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Am J Physiol Regul Integr Comp Physiol 274: R626-R634, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 3, R626-R634, March 1998

Determinants of cardiac tyrosine hydroxylase activity during exercise-induced sympathetic activation in humans

Graeme Eisenhofer1, Bengt Rundqvist2, and Peter Friberg3

1 Clinical Neuroscience Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892; and Departments of 2 Cardiology and 3 Clinical Physiology, University of Göteborg, S-413 90 Göteborg, Sweden

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References

This study assessed whether the mechanisms regulating cardiac norepinephrine (NE) synthesis with changes in NE release are influenced by functions of sympathetic nerves affecting transmitter turnover independently of transmitter release. Differences in arterial and coronary venous plasma concentrations of NE and its metabolites and of dihydroxyphenylalanine (DOPA), the immediate product of tyrosine hydroxylase (TH), were examined before and during cycling exercise. Relative increases during exercise in cardiac tyrosine hydroxylation (as reflected by the %increase in cardiac DOPA spillover) matched closely corresponding increases in NE turnover, but were much lower than increases in NE release. The much larger relative increases in release than turnover of NE were largely attributable to the extensive contribution to transmitter turnover from intraneuronal metabolism of NE leaking from storage vesicles. This contribution remains unchanged during sympathetic activation so that the relative increase in NE turnover is much smaller than that in exocytotic release of NE. To replenish the NE lost from stores during sympathetic activation, TH activity need increase only in proportion to the smaller increase in turnover rather than the larger relative increase in release. The ability to "gear down" increases in tyrosine hydroxylation relative to increases in NE release provides sympathetic nerves the capacity for a more extended range of sustainable release rates than otherwise possible.

dihydroxyphenylalanine; norepinephrine; sympathetic nerves; transmitter turnover

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References

DURING SYMPATHETIC ACTIVATION, such as occurs with exercise, increases in norepinephrine (NE) release are balanced by increases in synthesis so that transmitter stores do not become depleted (19, 27). Increases in NE synthesis are dependent on changes in the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis that converts tyrosine to 3,4-dihydroxyphenylalanine (DOPA). The contribution of exocytotic transmitter release to transmitter turnover (loss of previously synthesized transmitter) is modified by the actions of monoamine transporters. Removal of transmitter from sites of action by outer membrane transporters operates not only to inactivate released transmitter, but also serves, in sequence with the vesicular monoamine transporter, to return transmitter to storage vesicles. This minimizes the impact of transmitter release on turnover and the requirement for ongoing synthesis. The vesicular monoamine transporter also functions to counteract leakage of transmitter from stores into the axoplasm where the presence of monoamine oxidase (MAO) leads to production of deaminated metabolites. Other turnover is secondary to extraneuronal uptake and metabolism or loss into the circulation of transmitter that escapes neuronal and extraneuronal uptake.

The above considerations suggest that regulation of TH and NE synthesis with changes in NE release are influenced by functions of sympathetic nerves that affect or contribute to transmitter turnover independently of transmitter release. The present study examined this hypothesis by examining the mechanisms that determine cardiac NE turnover and synthesis in 11 normal volunteers studied in the cardiac catheterization laboratory at rest and during supine cycling exercise. Subjects received intravenous infusions of trace amounts of [3H]NE and epinephrine so that cardiac sympathetic function could be examined according to established methods (3, 4, 12). Blood samples were taken from an artery and the coronary sinus. Cardiac NE turnover was estimated from differences in rates at which NE and its metabolites entered and left the coronary circulation (4). The cardiac production of DOPA provided an index of tyrosine hydroxylation (4, 10). Rates of NE neuronal and extraneuronal uptake and vesicular-axoplasmic exchange of NE in cardiac sympathetic nerves were estimated using established methods (2-5, 8, 9).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References

Subjects

Eleven normal male volunteers aged 23-50 yr (mean 36 yr) gave their informed consent to participate in the study that was approved by the Ethics and Isotope Committees at Sahlgrenska University Hospital and by the Office of Human Subjects Research at the National Institutes of Health. Subjects refrained from smoking and from consuming caffeinated beverages for 12 h before studies. A cannula inserted under local anesthesia in a radial or brachial artery was used for arterial pressure monitoring and sampling of arterial blood. A thermodilution catheter advanced under fluoroscopic guidance into the coronary sinus was used for sampling coronary venous blood and determination of coronary sinus blood flow. A cannula inserted into a forearm vein was used for infusion of radiotracers and desipramine.

Experimental Protocol

Radiotracer infusions. Subjects received an intravenous infusion of [3H]NE and epinephrine (levo-[2,5,6-3H]NE and levo-N-methyl-[3H]epinephrine; New England Nuclear, Boston, MA) starting between 9:20 and 10:20 AM once all catheters were in place. Radiotracers were infused at a rate of 1.0-1.5 µCi/min. Because of the location of the 3H label on the NH2-terminal methyl group of [3H]epinephrine, there is no interference of this radiotracer with the production of [3H]DHPG from [3H]NE.

Blood sampling and blood flow measurements. Arterial and coronary venous blood samples (10-20 ml) were drawn simultaneously beginning at least 15 min after the start of radiotracer infusions. Samples were collected into ice-chilled tubes containing heparin. Plasma was separated by centrifugation and stored at -80°C until assayed for catechols and metabolites. Coronary sinus blood flow was measured by thermodilution immediately before each collection of blood.

Cycling exercise. After the first baseline blood samples were taken, subjects commenced supine cycling exercise at 50% of their previously determined maximum work capacity. Cycling exercise was performed for 18-20 min. Arterial and coronary venous blood samples were drawn during the last minutes of exercise.

Desipramine administration. A second set of resting arterial and coronary sinus blood samples was taken 33-52 min after the end of cycling exercise. Desipramine hydrochloride (Ciba-Geigy) was then administered by intravenous infusion into a forearm vein. Infusions of desipramine lasted 30 min during which time subjects received a cumulative dose of 0.5 mg/kg. Arterial and coronary venous blood samples were drawn within 30 min after completion of the desipramine infusion.

In one subject, the study was discontinued at the end of the exercise period. All procedures were completed in the other 10 subjects.

Analysis of Blood Samples

Plasma catechols. Plasma catechols were extracted from plasma (1 ml) and samples of infusate (10 µl) using alumina adsorption and separated by liquid chromatography according to a previously described method (28). Concentrations of total catechols were quantified by electrochemical detection. Timed collections of the eluant leaving the electrochemical cell allowed fractionation of [3H]DHPG, [3H]NE, and [3H]epinephrine into scintillation vials for counting by liquid scintillation spectroscopy. Interassay coefficients of variation were (in %) 8.4 for DHPG, 6.5 for NE, 5.9 for DOPA, 11.4 for epinephrine, and 11.6 for dihydroxyphenylacetic acid (DOPAC). Intra-assay coefficients of variation were (in %) 4.8 for DHPG, 1.9 for NE, 3.8 for DOPA, 3.0 for epinephrine, and 3.9 for DOPAC.

Plasma metanephrines. Plasma concentrations of normetanephrine and metanephrine were determined by liquid chromatography with electrochemical detection after extraction by cation-exchange chromatography (23). Plasma concentrations of [3H]normetanephrine and [3H]metanephrine were estimated as described above for catechols. Interassay coefficients of variation were 12.2% for normetanephrine and 11.2% for metanephrine. Intra-assay coefficients of variation were 4.2% for normetanephrine and 3.3% for metanephrine.

Plasma 3-methoxy-4-hydroxyphenylglycol. Plasma concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG) were also determined by liquid chromatography with electrochemical detection after extraction from plasma into ethyl acetate. The interassay coefficient of variation was 7.0% and the intra-assay coefficient of variation was 4.8%.

Calculations

Cardiac DOPA spillover. DOPA is produced from tyrosine by the actions of TH, and cardiac spillover of DOPA provides an index of TH activity in sympathetic nerves of the heart (4, 10, 21, 31). The human heart extracts insignificant amounts of [13C]DOPA from inflowing arterial plasma in vivo (16). Therefore, the increment in DOPA concentrations from inflowing arterial plasma to outflowing coronary venous plasma reflects the amount of DOPA produced locally that escapes further metabolism to enter the venous drainage of the heart. Cardiac spillovers of DOPA were therefore estimated from the product of the arterial-venous difference in plasma DOPA concentrations and coronary plasma flow as described elsewhere (4, 10, 16).

Cardiac turnover of NE. Cardiac NE turnover, the rate of depletion of previously synthesized stores of NE by net release and metabolism, before and during cycling exercise was estimated from the net difference in rates at which NE and its metabolites entered and left the coronary circulation (i.e., the product of arterial-coronary venous differences in plasma concentrations and blood or plasma flow). Previous findings showed that the human heart does not produce vanillylmandelic acid and that 92% of cardiac NE turnover reflects metabolism of NE to its deaminated glycol metabolites, 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) (4). Therefore, cardiac NE turnover was estimated using the arterial-coronary venous increments in these metabolites in addition to the generally smaller arterial-coronary venous differences in plasma NE and its O-methylated metabolite, normetanephrine. The sum of the products of these differences with coronary blood flow (for DHPG and MHPG) or plasma flow (for NE and normetanephrine) provided estimates of cardiac NE turnover. These estimates did not take into account the small amount of sulfate-conjugated DHPG formed in the normal human heart (4) and therefore may underestimate total cardiac NE turnover by ~6%. No other sulfate-conjugated catecholamine metabolites are produced by the human heart (4).

Cardiac spillovers of DHPG and MHPG. Cardiac spillovers of DHPG and MHPG, which show negligible extraction by the heart, were estimated from the product of the arterial-venous difference in plasma concentrations of each compound and the coronary blood flow as previously described (3, 4).

Fractional cardiac extractions of [3H]catecholamines. The fractional cardiac extractions of [3H]NE or [3H]epinephrine (F), the proportions of NE or epinephrine removed from plasma during their passage through the coronary circulation, were estimated using the equation
F = <FR><NU>[<SUP>3</SUP>H]C<SUB>A</SUB> − [<SUP>3</SUP>H]C<SUB>V</SUB></NU><DE>[<SUP>3</SUP>H]C<SUB>A</SUB></DE></FR> (1)
where [3H]CA and [3H]CV are the arterial and coronary venous plasma concentrations of [3H]NE or [3H]epinephrine [disintegrations per minute (dpm)/ml], respectively.

Cardiac spillovers of catecholamines. Cardiac spillovers of NE or epinephrine (S), the rates of entry into the coronary venous drainage of the NE or epinephrine released by cardiac tissues (in pmol/min), were estimated by the equation (12)
S = [(C<SUB>V</SUB> − C<SUB>A</SUB>) + (C<SUB>A</SUB> ⋅ F)] ⋅ Q<SUB>P</SUB> (2)
where CA and CV are the arterial and coronary venous plasma concentrations of NE or epinephrine (pmol/ml) and where Qp is the coronary sinus plasma flow (ml/min).

Cardiac spillovers of metanephrines. The cardiac spillovers of 3H-labeled and endogenous normetanephrine were estimated after correction for the amount extracted using the cardiac extraction of metanephrine, as described elsewhere (8) and using a method similar to that described for estimation of cardiac catecholamine spillovers (see Eq. 2).

Desipramine-sensitive cardiac removal of [3H]NE. The desipramine-sensitive cardiac removal of [3H]NE (U[3H]NE), the rate of entry of [3H]NE into cardiac sympathetic neurons (in dpm/min), was estimated from the difference in the cardiac fractional extraction of [3H]NE before and after desipramine according to the equation
U<SUB>[<SUP>3</SUP>H]NE</SUB> = Q<SUB>P</SUB> ⋅ [<SUP>3</SUP>H]NE<SUB>A</SUB> ⋅ (F − F<SUB>dmi</SUB>) (3)
where F and Fdmi are the cardiac fractional extractions of [3H]NE before and after desipramine and [3H]NEA represents the arterial plasma concentration of [3H]NE before desipramine (dpm/ml).

Desipramine-sensitive cardiac spillovers of 3H-labeled and endogenous DHPG. The cardiac spillover of [3H]DHPG derived from [3H]NE taken up by cardiac sympathetic neurons and metabolized before storage (S[3H]DHPG), i.e., not that derived from [3H]NE leaking from storage vesicles (in dpm/min), was estimated from differences in cardiac spillovers of [3H]DHPG immediately before and after desipramine according to the equation
S<SUB>[<SUP>3</SUP>H]DHPG</SUB> = (Q ⋅ [<SUP>3</SUP>H]DHPG<SUB>AV</SUB>) − (Q<SUB>dmi</SUB> ⋅ [<SUP>3</SUP>H]DHPG<SUB>AVdmi</SUB>) (4)
where Q and Qdmi are coronary blood flows (ml/min) before and after desipramine, and [3H]DHPGAV and [3H]DHPGAVdmi represent the arterial-coronary venous differences in plasma concentrations (dpm/ml) of [3H]DHPG immediately before and after desipramine. The desipramine-sensitive cardiac spillover of DHPG was estimated similarly by subtraction of the cardiac spillover of DHPG after desipramine from that of the spillover of DHPG before desipramine.

Extraneuronal cardiac removal of [3H]NE. Cardiac fractional extractions of [3H]NE in denervated hearts indicate that the cardiac fractional extraction of [3H]NE by extraneuronal uptake is ~13% (31). The product of this fraction with the rate of inflow of [3H]NE into the coronary circulation (i.e., the product of coronary sinus plasma flow and the arterial plasma [3H]NE concentration) provided an estimate of the rate of extraneuronal removal [3H]NE by the heart (dpm/min).

Statistics

Results are expressed as means ± SE. The significance of differences was determined by paired t-tests or analysis of variance where appropriate. Post hoc tests were carried out using Scheffé's method. Linear regression analysis was by least squares. The significance of correlations was determined using Pearson's correlation coefficient. Statistical significance was defined as P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References

Plasma Catechol and Metabolite Concentrations

Coronary venous plasma concentrations of DOPA, DHPG, MHPG, and DOPAC were consistently higher (P < 0.001) than arterial concentrations during all procedures (Table 1). Coronary venous plasma normetanephrine concentrations were higher (P < 0.04) than arterial concentrations at rest and during exercise, but not after desipramine. Coronary venous plasma NE concentrations were higher (P < 0.001) than arterial concentrations during exercise and after desipramine, but not at rest. In contrast, coronary venous plasma concentrations of epinephrine and metanephrine were consistently lower (P < 0.02) than arterial concentrations. There were no differences in arterial or coronary venous plasma concentrations of catechols and metabolites between the first and second rest periods.

                              
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  		Table 1.   Arterial and coronary venous plasma concentrations of catechols and metabolites at rest, during exercise, and after desipramine

Effects of Cycling Exercise

Supine cycling exercise increased (P < 0.025) arterial and coronary venous plasma concentrations of DOPA, NE, normetanephrine, epinephrine, and metanephrine as well as arterial but not coronary venous plasma concentrations of DHPG, MHPG, and DOPAC (Table 1).

At rest, almost all estimated cardiac turnover of NE could be accounted for by cardiac spillovers of DHPG and MHPG, whereas during supine cycling exercise these metabolites accounted for 78% of cardiac NE turnover (Table 2). The difference in these contributions was secondary to the large arterial-coronary venous increase in plasma concentrations of NE during exercise, but not at rest (Table 1). At rest, cardiac removal of arterial NE approximated cardiac spillover of NE so that arterial and coronary venous NE concentrations were similar.

                              
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  		Table 2.   Effects of dynamic exercise on plasma indexes of cardiac sympathetic function

Exercise increased (P < 0.001) cardiac spillover of DOPA by 2.4-fold, a similar proportional response to the 2.6-fold increase (P < 0.004) in cardiac NE turnover (Table 2). In contrast, NE spillover increased (P < 0.001) by 16.7-fold during exercise. The 2.4-fold increase (P < 0.001) in cardiac DHPG spillover and the 1.9-fold increase (P < 0.03) in DOPAC spillover were much closer to the proportional increases in cardiac DOPA spillover and NE turnover than that in NE spillover. Cardiac normetanephrine spillover showed a 3.3-fold increase (P < 0.001) during exercise, and cardiac epinephrine spillover showed an 18.8-fold increase (P < 0.003) that matched closely the 16.7-fold increase in cardiac NE spillover during exercise.

During exercise, relative increases in cardiac NE spillover were substantially more (P < 0.05) than those in cardiac NE turnover and cardiac spillovers of DOPA, DHPG, MHPG, normetanephrine, and DOPAC (Fig. 1). Relative increases in cardiac normetanephrine spillover during exercise were also larger (P < 0.05) than those in cardiac spillovers of DOPA and MHPG.


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  		Fig. 1.   Relative increases above values at rest in cardiac norepinephrine (NE) turnover or cardiac spillovers of dihydroxyphenylalanine (DOPA); NE; 3,4-dihydroxyphenylglycol (DHPG); 3-methoxy-4-hydroxyphenylglycol (MHPG); normetanephrine (NMN); and 3,4-dihydroxyphenylacetic acid (DOPAC) during submaximal cycling exercise. * A larger relative increase in cardiac NE spillover (P < 0.05) than in NE turnover or cardiac spillovers of DOPA, DHPG, MHPG, or DOPAC. dagger  A larger relative increase in cardiac spillover of NMN (P < 0.05) than in cardiac spillovers of MHPG or DOPA.

Relative increases in cardiac DOPA spillover during exercise were positively correlated with those in cardiac NE turnover and cardiac NE spillover (Fig. 2). Regression lines for both relationships intersected close to the origin, but slopes of regression lines differed considerably. The slope of the regression line for the relationship between DOPA spillover and NE turnover was close to unity with relative increases in DOPA spillover 93% those of NE turnover. In contrast, the slope of the regression line for DOPA versus NE spillovers indicated much lower relative increases in DOPA spillover (P < 0.001) of only 14% those in NE spillover.


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  		Fig. 2.   Relationships between exercise-induced relative increases in cardiac DOPA spillover and increases in cardiac NE turnover (top) or cardiac NE spillover into plasma (bottom). Relative increases in DOPA spillover show a close one-to-one relationship with relative increases in NE turnover, whereas relative increases in NE spillover are much greater than those in DOPA spillover.

Cardiac fractional extractions of [3H]NE and [3H]epinephrine and of endogenous metanephrine were all decreased (P < 0.001) during cycling exercise (Table 2).

Effects of Desipramine

Desipramine decreased (P < 0.001) arterial and coronary venous plasma concentrations of DOPA, DHPG, and DOPAC (Table 1). Desipramine also caused a small but significant (P < 0.005) decrease in arterial plasma concentrations of NE, but increased (P < 0.001) coronary venous plasma concentrations of NE and epinephrine. Plasma concentrations of normetanephrine and metanephrine were unaffected by desipramine.

Desipramine decreased (P < 0.001) cardiac fractional extractions of [3H]NE and [3H]epinephrine substantially by over 69%, but had no consistent effect on the cardiac fractional extraction of metanephrine (Table 3).

                              
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  		Table 3.   Effects of desipramine on plasma indexes of cardiac sympathetic function

The cardiac spillover of endogenous DHPG was decreased (P < 0.02) by 14% after desipramine, whereas the cardiac spillover of [3H]DHPG was decreased (P < 0.005) by 38% (Table 3). Cardiac spillovers of DOPA and DOPAC were also both decreased (P < 0.04) by 18-24%, and the cardiac spillover of epinephrine was decreased (P < 0.05) by over 80% after desipramine. In contrast, the cardiac spillover of NE was increased (P < 0.001) by 55% after desipramine.

Neuronal and extraneuronal disposition of [3H]NE. Comparison of the desipramine-sensitive cardiac removal of [3H]NE with the desipramine-sensitive cardiac spillover of [3H]DHPG (Table 4) indicates that only 4.5% of the NE entering the sympathetic axoplasm, and not sequestered into storage vesicles, is metabolized to DHPG that enters the coronary venous drainage (i.e., 22-fold more NE enters the sympathetic axoplasm than appears in coronary venous plasma as DHPG).

                              
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  		Table 4.   Neuronal (desipramine sensitive) and extraneuronal uptake of [3H]NE in the heart and corresponding spillovers of NMN and DHPG at rest

Comparison of the rate of cardiac removal of [3H]NE by extraneuronal uptake with the rate of [3H]normetanphine spillover (Table 4) indicates that only 3.5% of the NE removed by extraneuronal uptake in the heart is metabolized to normetanephrine that enters the coronary venous drainage (i.e., 28-fold more NE is removed by extraneuronal uptake than appears in coronary venous plasma as normetanephrine).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References

This study examines the mechanisms that influence or regulate NE turnover and TH activity independently of transmitter release. During sympathetic activation, increases in cardiac tyrosine hydroxylation match closely increases in cardiac NE turnover, presumably so that tissue stores of NE do not become depleted. However, relative increases in NE turnover and tyrosine hydroxylation are much lower than those in exocytotic release and spillover of NE into plasma. This reflects the modifying effects of NE reuptake and vesicular-axoplasmic exchange on NE turnover. The large contribution of NE leakage from storage vesicles to intraneuronal metabolism and turnover of NE "gears down" the requirement for large changes in exocytotic NE release to be matched by comparably large changes in TH activity.

Use of plasma DOPA as an index of TH activity in sympathetic nerves in vivo is based on considerable experimental data in animals and humans. Plasma concentrations of DOPA are reduced after inhibition of TH with alpha -methyl-para-tyrosine (18) or by treatments that raise axoplasmic NE concentrations (e.g., acute reserpine or NE infusion) to cause product inhibition of the enzyme (6). Activation of TH during increased nerve activity or other stimuli is secondary to phosphorylation of several serine residues on the enzyme (30). Thus, in rats, sympathetic activation and forskolin-induced phosphorylation of TH increase plasma DOPA, whereas inhibition of sympathetic outflow by administration of clonidine or ganglion blockers reduce plasma DOPA (6, 15, 22). Similarly, in humans, increases in sympathetic outflow are associated with increases in plasma DOPA, whereas decreases in sympathetic outflow decrease plasma DOPA (7, 18). The source of DOPA from sympathetic nerves of the heart and other organs is supported by studies showing abolishment of arterial-venous plasma increments and regional spillovers in DOPA after sympathetic denervation due to autonomic neuropathy (17, 29), organ transplant (21, 31), and surgical or chemical sympathectomy (18, 20).

The increases in cardiac spillover of DOPA during exercise-induced sympathetic activation observed here support the conclusion that changes in DOPA spillover reflect changes in tyrosine hydroxylation and NE synthesis. The increased plasma concentrations and cardiac spillover of DOPA are in agreement with findings of increased plasma concentrations of DOPA during exercise in a previous clinical study (1) and of increased cardiac spillover of DOPA in dogs during electrical stimulation of cardiac sympathetic nerves (10). The small but significant decreases in plasma concentrations and cardiac spillover of DOPA after desipramine are also consistent with previous observations of similar decreases in plasma DOPA after desipramine (6, 7); this is due to the drug's effect of markedly decreasing sympathetic nerve traffic (13), resulting in a proportionally smaller decrease in NE turnover (24, 32). The divergent desipramine-induced increases in cardiac spillover of NE and decreases in spillover of epinephrine are in agreement with previous findings in anesthetized dogs where these reciprocal changes were considered to be secondary to differences in the disposition of NE and epinephrine after release from nerves combined with marked reduction in sympathetic nerve traffic (11).

The present study extends previous work about plasma DOPA as an index of TH activity by establishing in the human heart how changes in tyrosine hydroxylation and NE synthesis, occurring secondary to changes in exocytotic release and turnover of NE, are modified by other functions of sympathetic nerves such as neuronal reuptake and vesicular-axoplasmic exchange of NE (Fig. 3). The model in Fig. 3 includes the cardiac spillovers from Table 2 of all significant catechols and their metabolites before and during exercise and is derived according to the calculations described in the APPENDIX. Calculated cardiac turnover rates of NE before and during exercise (1,076 and 2,819 pmol/min) are illustrated by the rates of synthesis of NE from dopamine (i.e., values for dopamine right-arrow NE) that would be required to maintain constant stores of transmitter. The preceding rates of DOPA decarboxylation and tyrosine hydroxylation reflect those estimated from sequential sums of NE synthesis rates and DOPAC and DOPA spillover rates as described in the APPENDIX.


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  		Fig. 3.   Model quantitatively illustrating processes of synthesis, vesicular-axoplasmic exchange, metabolism, release, neuronal and extraneuronal uptake, spillover, and turnover of NE for sympathetic nerves of the normal human heart at rest (left) and during submaximal cycling exercise (right). Numbers with each arrow represent rates of each process in picomoles per minute. Values for rates of entry of DHPG, DOPA, DOPAC, NE, NMN, and MHPG into the bloodstream compartment are from Table 2. Derivation of all other values is outlined in the APPENDIX. U1, neuronal uptake; U2, extraneuronal uptake; MAO, monoamine oxidase; COMT, catechol-O-methyltransferase; TYR, tyrosine; DA, dopamine.

Comparison of the cardiac DOPA spillover rate with the rate of tyrosine hydroxylation required to maintain constant stores of transmitter (Fig. 3) indicates at rest and during exercise that only 7% (94 of 1,317 and 222 of 3,315) of the DOPA synthesized in cardiac sympathetic nerves escapes further metabolism to spill over into the coronary venous drainage. The similar exercise-associated 2.4-fold increase in cardiac DOPA spillover and 2.5-fold increase in the required rate of tyrosine hydroxylation indicate that cardiac DOPA spillover provides an accurate index of local tyrosine hydroxylation. This conclusion is further supported by the close one-to-one relationship between relative increases in cardiac DOPA spillover and NE turnover during exercise (Fig. 2).

The close exercise-induced 2.4- and 2.6-fold increases in DOPA spillover and NE turnover contrast with the much larger 16.7-fold increase in cardiac NE spillover (Fig. 1); this difference reflects the additional modifying influences of neuronal uptake and vesicular-axoplasmic exchange of NE on transmitter turnover. As illustrated in the model (Fig. 3), at rest 91% (2,310 of 2,529) of the NE that is released is recaptured and 94% ([2,310 + 12,936]/14,305) of the NE entering the axoplasm is sequestered into storage vesicles rather than being metabolized to DHPG. These high efficiencies of neuronal uptake and vesicular sequestration minimize the impact of exocytotic NE release on the requirement for NE synthesis by recycling most of the NE released by nerves back into storage vesicles. Thus at rest and during sympathetic activation only ~10% of the NE released by exocytosis is ultimately lost from sympathetic storage vesicles and requires replenishment by NE synthesis. However, as illustrated by the model (Fig. 3), a much larger proportion of NE synthesis at rest is required to replace the NE lost due to leakage of transmitter from storage vesicles than that lost by exocytosis. This may seem a paradoxical negation of the gains obtained from NE reuptake and vesicular sequestration to minimize NE loss, but actually confers some advantage to sympathetic function by extending the range of exocytotic NE release capable of being sustained during prolonged periods of sympathetic activation.

Acute activation of TH by phosphorylation in vitro or by acute stimulation of sympathetic nerves in vivo causes limited increases in enzyme activity (19, 26, 27). For example, acute periods of exercise in rats cause less than twofold increases in TH activity among a variety of sympathetically innervated tissues (19, 27). This limited capacity of TH to increase its activity in response to acute increases in transmitter turnover impacts importantly on the ability of sympathetic nerves to maintain relatively constant stores of transmitter during sustained periods of sympathetic activation. During the 20 min of submaximal exercise performed in the present study, cardiac NE spillover increased by nearly 17-fold, reflecting a 10-fold increase in exocytotic release of NE. If changes in NE turnover were directly proportional to changes in NE release, a 10-fold increase in NE release would cause a 90% reduction in the half-life of NE stores from the normal 10 h at rest to 1 h during exercise. Under these circumstances, sympathetic stores of NE would soon become depleted unless NE synthesis also increased 10-fold. The large proportion of NE synthesis required to replenish NE metabolized after leakage from storage vesicles gears down the requirement for TH activity to increase in proportion to increases in NE release. This extends the gain of maximal NE release rates that may be sustained without causing depletion of NE stores secondary to the limited capacity of TH for matching increases in activity.

The large contribution of NE leakage from storage vesicles to NE turnover was recognized by Maas and colleagues (25) many years ago and has since been shown to occur in other catecholamine systems, such as dopaminergic neurons in the brain (14). Thus NE and other monoamine transmitters exist in storage vesicles in a highly dynamic rather than a static state, exchanging rapidly with transmitter in the axoplasm. The present analysis suggests that transmitter leakage from storage vesicles may not be without purpose, but may allow extended periods and ranges of sympathetic activation to occur without substantial depletion of NE stores.

In conclusion, the high rate of NE leakage from vesicles, which impacts considerably on NE turnover and synthesis, may seem inconsistent with cellular economy but actually enables sympathetic nerves to respond appropriately to stresses despite relatively limited capacity to increase TH activity. During sympathetic activation, such as occurs during exercise, the contribution of NE release to turnover increases, whereas leakage of transmitter remains unchanged. Thus the proportionate increase in turnover is smaller than that in exocytotic release. To maintain NE stores relatively constant, TH activity need increase only in proportion to the smaller increase in turnover than the larger increase in release. The ability to gear down increases in NE turnover and synthesis in relationship to increases in release provides sympathetic nerves with a capacity for a more extended range of sustainable release rates than would otherwise be possible.

Perspectives

Studies about what determines TH activity have been largely restricted to cell culture systems or measurements in isolated tissues and have concentrated on the mechanisms regulating TH gene expression or activity of the enzyme. These processes are often shown to be regulated by the same underlying mechanisms involved in regulating exocytotic release of catecholamines. From this, it is commonly assumed that the level of TH activity is closely related to the level of exocytosis, but few if any studies have attempted to quantitatively establish the validity of this relationship. By invoking the dependence of TH activity on exocytotic release of catecholamines, it is often concluded that catecholamine release is the primary if not sole determinant of catecholamine turnover (i.e., loss of previously synthesized catecholamine, balanced by synthesis of new catecholamine under the control of TH). From this it is commonly concluded that these processes are inextricably linked and may be regarded quantitatively as one and the same. The present study challenges this notion by showing that the principal determinant of cardiac NE turnover, and thus TH activity, is leakage of NE from vesicular stores, not exocytotic release of NE from nerves. This study also shows how examination of TH may be extended to clinical settings. Future studies about TH may benefit from a more integrative approach where regulation of the enzyme is considered not in isolation, but in relationship to the many ongoing processes in nerves or chromaffin tissue and as a part of the functioning of the organism as a whole.

    APPENDIX
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References

Derivation of the Model of Sympathetic Function in Fig. 3

Values for rates of entry of the various compounds into the bloodstream from cardiac sympathetic nerve endings or extraneuronal tissues (e.g., cardiac myocytes) are represented by those calculated for the cardiac spillovers of compounds before and during exercise (see Table 2).

In the model, it is assumed that cardiac NE stores are maintained constant by a rate of synthesis of new NE that equals the rate of loss from nerves of previously synthesized NE. Thus rates of NE synthesis from dopamine (i.e., values for dopamine right-arrow NE in Fig. 3) are determined to be the same as cardiac NE turnover rates (see Table 2).

There is no cardiac spillover of homovanillic acid or dopamine-sulfate and neglible spillover of dopamine (4). Therefore calculations of dopamine synthesis (i.e., values for DOPA right-arrow dopamine) are derived from the sum of the cardiac spillovers of DOPAC and rates of NE synthesis.

Rates of tyrosine hydroxylation (i.e., values for Tyr right-arrow DOPA in Fig. 3) are calculated from the sum of cardiac DOPA spillovers and synthesis rates of dopamine.

Rates of NE reuptake and NE leakage from storage vesicles and sequestration back into storage vesicles are calculated according to established methods that depend on examination of the formation of [3H]DHPG from intravenously infused [3H]NE (3-5, 9). Comparison of the desipramine-sensitive cardiac neuronal removal of [3H]NE (41,379 dpm/min) with the desipramine-sensitive cardiac production of [3H]DHPG (1,862 dpm/min) shown in Table 4 indicates that the rate of entry of NE into the cardiac sympathetic axoplasm is 22-fold (41,379/1,862) more than the corresponding spillover of DHPG derived from the NE entering the sympathetic axoplasm (693 pmol/min). Since the DHPG appearing in plasma has a neuronal source (3-5), the total rate of entry of NE into the sympathetic axoplasm at rest can therefore be calculated to be 22-fold more than the cardiac spillover of DHPG (22 × 693 = 15,246 pmol/min).

The rate of entry of NE into the sympathetic axoplasm reflects entry from two sources: neuronal uptake of NE and leakage of NE into the sympathetic axoplasm from storage vesicles (3, 4). Use of desipramine to block NE reuptake and inhibit sympathetic outflow, thereby decreasing release and reuptake, provides a pharmacological tool to separate the contributions of NE reuptake and leakage to DHPG formation (3, 4, 9). The desipramine-sensitive cardiac spillover of DHPG (105 pmol/min) reflects the amount of DHPG derived from reuptake as distinct from leakage of NE from storage vesicles (see Table 4). The rate of neuronal uptake of NE at rest can therefore be estimated to be 22-fold (41,379/1,862, see above) more than the desipramine-sensitive cardiac spillover of DHPG (22 × 105 = 2,310 pmol/min). The difference between this and the total rate of entry of NE into the axoplasm (15,246 - 2,310 = 12,936 pmol/min) reflects the rate of leakage of NE from storage vesicles. The rate of vesicular sequestration of NE is estimated by subtraction of the DHPG production rate from the total entry rate (15,246 - 941 = 14,305 pmol/min).

Rates of extraneuronal uptake of NE are estimated by an established method that depends on measurements of formation of [3H]normetanephrine from intravenously infused [3H]NE (2, 8). Comparison of the extraneuronal removal of [3H]NE (8,895 dpm/min) with the cardiac spillover of [3H]normetanephrine (315 dpm/min) shown in Table 4 indicates that the rate of extraneuronal uptake of NE is 28.2-fold (8,895/315) more than the corresponding spillover of normetanephrine derived from this NE (5.1 pmol/min). Since normetanephrine has only an extraneuronal source (8), the extraneuronal uptake rate of NE at rest is 144 pmol/min (28.2 × 5.1).

Release of NE from nerves is then estimated from the sum of rates of NE spillover and neuronal and extraneuronal NE uptake (i.e., at rest NE release = 75 + 2,310 + 144 = 2,529 pmol/min).

The rate of MHPG production derived from extraneuronal metabolism of normetanephrine (i.e., value for normetanephrine right-arrow MHPG in Fig. 3) is calculated at rest from the difference in extraneuronal uptake of NE and the spillover of normetanephrine (144 - 5 = 139 pmol/min). From this, the extraneuronal production of MHPG from DHPG produced in neurons (i.e., value for DHPG right-arrow MHPG) is calculated from the difference in MHPG spillover and MHPG production from normetanephrine (387 - 138 = 248 pmol/min). The rate of intraneuronal production of DHPG (i.e., value for NE right-arrow DHPG) is then calculated from the sum of spillover of DHPG and the metabolism of DHPG to MHPG before entry into the bloodstream (693 + 248 = 941 pmol/min).

Similar calculations to those above are used for estimation of intraneuronal and extraneuronal rates of processes during exercise with additional assumptions and limitations outlined below.

Assumptions and Limitations of the Derivation

The validity of estimates of synthesis of NE, dopamine, and DOPA depends on the assumption of balanced rates of NE synthesis and NE loss (i.e., NE turnover) and negligible local O-methylation of DOPA. However, the validity of these assumptions does not impact on any of the main conclusions of this article, except estimates of proportions (7%) of DOPA produced in neurons that escape metabolism to dopamine and spillover into plasma.

Radiotracer-dilution estimation of rates of neuronal and extraneuronal uptake of NE depends on the key assumption that the proportion of NE removed by an uptake process to the amount of metabolite appearing in plasma is the same for infused and endogenously released NE. The validity of this and other assumptions involved in these measurements has been discussed in depth previously (2, 5). Accuracy of estimated relative contributions of neuronal uptake and vesicular leakage to DHPG formation and NE turnover also depends on the degree of inhibition of neuronal uptake by desipramine. Since ~13% of the cardiac extraction of [3H]NE reflects extraneuronal removal (31), the 69% decrease in cardiac [3H]NE extraction after desipramine indicates that neuronal uptake was inhibited by at least 82% ([86.4 - 26.4]/[86.4 - 13]). Thus the rate of neuronal NE uptake at rest (2,310 pmol/min) represents at most an 18% underestimation, whereas the rate of NE leakage from storage vesicles represents at most a 3.2% overestimation.

Calculations of NE reuptake and leakage of NE from vesicles and sequestration of NE back into vesicles during exercise assume that the rates of vesicular leakage of NE do not change during exercise. Previous findings of constant rates of formation of cardiac DHPG during sympathetic activation in desipramine-treated dogs establish that vesicular leakage of NE remains unaffected by sympathetic activation (9). Calculations also assume that the proportions of NE removed by neuronal uptake to DHPG entering the coronary venous outflow do not change during exercise. It is, however, possible that during exercise-induced sympathetic activation MAO might compete more effectively with the vesicular transporter for the increased amounts of axoplasmic NE. Also, the proportionally smaller exercise-induced increase in MHPG than in DHPG suggests that less of the DHPG produced intraneuronally is metabolized to MHPG. Both effects would lead to an overestimation of the rates of NE reuptake during exercise shown in the model (Fig. 3).

    FOOTNOTES

Address for reprint requests: G. Eisenhofer, Bldg. 10, Rm. 4D20, National Institutes of Health, 10 Center Dr., MSC 1424, Bethesda, MD 20892-1424.

Received 18 August 1997; accepted in final form 13 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References

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AJP Regul Integr Compar Physiol 274(3):R626-R634


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