Selective sympathetic neural changes in hypertrophied right ventricle

PHILLIP G. SCHMID, DONALD D. LUND, JANINE A. DAVIS, CAROL A. WHITEIS, RANBIR K. BHATNAGAR, AND ROBERT ROSKOSKI, JR. Veterans Administration Medical Center, Cardiovascular Center, and Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52240

SCHMID,PYILLIP G.,DONALD D. LUND,JANINE A. DAVIS, CAROL A. WHITEIS, RANBIR K. BHATNAGAR, AND ROBERT ROSKOSKI, JR. Selective sympathetic neural changes in hyper- trophied right ventride. Am. J. Physiol. 243 (Heart Cim. Phys- iol. 12): H175-HMO, 1982.-Selective pressure overload of the right ventricle in guinea pigs resulted in early and sustained reductions in tyrosine hydroxylase and dopamine+hydroxyl- ase activities in the right ventricle. No changes in tyrosine hydroxylase activity were detected in stellate ganglia, sinoatrial (SA) nodal region, atrioventicular (AV) nodal region, or left ventricle. Reductions in tyrosine hydroxylase activity in stressed right ventricle were similar regardless of duration of pulmonary artery constriction, extent of hypertrophy, presence or absence of hepatic congestion, and preservation or depletion of catecholamines. The changes may represent localized loss of sympathetic nerve fibers; factors involved directly in the process of pressure-overload-induced hypertrophy may be responsible. However, sympathetic nerves remaining in hypertrophied ven- tricle respond normally to cold-induced sympathetic activation. The reduction in tyrosine hydroxylase activity and the main- tenance of norepinephrine turnover in residual innervation to hypertrophied right ventricle support the concept that sympa- thetic neural regulation of hypertrophied cardiac tissue is al- tered but not lost.

catecholamines; tyrosine hydroxylase; dopamine+hydroxyl- ase; choline acetyltransferase; pulmonary artery constriction; right ventricular hypertrophy; right heart failure; guinea pigs

CARDIAC TYROSINE HYDROXYLASE activity decreases in certain types of heart disease (8, 16). Since this enzyme catalyzes the conversion of tyrosine to dihydroxyphen- ylalanine and initiates the biosynthesis of the sympa- thetic neurotransmitter, norepinephrine, a reduction in tyrosine hydroxylase activity has been causally linked to catecholamine depletion and impaired neural regulation of the hypertrophied and failing heart (8, 16).

The mechanisms responsible for a decrease in tyrosine hydroxylase activity in the hypertrophied heart are un- known. The present study was intended to investigate two possibilities: first, that a decrease in tyrosine hydrox- ylase activity might represent an overall reduction in sympathetic neural function, and, second, that a change might represent localized alteration in postganglionic sympathetic nerves. A general change might be expected

to affect tyrosine hydroxylase activity throughout heart and thoracic sympathetic ganglia as well as choline ace- tyltransferase activity in thoracic sympathetic ganglia. This latter enzyme catalyzes the biosynthesis of acetyl- choline, the preganglionic neurotransmitter. A local change, on the other hand, might affect only tyrosine hydroxylase activity in specific regions of the heart. A distinction between localized and generalized alterations in tyrosine hydroxylase activity could provide important new insights about the causes of abnormal neural regu- lation of the diseased heart.

METHODS

Eighty-seven male guinea pigs were studied. They were anesthetized with halothane (Fluothane, Aye&) and pentobarbital sodium (35 mg/kg), paralyzed with succi- nylcholine (1 mg), and ventilated through an endotra- cheal tube using a Harvard rodent respirator. In 54 guinea pigs, the pulmonary artery was exposed via a left thoracotomy and constricted with a 1.8.mm (ID) band. Twenty-eight guinea pigs had a sham operation identical to that of the experimental group except that, instead of a band, a suture was temporarily placed around the pulmonary artery. Five normal guinea pigs also were studied. This experimental animal model has been de- scribed in detail (22).

Samples were taken from right and left ventricles and regions that Anderson previously identified in the guinea pig as containing the sinus (SA) node and the atrioven- tricular (AV) node (1,20). Additional samples were taken from the stellate ganglia. Samples were frozen immedi- ately in liquid nitrogen and stored until analysis.

Hemodynamic determinations. Cardiac output was determined in 17 guinea pigs by an indicator dilution technique (4). Systemic arterial and central venous pres- sures were measured via cannulas in the carotid artery and jugular vein, respectively. Heart rate was determined from the record of pulsatile pressure.

Biochemical det&minati&s. Total tissue norepineph- rine was measured by modification of the Anton and Sayre alumina-trihydroxyindole procedure (2, 19). Do- pamine, norepinephrine, and epinephrine were measured in selected samples by a radioenzymatic method (18).

H176

Frozen tissue samples for determination of total tissue norepinephrine were crushed in a stainless steel pulver- izing apparatus cooled to the temperature of liquid nitro- gen, followed by homogenization in a 0.4 N perchloric acid, absorption onto alumina at a pH of 8.6, and elution with 5 ml of 0.05 N perchloric acid. The fluorescent trihydroxyindole derivative, formed by oxidation with potassium ferricyanide and subsequent rearrangement in strong-base, was measured in a fluorescence spectropho- tometer (Perkin-Elmer MPF-2A) at an excitation wave- length of 396 nm and an emission wavelength of 500 nm.

For tyrosine hydroxylase and dopamine+-hydroxylase activity determinations, the stored tissues were homog- enized (20 vol of ice-cold 5 mM potassium phosphate, 0.1 mM ethylenediaminetetraacetic acid, pH 7:4, per gram wet weight tissue) with four 10-s bursts with a Tekmar Tissumizer at a setting of 70. Pilot studies had shown that this method of tissue dispersion was equivalent to pulverization at liquid nitrogen temperatures as previ- ously reported (14). Tyrosine hydroxylase activity in heart and thoracic ganglia was measured by the proce- dure of Coyle (5) using a lo-min incubation at 37OC. With guinea pig heart, the rate was linear over 20 min and proportional to the amount of protein between protein concentrations of 1 and 5 mg/ml. Protein concentration of 5 mg/ml was used during the incubation. The final concentrations of tyrosine and 2-amino-4-hydroxy-6, 7- (dimethyl-tetrahydro)pteridine (DMPH4) were 0.2 and 1.0 mM, respectively. Determinations of K, for tyrosine and DMPH4 were carried out on selected samples using five concentrations of each substrate and Lineweaver- Burk analysis (15, 25, 26). Dopamine+-hydroxylase in heart was measured by the procedure of Coyle and Axelrod (6) using a 20-min incubation at 37°C. Protein was determined by the method of Lowry and co-workers (13) with bovine serum albumin as the standard. The activity of choline acetyltransferase in thoracic ganglia was determined by a radiochemical method (14,17,20).

Norepinephrine turnover determinations. An ap- proach for determining rate constants of norepinephrine turnover has been validated in our laboratory (9, 19). The method involves separate infusions of- [3H]tyrosine and [14C]tyrosine during I) control and 2) test periods, respectively. The mathematical formulation, specific ac- tivities of plasma tyrosine over time, and [3H]norepi- nephrine and [14C]norepinephrine determined in heart regions after the guinea pigs were killed are used to calculate base-line &*a and activated KNEW. These are rate constants of neurotransmitter turnover and are cal- culated to obtain estimates of changing regional sympa- thetic activity in the same animal under 1) basal and 2) stimulated conditions (9).

The radiolabeled precursors C3H]tyrosine and [ 14C]ty- rosine are obtained from New England Nuclear. Working solutions of 0.25 mCi/ml C3H]tyrosine and 0.05 mCi/ml f’4C]tyrosine are prepared by dilution of the solutes obtained from New England Nuclear in appropriate vol- umes of normal saline and titration to pH 6 with 1 N NaOH. This solution is then calibrated on an amino acid analyzer; 0.988 mCi l kg-’ . h-l [3H]tyrosine amounting to 0.26 pm01 l kg-’ . h-’ is infused first, and 0.198 mCi. kg-’ l

h-l r14Cltvrosine amounting to 0.43 Dmol* kg-’ i h-l is

SCHMID ET AL.

infused second. In these studies, [3H]tyrosine was infused from t = O-60 min. At t = 90 min in the experiment the guinea pig was moved from room temperature (basal condition) to a cold room at 4OC, which served as a sympathetic stimulus (stimulated condition). Then [ 14C] tyrosine was infused from t = 120-180 min. Blood samples (0.8 ml) were obtained at 20-min intervals. The red blood cells in an appropriate volume of saline were returned to the animal to minimize changes in total vascular volume. Animals were killed at t = 180 min, and tissue samples were obtained for catecholamine determination using high-performance liquid chromatography (10, 11).

Extraction of the catechols from heart samples was carried out on alumina using the procedure of Anton and Sayre (2, 19). The high-performance liquid chromato- graph consists of a solvent delivery system (model 6OOOA) and U6 K injector from Waters Associates (Milford, MA), a Cl8 column (Biophase ODS, 5-pm particle size range, 250 x 4.6 mm), guard column (Biophase ODS, 5 pm), and an LC-17 electrochemical detector with LC-3 ampero- metric controller from Bioanalytical Systems (West La- fayette, IN).

The mobile phase is composed of three parts of 0.1 M citric acid and two parts 0.1 M sodium phosphate dibasic and 0.1 M sodium octylsulfate. The detector potential is set at +0.75 V vs. Ag/AgCl reference electrode. The mobile phase is pumped at a rate of 1.3 ml/min at an ambient temperature (11).

One hundred microliters of each sample are injected onto the analytical column. The retention time is 5.4 min for norepinephrine and 10.2 min for dihydroxybenzoic acid, an internal standard. Catecholamines in the eluate are quantitated by comparing peak areas of the unknown samples with those of comparable standards. The repro- ducibility of repeated injections does not vary more than 3.6% (SD) for samples and 1.7% (SD) for standards. The percent recovery averages 75.5% (n = 14).

The linear relationship between increasing concentra-. tions of injected standards and detector response is made by using peak area. A good linear correlation (r = 0.999) has been demonstrated between the amount of injected norepinephrine and resulting ‘peak area. The minimal detectable amount of norepinephrine is 50 pg, and the response is linear between 50 pg and 100 ng with a 2.5% (SD) error.

Statistical analysis. Analysis of variance and appro- priate tests for comparing multiple group means were used in the statistical analysis (24).

RESULTS

Hemodynamic data (Table 1). Guinea pigs with pul- monary artery constriction maintained cardiac output and systemic arterial pressures despite marked increases in the ratio of right ventricular weight to body weight. Central venous pressure was increased in those guinea pigs with pleural fluid, ascites, and hepatic congestion. Heart rates were similar in normal, sham-operated, and pulmonary-artery-banded guinea pigs.

Biochemical data (Tables 2-4, Figs. 1,2). The concen- trations of dopamine and norepinephrine, but not epi- nephrine, were decreased (P < 0.05) in the h-vpertrophied

SYMPATHETIC NERVES IN HYPERTROPHIED RIGHT VENTRICLE H177

TABLE 1. Hemodynamics in normal, sham-operated, and pulmonary artery-constricted guinea pigs

Type of Guinea Pig n RV/BW, Cardiac Output, MAP, HR CVP, g/k mlomin-l-kg-l =Hg beats/min -Hg

Normal + sham Pulmonary artery-constricted

RVH RVH + hepatic congestion

7 0.69 -+ 0.04 172 k 12 69 of= 5 210 * 17 1.9 * 0.3

6 1.26 AZ 0.05" 137 t,18 72 t 6 214 AZ 10 3.3 zk 0.3 4 1.29 * 0.08” 142 k 19 70 dz 5 213 zk 20 7.7 k 2.1

Values are means & SE for normal unoperated (n = 5), sham-operated ( IC = 2), and pulmonary artery-constricted guinea pigs with right ventricular hypertrophy (RVH) alone and right ventricular hypertrophy plus hepatic congestion. Sham and RVH groups were studied approximately 7-9 wk after surgery. Values from normal and sham-operated animals were combined. RV/BW, right ventricle-to-body wt ratio; MAP, mean arterial pressure; HR, heart rate; CVP, central venous pressure. * Values differ from those in normal and sham animals, P c 0.05. Groups were compared using analysis of variance (24).

TABLE 2. Cardiac norepinephrine, epinephrine, and dopamine activity in guinea pigs with sham surgery and pulmonary artery constriction

n Duration, NE wk

Epi DA

TYROSINE HYDROXYLASE ACTIVITY (n-moi,vent -l,hr-l)

100

80

60

40

20

0

m SHAM

(lo) PULMONARY m

* peo.05

ARTERY CONSTRICTION PW Right ventricle

Sham, 10 2-3 956t 6 ngeg-l 14 7-9 987*48

PAC, 15 2-3 532+52* ngog-’ 29 7-9 481t 50"

Sham, 10 2-3 46Ok 36 ng . ventricle-’ 14 7-9 4Olk 22

PAC, 15 2-3 38Ok 32 ng ventricle-’ 29 7-9 299 Ik 29

1 19.6 & 1.2 32.8 k 2.0

24.0 AI 2.0"

15.6 -+ 1.6

17.2 d: 1.6

1 24.4 k 4.0

9.6 t, 0.8

17.6 k 2.8

2-3 Weeks 7-9 Weeks Left ventricle Sham, 10 2-3 912 & 60

ngwg-’ 13 7-9 725t 39 PAC, 15 2-3 912t 28

ngeg-’ 27 7-9 775k 59 SlilUll, 10 2-3 1,264 k 88

ng . ventricle-’ 13 7-9 942t 57 PAC, 15 2-3 1,228 t 48

ng ventricle-’ 27 7-9 8702 57

22.4 I!Z 2.4 31.2 k 1.6

24.0 t 2.4 39.6 t 2.0*

31.2 z!z 2.4 46.0 -+ 5.2

32.4 I!Z 3.2 53.0 k 4.0

FIG. 1. Tyrosine hydroxylase activity in right ventricle. Determi- nations were carried out 2-3 or 7-9 wk after surgery. Bars represent mean values & SE; no. of animals are in parentheses.

TABLE 3. Tyrosine hydroxylase activity in stellate ganglia, SA node, A V node, and left ventricles in guinea pigs with sham surgery and pulmonary .artery constriction Values represent means & SE; n, no. of animals. NE, norepinephrine;

EPi, epinephrine; DA, dopamine; PA-C, pulmonary artery constriction. * P < 0.05 PAC vs. sham. TH Activity

,

2-3 Wk 7-9 Wk

Sham PAC Sham PAC (10) (1% 03) (10)

right ventricles of guinea pigs with pulmonary artery constriction (Table 2) although the content of norepi- nephrine per right ventricle was not significantly changed. No reductions in catecholamines were detected in the left ventricle; dopamine concentration, but not total content, was increased (Table 2).

Pulmonary artery constriction (PAC) for 2-3 wk and for 7-9 wk was associated with significant decreases (P < 0.05) in total right ventricular tyrosine hydroxylase activity (Fig. 1). No changes occurred in tyrosine hydrox- ylase activity in stellate ganglia (Table 3). Choline ace- tyltransferase activity (nmol l mg protein-‘, h-l), a marker of the preganglionic sympathetic terminals in ganglia, also remained unchanged after pulmonary artery con- striction for 2-3 wk (sham 73 t 7 vs. PAC 88 t 6) and for 7-9 wk (sham 72 t 3 vs. PAC 70 t 4). Tyrosine hydrox- ylase activity was unchanged in sinoatrial (SA) node, ‘atrioventricular (AV) node, and left ventricle (Table 3). Decreases in right ventricular tyrosine hydroxlyase activ- ity were similar regardless of duration of pulmonary artery constriction, extent of increase in the right ventri- cle-to-body weight ratio, and presence or absence of hepatic congestion (data not shown), Decreases in right

Stellate ganglia, nmol . 129 * 9 135 k 6 113 * 7 108 * 5 mg protein-’ l h-’

SA node, nmol l g-’ l h-’ 424k42 402=t30 54h90 480+45 AV node, nmol l g-’ . h-’ 156& 11 150 k 13 177 k8 204*20 Left ventricle, nmol . 191* 18 M&15 101*5 104~9

ventricle-’ l h-’

Values represent means & SE with no. of animals in each group in parentheses.TH, tyrosine hydroxylase; SA, sinoatrial; AV, atrioventric- ular; sham, sham surgery; PAC, pulmonary artery constriction. There are no significant differences (P 2 0.05) between sham and PAC groups.

ventricular tyrosine hydroxylase activity also were simi- lar regardless of preservation or depletion of catechol- amines in the stressed right heart chamber of guinea pigs in the 7- to 9-wk group (Fig. 2).

To determine whether the apparent change in tyrosine hydroxylase activity was related to alterations in appar- ent affinity of enzyme for substrate, the Km’s for tyrosine or cofactor were determined by Lineweaver-Burk analy- sis (n = 5). This analysis revealed no detectable changes (P > 011) in the &, (mM) for tyrosine (sham 0.14 t 0.006

H178

Tyrosine Hydroxylase Activity

m PAC and Normal tissue Catecholamines

m PAC and Depleted tissue Catecholamines

* peo.05

SA Node AV Node RV LV

500 T (4)

0 SHAM

(nmol dopa gm -l hr -I) (nmol dopa vent -l hr -I) FIG. 2. Tyrosine hydroxylase activity in various tissues of guinea

pigs with pulmonary- artery constriction (PAC) and sham surgery. Tissue catecholamines were considered normal if right ventricular values were greater than 900 rig/g and depleted if values were less than 700 w/g.

TABLE 4. Rate constant of norepinephrine turnover in right ventricles of guinea pigs with sham surgery and pulmonary artery constriction during 25 and 4’C

km, h-’ n A km, h-’

25°C 4°C

Sham 8 0.029 zt 0.005 0.128 zk 0.025* 0.099 zt 0.028 PAC 7 0.032 =f: 0.008 0.093 zt 0.012* 0.061 zt 0.016

Values represent means * SE; n, no. of animals. &, rate constant of norepinephrine turnover; sham, sham surgery; PAC, pulmonary artery constriction. * P 5 0.05 between 25°C (basal condition) and 4OC (activated sympathetic condition). There were no significant dif- ferences between sham and PAC groups.

vs. PAC 0.16 & 0.007) or the DMPHd cofactor (sham 0.8 t 0.02 vs. PAC 0.71 t 0.06). There was, however, a significant reduction (P < 0.05) in the maximal conver- sion rate ( urnaX) (nmol l g-’ l h-‘) for both tyrosine (sham 715 t 47 vs. PAC 245 t 21) and DMPH4 (sham 910 -+ 90 vs. PAC 470 t 52).

In parallel with tyrosine hydroxylase in the 2- to 3-wk pulmonary artery-constricted groups, the activity of do- pamine-P-hydroxylasc (nmol l ventricle-’ l h-‘) was signif- icantly decreased (P < 0.05) in the hypertrophied right ventricles of pulmonary artery-constricted guinea pigs when compared with the right ventricles of the sh-am- operated controls (sham 311 t 17 vs. PAC 220 t 14). Corresponding values for left ventricles displayed no difference (P > 0.1) between the two groups (sham 1,050 t 98 vs. PAC 1,012 t 59). The groups also did not differ with respect to dopamine-P-hydroxylase activity in the SA and AV node.

In a separate set of experiments the rate constant of norepinephrine turnover (km), a functional index of sym- pathetic neuronal activity, was determined in the right ventricle of control and pressure-hypertrophied right ventricles. The k NE was first determined in each con- scious animal under basal, resting conditions (25°C) and again during 1 h of cold stress (4”C), which stimulates sympathetic neuronal activity. In the pressure-h-vpertro-

SCHMID ET AL.

phied right ventricles, basal kNE averaged 0.032 t 0.008 (Table 4), while k NE during cold stress increased signifi- cantly to 0.098 t 0.012. Corresponding values in sham animals were also noted (Table 4). Therefore, norepi- nephrine turnover in sympathetic terminals in the right ventricle can increase from a normal basal level that replaces approximately 3% of the neurotransmitter per hour to a level that replaces over 9% of the neurotrans- mitter per hour in the hypertrophied heart and 12% in the control animal. There was no difference in the km in right ventricle between the control and pulmonary ar- tery-constricted animals.

DISCUSSION

In an earlier study reported from this laboratory (18), pulmonary artery constriction to the same extent as employed in the present study resixlted in an acute re- duction in cardiac output without detectable changes in arterial and venous pressures. After 7-9 wk, pulmonary artery-banded guinea pigs undergoing hemodynamic de- terminations in the present study had cardiac outputs in the normal range and normal arterial blood pressures. Only those guinea pigs with gross evidence of fluid reten- tion had elevated venous pressures. Therefore, the ani- mals utilized for this study probably represent compen- sated right ventricular pressure overload, right ventricu- lar hypertrophy, and, in a limited number of animals, gross fluid retention.

Selective hypertrophy of the right ventricle in guinea pigs with pulmonary artery constriction was associated with localized decreases in tyrosine hydroxylase and do- pamine+hydroxylase activities. Changes in tyrosine hy- droxylase and dopamine+hydroxylase were not de- tected in tissues from SA and AV node regions, which should have been subjected to some degree of pressure overload. In addition, no changes were detected in the nonstressed left ventricle or in the sympathetic chain adjacent to right or left stellate ganglia, which contain the perikarya of individual sympathetic fibers that inner- vate the heart. A similar trend of more pronounced reductions in tyrosine hydroxylase activity in hypertro- phied right ventricles of dogs was reported by Pool and co-workers (16).

Sham operation did not result in the changes noted in the groups with pulmonary artery constriction. In addi- tion, tyrosine hydroxylase activity was not decreased in SA and AV nodal regions or in nonstressed left ventricles of the pulmonary artery-constricted animals. These re- sults suggest that damage to nerves during surgical prep- aration could not account for the changes.

The decrease in tyrosine hydroxylase activity would not appear to be the result of overall changes in sympa- thetic activity, first, because decreases were localized, and, second, because decreases in right ventricular tyro- sine hydroxylase activity were not associated with de- creases in tyrosine hydroxylase and choline acetyltrans- ferase activities in stellate ganglia (14). These consider- ations suggest that decreases in tyrosine hydroxylase and dopamine+hydroxylase activities may represent a net loss of sympathetic nerve fibers to the stressed right ventricle.

Recently, investigation of tyrosine hydroxylase activity

SYMPATHETIC NERVES IN HYPERTROPHIED RIGHT VENTRICLE H179

has suggested that the enzyme is capable of undergoing changes during sympathetic activation (27). These changes are a possible mechanism for increasing neuro- transmitter synthesis (27). However, it is likely that in vitro measurements of tyrosine hydroxylase activity as carried out in the present study (5) represent assessment of total enzyme content, inasmuch as our high cofactor concentration would obscure any effect of adenosine 3’,5’- cyclic monophosphate (CAMP)-dependent phosphoryla- tion (12, 15, 27). If this is the case, parallel decreases in tyrosine hydroxylase activity and dopamine+hydroxyl- ase activity reported ‘here may reflect loss of enzyme molecules and nerve mass rather than impairment of function in individual nerve fibers that remain (23).

Kinetic analysis of tyrosine hydroxylase activity in hypertrophied right ventricle also supports the conclu- sion that reduced enzyme activity resulted from a de- crease in the number of enzyme molecules rather. than an alteration in the functional characteristics of the en- zyme. The determinations of Km for substrates, tyrosine and DMPH4, were carried out using five concentrations of each. The average K, using DMP& as substrate was 0.80 t 0.02 (SE) mM for tyrosine hydroxylase in right ventricles from sham guinea pigs and 0.71 t 0.06 for the enzyme in stressed right ventricles. These values were not different statistically (P > 0.1). Therefore, the present values for & of cardiac tyrosine hydroxylase for cofactor are within the range of values obtained by other investi- gators (15, 25) and also in agreement with values of & for brain tyrosine hydroxylase reported from our own laboratory (26). The u max for tyrosine hydroxylase activity from the right ventricle of pulmonary-banded animals was significantly lower than controls for both tyrosine and the cofactor, DMPH4. As reconstitution experiments excluded the possibility of inhibitors of tyrosine hydrox- ylase, we conclude that the decrease in urnax in the right ventricular tissue from guinea pigs with heart failure represents a decrease in the quantity of enzyme per unit weight. This is consistent with a loss of sympathetic nerve fibers.

Borchard (3) has performed extensive investigations of adrenergic nerves of normal and hypertrophied hearts using morphometric techniques. His data clearly support a decrease in the density of sympathetic fibers greater than can be accounted for by dispersion of nerves by enlarging myocytes. Borchard (3) therefore has proposed that pressure-induced cardiac hypertrophy results in a net loss of sympathetic nerve fibers to the heart. The present results support this concept.

A new finding in this study was the ability of sympa- thetic fibers remaining in hypertrophied right ventricle to respond with regard to increasing the rate constant of norepinephrine turnover during cold stress. This obser- vation is consistent with kinetic. data indicating that functional characteristics of tyrosine hydroxylase were not altered in hypertrophied right ventricle. We can only

REFERENCES

1. ANDERSON, R. H. The disposition, morphology, and innervation of cardiac specialized tissue in the guinea pig. J. Anat. 111: 453-468, 1972.

2. ANTON, A. H., AND D. F. SAYRE. A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of

speculate about the significance of this finding with re- gard to neuroregulation of the heart. If density of fibers is decreased and synaptic contacts with cells are altered, there could be alterations in neural control despite intact functional responses in individual neurons.

In the present study, catecholamine content in hyper- trophied right ventricle was maintained and not reduced as reported previously in dogs (16) and humans (8). We have previously reported that catecholamine content in hypertrophied right ventricle of guinea pigs was main- tained in association with high rate constants of norepi- nephrine turnover and reduced in association with low rate constants of norepinephrine turnover (21). If nor- epinephrine content is maintained and neuronal mass is reduced, this may signify that the content of norepineph- rine per nerve terminal is increased or that there is an alteration in the nature of the sympathetic neurons, in which elements with high norepinephrine and low en- zyme content are selectively preserved. One possibility is that there are fewer nerve terminals and more preter- minal axons. Another possibility is that extraneuronal norepinephrine is increased. It is doubtful that the changes mean higher levels of norepinephrine in nerve terminals because generally higher neurotransmitter turnover is associated with normal or lower steady-state levels of neurotransmitter. Regardless of the precise mechanism, which must remain speculative, our data suggest that continual activation of sympathetic inner- vation to hypertrophied heart in guinea pigs may result in normal total content of neurotransmitter despite an apparent net loss of sympathetic nerve terminals and possibly other changes (7, 23).

The major finding in this study is that pressure-in- duced hypertrophy of the right ventricle is associated with selective reductions in tyrosine hydroxylase and dopamine+hydroxylase activities. These enzyme changes are detected in contractile regions of pressure- overloaded heart chamber, but not specialized regions (SA and AV nodes, for example). If decreases in tyrosine hydroxylase and dopamine#-hydroxylase activity rep- resent attrition of sympathetic innervation to contractile tissue in stressed heart, the factor(s) responsible may be localized to contractile regions actively involved in the process of hypertrophy. The data on kinetic character- istics of tyrosine hydroxylase activity and norepinephrine turnover in hypertrophied right ventricle indicate that the residual sympathetic innervation can respond to ac- tivation.

We thank Albert0 Subieta and Robert P. Oda for technical assistance and Mary Jo Thomann, Rita Yeggy, and Lisa Jo Lowenberg for secretarial assistance.

This work was supported by National Heart, Lung, and Blood Institute Grants HL-20768, HL-05529, HL-24246, and HL-24791, by Project Grant HL-14388, and by the Veterans Administration.

Received 23 January 1981; accepted in final form 15 March 1982.

catecholamines. J. Pharmacol. Exp. Ther. 138: 360-374, 1962. 3. BORCHARD, F. The adrenergic nerves of the normal and hypertro-

phied heart. In: Normal and Pathological Anatomy, edited by W. Bargmann and W. Doerr. Littleton, MA: P.G.S., 1978, vol. 33, p. l- 68.

HMO SCHMID ET AL.

4.

5.

6.

7.

COLEMAN, J. G. Cardiac output by dye dilution in the conscious rat. J. Appl. Physiol. 37: 452-454, 1974. COYLE, 3. T. Tyrosine hydroxylase in rat brain: cofactor require- ments, regional and subcellular distribution. Biochem. Pharmacol. 21: 193~1944,1972. COYLE, J. T., AND J. AXELROD. Dopamine-/?-hydroxylase in rat brain: developmental characteristics. J. Neurochem. 19: 449-459, 1972. DECHAMPLAIN, J., L. R. KRAKOFF, AND J. AXELROD. Relationship between sodium intake and norepinephrine storage during the development of experimental hypertension. Circ. Res. 23: 479-491, 1968’

8.

9.

10.

11.

12.

13.

14.

15.

DEQUATTRO, V., T. NAGATSU, A. MENDEZ, AND J. VERSKA. Deter- minants of cardiac noradrenaline depletion in human congestive failure. Cardiovasc. Res. 7: 344-350, 1973. DYKSTRA, R. H., P. G. SCHMID, R. P. ODA, AND H. E. MAYER. Norepinephrine turnover before and after interventions: a mathe- matical approach for these determination in individual animals. J. Pharmacol. Methods 1: 277-297, 1978. FELICE, L. J., J. D. FELICE, AND P. T. KISSINGER. Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography. J. Neurochem. 31: 1461-1465, 1978. KELLER, R., A. ORE, I. MEFFORD, AND R. N. ADAMS. Liquid chromatographic analysis of catecholamines routine assay for re- gional brain mapping. Life Sci. 19: 995-1004, 1976. LOVENBERG, W., E. A. BRUNKWICH, AND I. HANBAUER. ATP, cyclic AMP and magnesium increase the affinity of rat striatal tyrosine hydroxylase for its cofactor. Proc. NatZ. Acad. Sci. USA 72: 2955-2958, 1975. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275,195l. LUND, D. D., M. M. KNEUPFER, M. J. BRODY, R. K. BHATNAGAR, P. G. SCHMID, AND R. ROSKOSKI, JR. Comparison of tyrosine hydroxylase and choline acetyltransferase activity in response to sympathetic nervous system activation. Brain Res. 156: 192-197, 1978. MORITA, K., E. TACHIKAWA, M. ORA, AND T. OHUCHI. Activation of adrenal tyrosine hydroxylase by ATP and other nucleotides. FEBS Lett. 84: 101-104, 1977.

16. POOL, P. E., J. W. COVELL, M. LEVITT, J. GIBB, AND E. BRAUN-

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

WALD. Reduction of cardiac tyrosine hydroxylase activity in exper- imental congestive heart failure. Circ. Res. 20: 349-353,1967. ROSKOSKI, R., JR., H. E. MAYER, AND P. G. SCHMID. Choline acetyltransferase activity in guinea pig heart in vitro. J. Neuro- them. 23: 1197-1200, 1974. SCHMIDT, R. H., AND R. K. BHATNAGAR. Regional development of norepinephrine, dopamine+hydroxylase, and tyrosine hydroxylase in rat brain subsequent to neonatal treatment with subcutaneous 6-hydroxydopamine. Brain Res. 166: 293-308,1979. SCHMID, P. G., R. H. DYSKTRA, H. E. MAYER, R. P. ODA, AND J. J. DONNELL. Evidence of nonuniform sympathetic neural activity in heart regions in guinea pigs. Am. J. Physiol. 237 (Heart Circ. Physiol. 6): H606-H611, 1979. SCHMID, P. G., B. J. GREIF, D. D. LUND, AND R. ROSKOSKI, JR. Regional choline acetyltransferase activity in the guinea pig heart. Circ. Res. 42: 657-660, 1978. SCHMID, P. G., D. D. LUND, AND R. ROSKOSKI, JR. Efferent auto- nomic dysfunction in heart failure. In: Disturbances in Neurogenic ControZ.of Circulation, edited by F. M. Abboud, H. A. Fozzard, J. P. Gilmore, and D. J. Reis. Bethesda, MD: Am. Physiol. Sot., 1981, p. 33-49. SCHMID, P. G., H. E. MAYER, A. L. MARK, D. D. HEISTAD, AND F. M. ABBOUD. Differences in the regulation of vascular resistance in guinea pigs with right and left heart failure. Circ. Res. 41: 85-93, 1977. SPANN, J. F., JR., C. A. CHIDSEY, P. E. POOL, AND E. BRAUNWALD. Mechanisms of norepinephrine depletion in experimental heart failure produced by aortic constriction in the guinea pig. Circ. Res. 17: 312-321,1965. STEEL, R. G. D., AND J. H. TORRIE. Principles and Procedure of Statistics. New York: McGraw, 1960, p. 67-87. VULLIET, P. R., T. A. LANGAN, AND N. WEINER. Tyrosine hydrox- ylase: a substrate of cyclic AMP-dependent protein kinase. Proc. NatZ, Acad. Sci. USA 77: 92-96,198O. VRANA, K. E., C. L. ALLHISER, AND R. ROSKOSKI, JR. Tyrosine hydroxylase activation and inactivation by protein phosphorylation conditions. J. Neurochem. 36: 92-100,198l. WEINER, N., F. L. LEE, E. DREYER, AND E. BARNES. The activation of tyrosine hydroxylase in noradrenergic neurons during acute nerve stimulation. Life Sci. 22: 1197-1216, 1978.