of cAMP in the ganglion (20, 31). It is tempting to speculate that the noncholinergic component of nerve stimulation
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may be mediated by the release of a vasoactive intestinal peptide-like or secretin-like peptide from the preganglionic nerve terminals, which in turn leads to an increase in the content of cAMP and the activation of cAMP-dependent protein kinase in the ganglion. We have, however, no direct evidence in support of this idea. Although muscarinic agonists can increase the activity and the phosphorylation of tyrosine hydroxylase in the SCG (19, 21), there was no detectable muscarinic component to the effect of nerve stimulation on tyrosine hydroxylase activity (16) or on tyrosine hydroxylase phosphorylation. It may be that, under other stimulation conditions, muscarinic receptors also play a role in the regulation of tyrosine hydroxylase activity in the ganglion. Because depolarizing agents such as high Ki concentration and veratridine increase the activity (16, 32) and the phosphorylation of tyrosine hydroxylase in the SCG (21), we tested the effects of direct electrical depolarization on the phosphorylation of tyrosine hydroxylase. Antidromic stimulation increased the phosphorylation of tyrosine hydroxylase to almost the same extent as did orthodromic stimulation. The efficacy of antidromic stimulation raised the concern that the effects of preganglionic stimulation might also be due to direct depolarization of the principal neurons of the ganglion. However, in all these experiments, we placed the stimulating electrode on the preganglionic nerve serveral mm away from the ganglion to avoid the direct stimulation of the principal neurons. Moreover, the inhibition of the effect of preganglionic stimulation on tyrosine hydroxylase phosphorylation by hexamethonium and by a low Ca2+/high Mg2+ medium indicates that this process requires synaptic transmission. We believe, therefore, that the effects of preganglionic nerve stimulation are mediated by the release of neurotransmitters from the preganglionic nerve terminals. Preganglionic nerve stimulation increased the phosphorylation of at least six proteins other than tyrosine hydroxylase in the SCG. We have not identified any of these other phosphoproteins. Nonetheless, it is likely that these other phosphoproteins play a role in some of the other metabolic effects of nerve stimulation on the ganglion (33). In addition, preganglionic stimulation is known to increase the phosphorylation of the synaptic protein, synapsin I, in the SCG (34). We have not observed the phosphorylation of synapsin I in our experiments. This protein is apparently a minor phosphoprotein in the ganglion, and its isoelectric point of 10.2 (35) is outside the range that we examined. Most or all of the proteins that are phosphorylated in response to preganglionic stimulation are also phosphorylated in response to the nicotinic agonist dimethylphenylpiperazinium (unpublished observations). Thus, the results of these experiments are consistent with the hypothesis that preganglionic nerve stimulation increases protein phosphorylation in the SCG at least in part by a nicotinic mechanism. The phosphorylation of tyrosine hydroxylase is increased by stimulation frequencies between 2 and 20 Hz; at 20-Hz stimulation, enzyme phosphorylation is increased within 30 sec. These frequencies and this duration of stimulation are within the range over which neurons in the SCG are active (23). Thus, the phosphorylation of tyrosine hydroxylase in response to nerve stimulation may play a role in the regulation of tyrosine hydroxylase activity in vivo. We thank Drs. N. Weiner and A. W. Tank for generously providing antityrosine hydroxylase serum, and Dr. D. A. McAfee for the electrical stimulation chamber and superfusion pump and for his help and advice in carrying out these experiments. This research was supported in part by research Grant HL29025 from the National Institutes of Health and by a grant from the Earl M. Bane Charitable Trust.
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