The activation of tyrosine hydroxylase in noradrenergic neurons during acute nerve stimulation

Life Sciences, Vol . 22, pp . 1197-1216 Printed is the U .S .A .

Pergamon Press

THE ACTIVATION OF TYROSINE HYDROXYLASE IN NORADRENERGIC NEURONS DURING ACUTE NERVE STIMULATION Norman Weinar, Fu-Li Lee, Elisabeth Drayer and Ellen Barnes Department of Pharmacology, University of Colorado School of Medicine Denver, Colorado 80262

SUMMARY Tyrosine hydroxylase present in the adrenergic neurons of the vas deferens preparation of the guinea pig appears to exist in two forms ; a less active form with a relatively low affinity for pterin cofactor ; and a more active state in which the affinity for pterin cofactor is enhanced . Incubation of the vas deferens in the absence of stimulation is associated with conversion of the more active, high affinity enzyme form to the less active form, which exhibits reduced affinity for cofactor . Electrical stimulation of the hypogastric nerve to the vas deferens preparation for periods as short as one min at 25 Hz results in almost complete activation of the enzyme to the high affinity form . Incubation of intact vasa deferentia in the presence of 8-methylthio cyclic AMP also is associated with enhanced tyrosine hydroxylase activity in situ and results in similar kinetic changes in the soluble enzym~ich is prepared from these organs . Analogous changes in the kinetics of soluble tyrosine hydroxylase prepared from vasa deferentia can be achieved by incubation of the less active form of the enzyme in the presence of cyclic AMP, ATP and Mg ++ . The effects of electrical stimulation on the activation of tyrosine hydroxylase can be potentiated by stimulating the hypogastric nerve vas deferens preparation in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor . These results are consistent with the notion that increased tyrosine hydroxylase activity in situ which is associated with nerve stimulation results from acyclic AMP-dependent protein phosphorylating reaction . It has not yet been determined whether this cyclic AMP-dependent activation of tyrosine hydroxylase is a consequence of direct phosphorylation of the enzyme or phosphorylation of an activator or inhibitor of the enzyme . It is conceivable that the activation of tyrosine hydroxylase by nerve stimulation, by the cyclic AMP-dependent phosphorylating system, by polyanions, by anionic phospholipids and by limited proteolysis is mediated by a fundamentally similar mechanism . There is ample evidence to support the thesis that prompt enhancement of norepinephrine synthesis accounts for the maintenance of tissue norepinephrine levels both in vivo during stressful periods when sympathetic nervous activity is enhanced (1-4),and in vitro , following acute adrenergic nerve stimulation (5,6) . Initial evidencéfor enhanced norepinephrine synthesis during nerve stimulation in vitro was obtained from investigations on the isolated hypogas tric nerve-vâs deferens preparation of the guinea pig (5,6) . The increase in norepinephrine synthesis during acute nerve stimulation appears to be largely the result of an increase in the activity of tyrosine hydroxylase in the 0300-9653/78/0410-1197$02 .00/0 Copyright Q 1978 ~ Pergamon Press

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Regulation of Tyrosine Hydroxylase Activity

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tissue rather than an increase in the amount of the enzyme present (7-10) . Although Udenfriend and coworkers originally demonstrated that tyrosine hydroxylase activity can be modulated by end-product feedback inhibition (1113), the increase in norepinephrine synthesis associated with nerve stimulation apparently cannot be explained simply by a reduction in end-product feedback inhibition as a consequence of reduced norepinephrine levels in the neuron (14,15) . More recently, it has been observed that rat striatal tyrosine hydroxylase can be activated in vivo consequent to the administration of dopamine receptor blocking agents (36'£Or Presumably the blockade of dopamine receptors by neuroleptic agents results in an increase in neuronal activity in the nigrostriatal pathway and an increase in tyrosine hydroxylase activity and dopamine synthesis . Examination of the properties of soluble tyrosine hydroxylase prepared from striatum of rat treated with neuroleptic agents reveals that the enzyme exhibits an increased affinity for pterin cofactor and a reduced affinity for dopamine (18,21) . These affects can be mimicked by cyclic AMP and activation of protein kinase (20,22,23) . Roth and coworkers have reported that electrical stimulation of adrenergic neurons to the rat hippocampus (9,24) and guinea-pig vas deferens (8) results in an increased affinity of tyrosine hydroxylase for both reduced pterin cofac tor and tyrosine and a reduced affinity of the enzyme for norepinephrine . They concluded that activation of the enzyme following electrical stimulation is a consequence of a direct interaction of the enzyme with calcium, whose uptake is enhanced during depolarization (8,9) . They also claimed that similar changes in tyrosine hydroxylase kinetics could be elicited by exposure of the soluble enzyme prepared from unstimulated tissues to calcium chloride (8,25) . However, other investigators have failed to demonstrate a stimulating effect of calcium on tyrosine hydroxylase prepared from either central (21,26) or peripheral (10) noradrenergic tissue or from adrenal medulla (21) . We have been able to demonstrate activation of tyrosine hydroxylase following stimulation of the hypogastric nerve of the guinea pig vas deferens preparation . However, in our studies only an increased affinity of pterin cofactor for tyrosine hydroxylase was demonstrable . We were unable to demonIn the present studies, strate any change in the affinity for tyrosine (10) . we have attempted to elucidate further the mechanism of the activation of tyrosine hydroxylase consequent to nerve stimulation . Our results indicate that the effects of nerve stimulation on tyrosine hydroxylase activity may be due to the activation of a cyclic AMP-dependent protein kinase system . These effects are seen when the enzyme is assayed in the presence of either 6-methyltetrahydropterin (6-McPtH4) or the putative natural cofactor, tetrahydrobiopterin (biopterin-H4) . METHODS In these studies, male guinea pigs weighing between 450-650 grams were employed . Animals were killed by a blow on the head and vasa deferentia with attached hypogastric nerves were dissected out according to the procedure of HukoviE (27) . Assay of tyrosine hydroxylase . Vasa deferentia, either freshly removed from guinea pigs or following incubation of the intact tissue as described below, are rapidly withdrawn from the incubation medium, quickly rinsed in cold saline and the hypogastric nerve and adherent adipose and connective tissues are dissected from the organ . Each organ is then quickly frozen on dry Two or three organs ice, weighed, and stored frozen until homogenization .

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which have been stimulated and the corresponding number of paired, contralateral unstimulated preparations are homogenized in 5-6 volumes of 15 mM KC1 and centrifuged at 40,000 x g for 30 minutes at 4 ° C . Approximately 25 ul of supernatant, which contains all of the detectable tyrosine hydroxylase in the vas deferens preparation, is used in the assay . Tyrosine hydroxylase is assayed by the coupled decarboxylase method of Waymire et al (28), employing dihydropteridine reductase, NADPH and catalase in the reaction system (18,29) . The reaction is carried out in a total volume of 100 ul . The assay medium contains 20 mM Tris acetate buffer, pH 6 ; 10 mM sodium phosphate buffer, pH 7 ; 90 mM potassium phosphate buffer, pH 6 .2 ; 0 .5 mM NADPH ; 1,000 international units of catalase ; 0 .1 mM 1-14C-L-tyrosine, specific activity 10 mCi/mmol ; and an amount of L-aromatic amino acid decarboxylase plus pyridoxal phosphate sufficient to decarboxylate quantitatively the 3,4dihydroxyphenylalanine (dope) formed in the reaction ; sheep liver dihydropteridine reductase ;and either 6-McPtH4 or biopterin-H4, in concentrations specified in Results . The final pH of the reaction mixture is 6 .2 . In kinetic studies, either the tyrosine concentration or the concentration of pterin cofactor is varied . The pterin cofactor is dissolved in sodium phosphate buffer and NADPH is dissolved in potassium phosphate buffer immediately prior to the assay . Sheep liver dihydropteridine reductase is prepared according to the procedure of Kaufman (30) through the second ammonium sulfate step and is dialyzed overnight against 10 mM Tris HC1 buffer, pH 7 .4 . Approximately 14 mg protein per ml is present in the final preparation . Hog kidney aromatic amino acid decarboxylase is prepared according to the procedure of Waymire et al (28) and is dialyzed overnight in 0 .1 M imidazole acetate buffer, pH 7, in 10$ glycerol . To the enzyme preparation is added 50 ug pyridoxal phosphate per ml and the enzyme is divided into small aliquots and stored at -80 ° C . The reaction is carried out in small test tubes, each of which is capped with a rubber stopper from which is suspended a plastic well containing a filter paper wick and 0 .2 ml of organic amine base (NCS tissue solubilizer) . The reaction is carried out at 37° C for 30 min in air and is terminated with the addition of 0 .1 ml 0 .8 M HC104 to the incubation medium . C02 is quantitatively collected and trapped by incubation of the tubes with shaking for an additional hour at 37 ° . The C02 trapped in the NCS sôlubilizer is transferred to counting vials containing scintillation fluid and is counted by liquid scintillation spectrometry . The scintillation fluid is composed of 4g 2,5-diphenyloxazole (PPO) ; 0 .5g p -bis-[2-(4-methyl-5-phenyloxazolyl] benzene (dimethyl POPOP) and 5 ml ethanol per liter of toluene . Efficiency of counting, generally 80-90$, is determined by the external standardization technique and the values are converted to disintegrations per minute . Results are expressed as nmols C02 produced per hr per g tissue . The enzyme reaction is linear for a period of 40 min, and the amount of enzyme activity is directly proportional to the volume of supernatant enzyme employed . Assay of tyrosine hydroxylase in intact vas deferens-hypogastric nerve preparations . Each hypogastric nerve-vas deferens preparation is placed in the inner chamber of a double chambered glass vessel which contains 1 .5 ml Krebs Ringer bicarbonate medium composed of 118 mM NaCl ; 4 .7 mM KC1 ; 2 .5 mM CaC12 ; 25 mM NaHC03 ; 0 .1 mM MgS04 ; 1 .4 mM KH2P04 ; 8 mM glucose and 5 x 10 - 5 M 1-14C-L-tyrosine, specific activity 10 or 20 mCi/mmol . The incubation medium is continuously bubbled with 95$ 02 - 5$ C02 . The pH of the medium is 7 .2 following equilibration with 95$ OZ - 5$ C02 . When stimulation of the hypogastric nerve is performed, the nerve is threaded through an aperture in the wall of the inner chamber of the double

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Regulàtion of Tyrosine Hydroxylaee Activity

Vol . 22, No .e 13-15, 1978

chambered vessel and is draped over a pair of platinum electrodes . The outer chamber of tha glass vessel is filled with mineral oil in order to cover the nerve and prevent its desiccation during the incubation period . The platinum electrodes traverse the outer chamber and penetrate the outer chamber glass wall where they are affixed to two leads . The leads in turn are connected to a Grass S-4 stimulator which is linked to an automatic timer for automatic programming of the stimulation . In order to trap quantitatively the 14C02 produced as a consequence of decarboxylation of the dopa which is formed from tyrosine, the double chambered vessel is capped with a rubber stopper and the 95$ 02 - 5$ C02 is continually bubbled through the incubation medium via an inlet port which pierces the rubber stopper . The exit tube from the vessel conducts the 95~ 02 - 5$ C02 into 5 ml scintillation fluid containing 1 ml of NCS solubilizer, which quantitatively traps the C02 . The collection tube containing scintillation fluid is changed every 10 min . At the end of the stimulation period, the entrance and exit ports conducting the gas are closed and 0 .2 ml of NCS solubilizer is injected through the rubber stopper into a small plastic well which is suspended from the stopper . The plastic well contains a filter paper wick in order to increase the surface area for C02 absorption . Immediately thereafter, 0 .3 ml of 50~k trichloroacetic acid is injected into the inner chamber to acidify the organ and medium and initiate the quantitative evolution of the C02 from the reaction system . The glass vessel is shaken at 37 ° C for an additional hour in order to collect all of the C02 in the system, At the completion of the collection period, the contents of the plastic cup are transferred to a vial containing scintillation fluid . The C02 trapped during and at the end of the incubation period is counted by liquid scintillation spectrometry as described above . The values are summed and converted to pmoles C02 produced per hour per organ (31) . The program of stimulation employed throughout these studies, unless otherwise indicated, is : 25 Hz at supramaximal voltage, usually 8-10 volts ; biphasic pulses of 5 cosec duration ; 10 sec every 20 sec for different times (standard program of stimulation) . The contralateral vas deferens preparations are treated identically, except that the nerve is not stimulated during the incubation period . Assay of tyrosine hydroxylase activated by cyclic AMP-dependent protein phosphorylating system . When the effects of the cyclic AMP-dependent protein kinase system on tyrosine hydroxylase are determined, the assay for soluble tyrosine hydroxylase, as described above, is employed . In addition to the ingredients cited above, enzyme activity is determined in the presence of 0 .5 mM ATP, 0 .8 mM theophylline ; 20 mM NaF ; 20 mM Mg(C2H302)2 ; 0 .12 mM ethylene glycol bis (~-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and O .1 .mM adenosine-3',5'-monophosphate (cyclic AMP) (complete "CAMP mix") (19) . Exogenous protein kinasé is not required when the crude supernatant vas deferens preparation is employed, since addition of muscle protein kinase does not increase the rate of the enzymâtic reaction . Materials . 14 C-1Yrosine and 14 C-dope were obtained from New England Nuclear ; 6-McPtHq, 2-mercaptoethanol and tyrosine were obtained from Calbiochem ; catalase was obtained from Boehringer-Mannheim ; Tris buffer, NADPH, 3-iodotyro sine, cyclic AMP, ATP, EGTA, pyridoxal phosphate, dopa and norepinephrine were obtained from Sigma ; and NCS solubilizer, dimethyl POPOP and PPO were obtained from Amersham . Biopterin was obtained from Regis Chemical Company or was generously provided by Dr . Martin Gal, University of Iowa . The compound was reduced to the tetrahydro form by dissolving 10 mg in 3 ml 0,1 N HC1 . The mixture was flushed with hydrogen gas in the presence of platinum oxide for approximately 2 hr . The solution containing reduced biopterin was filtered

Vol . 22, No .a 13-15, 1978

Regulation of Tyroaiae Hydroxylaee Activity

1201

through a glass filter and aliquots were placed in small tubes . Each tube was gassed with N2, capped, and frozen at -80° until use . The ultraviolet spectrum of the pterin compound revealed that it was virtually completely in the tetrahydro form and, in the frozen state, the biopterin-H4 was stable for at least four weeks . 1-Methyl-3-isobutylxanthine (IBMX) was generously provided by Roger L . Bergstrom, G .D . Searle and Co ., Chicago, Illinois . All other chemicals were obtained from the highest quality commercial sources available . RESULTS Activation of tyrosine hydroxylase in situ consequent to nerve stimulation When the isolated hypogastric nerve vas deferens preparation of the guinea pig was stimulated according to the standard program of stimulation (sae 'Methods") for 40 min in the presence of 1- 14 C-L-tyrosine, approximately a 3-fold increase in tyrosine hydroxylase activity was observed, compared with the contralateral unstimulated preparation (Figure 1) . Stimulation at either lower frequencies or for shorter periods of time was associated with a proportionately smaller enhancement of tyrosine hydroxylase activity in situ . No change in dopa decarboxylase activity in the intact tissue was ~monstrable during nerve stimulation .

1000 T a+ > ~ Y C ' O Q l0 OI O

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800 600 400

ao

0 T

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S

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FIG . 1 Effect of stimulation of the hypogastric nerve on tyrosine hydroxylase activity in the intact vas deferens preparation . Organs were stimulated for 40 min, as outlined in Methods, in the presence of 1- 14C-L-tyrosine . In C = unstimulated contralateral control organ . S = stimulated organ . some studies, the organs were incubated in the presence of either 0 .5 mM In the right panel are or 10 mM 8-methylthio cyclic AMP (8-McSH-cAMP) . shown results of analogous experiments conducted in Krebs-Ringer medium in which the CaC12 was replaced by 1 mM EGTA . Number of experiments is noted on the graph .

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Regulation of Tyrosine Hydro~ylase Activity

Vol . 22, No .s 13-15, 1978

Chan es in activit of soluble t sine h dro lass re ared from stimulated vase e erent a. Kinetic analysis o solu le tyrosine y roxylase rom control vase de erentia in the presence of different concentrations of 6-McPtHq indicated that the enzyme did not appear to obey Michaelis-Menton kinetics (Figure 2) . When a Lineweaver-Bark plot of these data was constructed, the enzyme activity appeared to be unexpectedly high at higher cofactor concentrations, as indicated by the downward deviation of the reciprocal enzyme velocity-reciprocal cofactor concentration relationship . These results suggest that two forms of the enzyme may coexist in the unstimulated tissue, one with a relatively high affinity and the second with a relatively low affinity for pterin cofactor (32) . Analogous results have been obtained for the high Km and the low Km forms of cyclic AMP phosphodiesterase (33) . The conclusion that two forms of the enzyme may coexist is supported by kinetic analysis of enzyme prepared from unstimulated tissues in the presence of different concentrations of tyrosine . Under these circumstances the enzyme appeals to obey Michaelis-Menton kinetics . A Lineweaver-Bark analysis of the data with variable substrate concentration yields a straight line, suggesting that the entire population of enzyme molecules exhibits an identical affinity for tyrosine . In the presence of subsaturating concentrations of either 6-McPtHq or biopterin-Hq, soluble supernatant tyrosine hydroxylase prepared from vase deferentia which had been stimulated for 30 min exhibited a considerably greater activity than that seen with enzyme prepared from the contralateral unstimulated preparations .

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FIG . 2 Lineweaver-Bark plot of tyrosine hydroxylase activity in the presence of Tissues were incubated for 30 min different concentrations of 6-McPtHq . without stimulation and soluble supernatant enzyme was prepared and Each point is the mean of six experiassayed as described in Methods . ments .

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Regulation of Tyrosine Hydroaylase Activity

1203

The kinetic analysis of tyrosine hydroxylase in the presence of different concentrations of 6-McPtHq following stimulation of the intact vas deferens preparation for 30 min reveals that virtually all of the enzyme is present in the high affinity, low Km form . Thus, nerve stimulation appears to be associated with a conversion of the low activity, low affinity form of the enzyme to the more active state . The shift in the affinity of enzyme for pterin cofactor as a consequence of neurally mediated activation of the enzyme is approximately eight-fold, from approximately 0 .5 mM 6-McPtHq to approximately 0 .06 mM 6-McPtH q . (Figure 3) .

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Effect of hypogastric nerve stimulation on vas deferens tyrosine hydroxylase activity . After stimulation of the nerve for 30 min, supernatant enzyme was prepared and assayed in the presence of different concentrations of 6-McPtHq . Lower panel : Direct cosubstrate concentration-enzyme velocity plot . Results are the means of eight experiments t S .E . Upper panel : Lineweaver-Burk plot of these data .

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Regulation of Tyrosine Hydroxylase Activity

Vol . 22, No .s 13- 15, 1978

When the hypogastric nerve of the vas deferens preparation was stimulated for different durations according to the standard program, it was observed that stimulation for brief periods of time (as short as one min) was associated with conversion of almost the entire population of enzyme molecules to the more active form . Tyrosine hydroxylase prepared from vasa deferentia which had been stimulated for periods ranging from 1 min to 30 min exhibited very similar kinetics with respect to the pterin cofactor . More dramatic changes in the kinetics of tyrosine hydroxylase were seen in the contralateral unstimulated preparation which were incubated for corresponding periods of time . In these preparations, with increasing duration of incubation, there appeared to be a progressive conversion of the enzyme to the less active form (Figure 4) . Based upon the analysis of these data, and assuming the enzyme exists in two forms with different affinities for pterin cofactor but with identical intrinsic specific activities (identical values of Vmax), one can calculate the proportion of the two enzyme forms according to the method of Reiner (32) . Based upon these computations, it appears that approximately 35$ of the enzyme present in freshly removed vasa deferentia is in the active form, whereas after 30 min of incubation in the absence of stimulation only approximately 8$ of the enzyme is present in the active form . Under apparently similar experimental conditions, the proportions of less active and more active forms of the

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FIG . 4

Effect of duration of nerve stimulation and organ incubation on tyrosine hydroxylase activity in vas deferens . Organs were either stimulated or incubated without stimulation for the times noted . Supernatant enzyme was assayed in the presence of 0 .1 mM 1 qC-tyrosine and 0 .05 mM 6-McPtHq . Values below experimental points indicate the enzyme activity at that time, expressed as the fraction of activity which was observed following one min of stimulation or incubation . Percent values indicate the percent increase in enzyme activity in the stimulated organs above that of the corresponding control group . Note the more rapid decline in activity seen with enzyme from the unstimulated organs as the incubation duration is increased . n = 4-6 .

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Regulation of Tyrosine Hydroxylase Activity

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enzyme vary among different experiments (compare figures 2 and 3) . In order to assess more accurately the effects of nerve stimulation on the activation of tyrosine hydroxylase, in subsequent experiments intact tissues were preincubated for 30 min to allow the bulk of the enzyme present in the organ to revert to the less active form. Under these circumstances, stimulation at 25 Hz for periods as brief as 10-20 sec is associated with a significant increase in the activity of the supernatant. enzyme prepäred from these organs . Stimulation for approximately 1 min is associated with virtually complete activation of the enzyme to the more active form . Similar results were obtained when the putative natural cofactor, biopterin-Hq (34), was employed in these studies . After incubation of the tissue for 30 min, stimulation of the hypogastric nerve for S min was associated with a considerable increase in tyrosine hydroxylase activity and approximately a ten-fold increase in the affinity of the enzyme for this pterin cofactor . The affect of the cyclic AMP-dependent protein phosphorylating system on tyrosine hydroxylase activity . Vase deferentia were incubated for 30 min in the absence of stimulation to allow the enzyme to revert to the less active form . Soluble tyrosine hydroxylase prepared from these organs was then incubated in the presence of the complete cyclic AMP-dependent protein phosphorylating system . In the presence of subsaturating concentrations of pterin cofactor, addition of the cyclic AMP-dependent protein phosphorylating system resulted in an approximately 5-fold increase in tyrosine hydroxylase activity (Table 1) . The increase was virtually completely dependent upon the presence TABLE 1 ACTIVATION OF VAS DEFERENS TYROSINE HYDROXYLASE BY CYCLIC AMP-DEPENDENT PROTEIN KINASE SYSTEM pmols x hr 1 x mg -1 No additions Complete system - ATP

~ Control

58

100

307

529

71

122

140

241

- Mg**

136

234

- Theophylline

343

591

- EGTA

316

545

- NaF

293

506

274

472

- cyclic AMP

- Theophylline, EGTA, NaF

Guinea-pig vas deferens supernatant was assayed by the pteridine reductase - catalase - NADPH coupled decarboxylase system in the presence of 100 yM 1- 14C-L-tyrosine and 0 .25 mM 6-McPtH4 . Complete system contains : 0 .5 mM ATP ; 0 .1 mM cyclic AMP ; 20 mM Mg (C2H302) 2 ; p,g ~ theophylline ; 0 .12 mM EGTA and 20 mM NaF . of ATP in the system and a marked reduction in the activation was seen when either cyclic AMP or Mg*+ was deleted from the system (Table 1) . The residual activation seen in the absence of cyclic AMP may be the result of the presence of either adenylate cyclase or some proportion of cyclic AMP-independent

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Regulation of Tyrosine Hydroaylase Activity

Vol . 22, No .s 1 3-15, 1978

protein kinase in the system . Similarly, the residual activity seen in the absence of Mg ++ may be due to the presence of endogenous Mg*+ in the tissue . Thus, the components of the "cyclic AMP mix" (10,19,20) which appear to be essential for full açtivation of tyrosine hydroxylase are ATP, cycliç AMP, Mg** and (endogenous) protein kinase . In the presence of the complete cyclic AMP-dependent protein phosphorylating system, activation of tyrosine hydroxylase is associated with an enhanced affinity of the enzyme for pterin cofactor (Figure 5) . No change in the affin ity of the enzyme for tyrosine is demonstrable . The kinetic changes observed following electrical stimulation of the hypogastric nerve to the organ and following incubation of the soluble enzyme with the cyclic AMP-dependent protein phosphorylating system are similar . As with nerve stimulation, considerable changes in the affinity of tyrosine hydroxylase for pterin cofactor were seen in the presence of the putative natural cofactor, biopterin-Hq . The Km for the enzyme is approximately 0 .2 mM biopterin-H4 in preparations from unstimulated organs . Following stimulation, the Km is reduced to approximately 0 .04 mM .

Lineweaver-Burk plot of enzyme activity vs . 6-McPtH4 concentration in the presence and absence of the complete cyclic AMP-dependent protein phosphorylating system ("CAMP mix") . Note the lower enzyme activity and the greater proportion of enzyme in the high Km form when organs are preincubated for 30 min . Activities of the soluble enzyme in the presence of "CAMP mix" are comparable for both "fresh" tissue and tissues incubated for 30 min, suggesting that the preincubation results in a reversible reduction in enzyme activity . Results are means ± S .E . of 4 experiments .

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Regulation of Tyrosine Hydroaylase Activity

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Effect of 8-meth lthio c clic AMP on t sine h dro lass activit in intact tissue . Incubation of intact vasa de erentia wit 8-met ylt io cyclic AMP was associated with a significant increase in tyrosine hydroxylase activity in the intact tissue . With 5-10 mM 8-methylthio cyclic AMP, the enhancement of tyrosine hydroxylase activity is approximately five-fold (Figure 1) . No change in the rate of decarboxylation of carboxyl-labeled dope to dopamine is seen in the presence of this cyclic nucleotide analog, suggesting that, as with electrical stimulation of the hypogastric nerve, the effect of 8-methylthio cyclic AMP is restricted to the tyrosine hydroxylation reaction . The effect of 8-methylthio cyclic AMP on tyrosine hydroxylase activity in situ is independent of the presence of calcium in the medium (Figure 1) . Incubation of the intact tissue in the absence of calcium and in the presence of 1 mM EGTA is associated with a significant increase in tyrosine hydroxylase activity in situ which is comparable to that seen in the presence of normal Krebs-Ringer bicarbonate medium . The effect of incubation of intact vas deferens preparations with 8-methyl thio cyclic AMP on the kinetics of soluble tyrosine hydroxylase . Intact vasa deferentia were incubated in the presence of 5 mM 8-methylthio cyclic AMP for 8 min . The tissues were then rinsed rapidly several times in saline to remove residual 8-methylthio cyclic AMP, and the tissues were frozen, homogenized and assayed for tyrosine hydroxylase activity in the presence of different concentrations of either 6-McPtHq or tyrosine . As was seen following nerve stimulation, exposure of the intact tissue to 8-methylthio cyclic AMP is associated with a considerable increase in the activity of soluble tyrosine hydroxylase prepared from the tissue (Figure 6) . The apparent change in pterin cofactor Km is less pronounced in this series of experiments, chiefly because of the short incubation period to which the control organs were subjected .

8 min ± 8-McSH-CAMP

0

5

10

15

20

25

30

35

40

~S mM-1 8 McPtH4

FIG . 6

Lineweaver-Bark plot of enzyme activity vs . 6-McPtHq concentration for soluble tyrosine hydroxylase prepared from vas deferens which had been incubated for 8 min in the presence and absence of 5 mM 8-methylthio cyclic AMP .

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Regulation of Tyrosine Hydrorylaee Activity

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No change in the affinity of the enzyme for tyrosine was observed . These results are consistent with the notion that increased levels of cyclic AMP in the adrenergic neuron are associated with activation of tyrosine hydroxylase in situ . The effect of the phosphodiesterase inhibitor, - isobutylmethylxanthine , on the activation of tyrosine hydroxylase associated wit nerve stimulation . If the activation of tyrosine hydroxylase in the isolated hypogastric nerve vas deferens preparation of the guinea pig is a consequence of activation of adenylate cyclase and increased cyclic AMP levels in the neuron, one would expect that the activation might be potentiated by the presence of inhibitors In order to test this notion, the effects of the degradation of cyclic AMP . of suboptimal frequencies of nerve stimulation, in the presence and absence of isobutylmethylxanthine, on the activation of the soluble tyrosine hydroxylase prepared from these organs, was determined . The hypogastric nerve of the isolated vas de£èrens was stimulated at 5 Hz for 10 sec every 20 sec at supramaximal voltage for 5 min . The soluble enzyme prepared from these tissues exhibited an activity which was only slightly greater than that seen with soluble enzyme prepared from the contralateral unstimulated organ (Figure 7) . In the presence of isobutylmethylxanthine the effects of this program of stimulation on tyrosine hydroxylase activity were more pronôunced (Figure 8) . Under these circumstances, there was a significant increase in tyrosine hydroxylase activity associated with nerve stimulation . Kinetic analysis of these data revealed that a significantly greater proportion of the enzyme was present in the high affinity, low Km form consequent to nerve stimulation . These results suggest that isobutylmethylxanthine does indeed potentiate the effects of nerve stimulation on the activation of tyrosine hydroxylase activity in situ and lend support to the notion that neurally mediated activation of tyrosine hydroxylase involves a cyclic AMP-dependent mechanism . The effects of nerve stimulation on the ability of catecholamines to in hibit tyrosine hydroxylase . Morgenroth et al (8,25) and Roth et al (9,24) reported that the activation of tyrosine hydroxylase as a consequence of either calcium or electrical stimulation is associated with a marked reduction in Lerner et al the ability of catecholamines to inhibit tyrosine hydroxylase . (21) reported a significant, but more modest, increase in the inhibitor affinity constant (Ki) of dopamine for rat striatal tyrosine hydroxylase following administration of neuroleptics . We have attempted to determine whether the activation of tyrosine hydroxylase in the vas deferens which is associated with nerve stimulation is accompanied by a reduced ability of norepinephrine or dopa to inhibit tyrosine hydroxylase . Our studies suggest that the activation of tyrosine hydroxylase by nerve stimulation is not associated with a consistent increase in the Ki of either norepinephrine or dopa for the vas deferens enzyme . Thus, the major basis for the activation of tyrosine hydroxylase associated with nerve stimulation appears to be the enhanced affinity of the enzyme for pterin cofactor . Effects of nerve stimulation, 8-methylthio cyclic AMP or cyclic AMP-depen dent phosphorylating system on tyrosine hydroxylase activity at different pH values . The optimum pH for tyrosine hydroxylase activity is generally regarded as approximately 6 .0 - 6 .2 (29,35) . We have observed a similar pH optimum for soluble supernatant vas deferens tyrosine hydroxylase prepared from unstimulated vasa deferentia following 30 min of incubation of the intact tissue . Stimulation of the hypogastric nerve is associated with a broadening of the pH optimum for tyrosine hydroxylase and a shift of the optimum toward the more physiological pH range . Similar changes in the pH-activity characteristics of tyrosine hydroxylase are seen following incubation of the tissue with 8-. methylthio cyclic AMP and when the soluble enzyme is exposed to a cyclic AMPdependent protein phosphorylating system . Thus, these three procedures, which

Vol . 22, No .s 13-15, 1978

Regulation of Tyrosine Hydroxylase Activity

1209

200

Stl~rlulated > 100

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0

I

0.2

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FIG . 7

Effect of hypogastric nerve stimulation at 5 Hz on activation of vas Organs were incubated for 30 min and deferens tyrosine hydroxylase . stimulated for 5 min at 5 Hz, as described in Methods . Soluble enzyme from stimulated and contralateral control organs was assayed in the Lower panel : Direct presence of different concentrations of 6-McPtHq . cofactor concentration-enzyme velocity plot . Upper panel : LineweaverBurk graphical presentation of these data . Results are means ± S .E . n = 4. result in the activation of tyrosine hydroxylase, appear to produce similar changes in the pH-activity curve for this enzyme . Goldstein et al (22) reported a similar shift in the pH optimum for rat striatal tyrosine hydroxylase when the soluble enzyme was incubated with cyclic AMP, ATP, and Mg++ . DISCUSSION The present studies indicate that the kinetics of tyrosine hydroxylase are considerably modified by electrical stimulation of the hypogastric nerve of the guinea-pig vas deferens preparation . The affinity of the enzyme for pterin cofactor is significantly enhanced following nerve stimulation and

1210

Ragulatioa of Tyrosine Hydroaylaee Activity

0.06 -

a

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30 min incubation 5 min Stimulation , 5 Hz t IBMX

be

[8] B-McPtH4

mM

FIG . 8 Effect of 0 .5 mM isobutylmethylxanthine (IBMX) on activation of tyrosine hydroxylase prepared from control and stimulated (5 Hz) vasa deferentia . For details, see legend, Figure 7 . Note the greater activation of tyrosine hydroxylase consequent to nerve stimulation at 5 Hz in the presence of the phosphodiesterase inhibitor (Figure 7 vs . Figure 8) . the effects are more dramatic when the putative natural cofactor, tetrahydrobiopterin, is employed instead of the more commonly used synthetic cofactors, dimethyltetrahydropterin (47) or 6-McPtH4 . The changes in the kinetics of tyrosine hydroxylase associated with nerve stimulation are sufficiently dramatic to account fully for the increase in tyrosine hydroxylase activity which occurs in situ during nerve stimulation, assuming the concentration of pterin cofactorin the adrenergic neuron is considerably below the Km of this sub stance for the enzyme (34) . Tyrosine hydroxylase is generally regarded as the rate limiting enzyme in the biosynthesis of catecholamines . The activity of the enzyme appears to be subject to modulation by a wide variety of manipulations . Tyrosine hydroxylase is inhibited by catechol compounds, including dope, dopamine and

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norepinephrine (11-13) . This inhibition is competitive with the pterin cofactor (12) . A variety of anionic substances, . including heparin (36,37), polyglutamic acid (38,39), and anionic phospholipids such as phosphatidyl serine (38,40,41), are capable of activating striatal tyrosine hydroxylase . It has been reported, however, that peripheral noradrenergic tyrosine hydroxylase is not susceptible to anion activation (42) . Although there are a few reports in the liXerature which suggest that calcium is able to activate tyrosine hydroxylase (8,23,42), other studies indicate that the enzyme cannot be activated directly by this cation (10,21,26) . Limited proteolysis of tyrosine hydroxylase also appears to be associated with activation of the enzyme (29,43, 44,45) . Finally, a cyclic nucleotide-dependent protein kinase system is able to activate tyrosine hydroxylase from a wide variety of sources (10,19, 20, 22,23,46) . It is of interest that, following limited proteolysic digestion of bovine adrenal tyrosine hydroxylase, the enzyme is no longer susceptible to activation by either the cyclic AMP-dependent protein kinase mechanism or phospholipids (47) . Although the reported changes in the kinetics of tyrosine hydroxylase associated with the various procedures known to activate the enzyme differ among different laboratories, it appears that stimulation of tyrosine hydroxyl ase either by nerve stimulation, by anions such as phosphatidyl serine, by limited proteolysis and by the cyclic AMP-dependent protein kinase system, may involve similar molecular mechanisms . It is tempting to speculate that either a polypeptide component, or a regulatory subunit of the enzyme, or a distinctly separate inhibitory molecule is able to interact at or near the active site of tyrosine hydroxylase and inhibit the binding of the pterin cofactor to the active site region . Presumably this inhibitory component contains both a positively charged group and a hydrophobic region which interact with an anionic site and an adjacent hydrophobic region, respectively, at or near the active site of the enzyme at which pterin cofactor binds . Limited proteolysis may result in a selective removal of this peptide strand or subunit from the region of the pterin binding site, thus resulting in enzyme activation . Similarly, interaction of anionic phospholipids or polyglutamic acid with the cationic site may lead to a dissociation of the regulatory component from the region of the pterin cofactor binding site, resulting in enzyme activation . Finally, phosphorylation of the regulatory component at a serine or threonine residue adjacent to the basic groups on the molecule (49) could result in dissociation of the substance from the region of the pterin binding site . All of these chemical manipulations could lead to an enhancement in the affinity of the enzyme for pterin cofactor and, perhaps, reduced affinity for catecholamine inhibitor, and could thereby produce similar kinetic changes in the enzyme (Figure 9) . Purification and chemical characterization of the enzyme and its active site must be completed before it can be ascertained whether or not this model of the regulation of tyrosine hydroxylase is valid . It remains to be determined whether the activation of tyrosine hydroxylase by a cyclic AMP-dependent protein kinase system involves direct enzyme phosphorylation . Lovenberg et al (20) and Lloyd and Kaufman (50) attempted to determine whether activation of tyrosine hydroxylase involves phosphorylation, employing protein kinase, cyclic AMP and ATP-Y- 32p . They found no evidence for direct phosphorylation of the enzyme . In contrast, Raese et al (51) have been able to purify bovine striatal tyrosine hydroxylase several hundred fold . In the presence of cyclic AMP, magnesium and ATP-Y-32P, they obtained evidence which suggested direct phosphorylation of the purified enzyme . By chromatographic techniques these workers were able to identify several peaks of tyrosine hydroxylase activity which differ in apparent molecular weights .

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C

C FIG . 9 Hypothetical scheme for the activation of tyrosine hydroxylase by either (a) cyclic AMP-dependent protein phosphorylating (PK) system ; (b) limited proteolysis ; or (c) anionic phospholipids or other anions . The regulatory strand (R) may be either a component of the enzyme, an inhibitory molecule or a subunit of the enzyme which can become juxtaposed, by coulombic and hydrophobic interactions, to an anionic and hydrophobic region at or near the catalytic site of the enzyme . The regulatory component presumably binds in the region where reduced pterin binds and competes with reduced pterin for binding, (a) Phosphorylation of R neutralizes the coulombic interaction between R and the enzyme active site region . (b) Limited proteolysis either releases the regulatory strand or a regulatory subunit from the enzyme or inactivates the regulatory molecule by proteolysis . (c) Polyanions or anionic lipids prevent coulombic interaction between R and the active site region by "screening" the cationic and hydrophobic regions of the R unit . They conclude that tyroSome of these enzyme peaks were associated with 3zP . sine hydroxylase may exist in different molecular forms and that some of these (51),the patterns of forms are phosphorylated . However, in their studies elution for both 3 zP and tyrosine hydroxylase activity did not superimpose exactly, raising the possibility that some contaminant in the preparation, rather than the tyrosine hydroxylase molecule per se, was being phosphorylated . Letendre et al (52,53) incubated rat adrenal medullae and superior cervical ganglia in organ culture for 16-20 hours in the presence of inorganic 32p-phosphate . They isolated tyrosine hydroxylase from these organs and demonstrated that 3 zP was incorporated into the enzyme . According to their calculations, approximately 1 mole of phosphate was incorporated into each mole of enzyme . They concluded that phosphate may be a constitutive component of

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tyrosine hydroxylase . In contrast to these conflicting results on the phosphorylation of tyrosine hydroxylase, Abita et al (54) and Milstien et al (55) have obtained convincing evidence that phenylalanine hydroxylase can be phosphorylated by a cyclic AMP-dependent protein kinase system. They were able to demonstrate that approximately one mole of phosphate was present in each enzyme monomeric unit following exposure to cyclic AMP, ATP, magnesium and protein kinase . Activation of the enzyme with a cyclic AMP-dependent protein phosphorylation system was associated with an increase in the maximal velocity of the enzyme reaction without any associated changes in the Km for either phenylalanine or biopterin-H4 . Irrespective of the fundamental molecular mechanism by which tyrosine hydroxylase is activated, it seems clear that acute activation of tyrosine hydroxylase during nerve stimulation plays a primary role in maintaining the releasable norepinephrine (and dopamine) stores in adrenergic (and dopaminergic) neurons during sustained neuronal activity . The present results are consistent with the notion that newly synthesized norepinephrine is crucially important to norepinephrine release during intensive sympathetic nervous activity (56,57) . This work was supported by USPHS grants NS 07927 and NS 09199 . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . X 14 . 15 . 16 . 17 . 18 . 19 .

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C.