S65

Plasma Free Dopamine: Physiological Variabilit

Pathophysiological SignificanceY and

Yukio Miura * 1, Toshiya Watanabe * 2, Takao Noshiro * 2, Kazumasa Shimizu * 2, Taku Kusakari * 2, Hiroyoshi Akama * 2, Satoru Shibukawa * 2, Wakako Miura * 2, Takashi Ohzeki * 2, Masaki Takahashi * 3, and Naoki Sano * 4

Dopamine (DA) is the most abundant catecholamines in human plasma and exists mostly in the sulfo-conjugated form (DA sulfate), a biologically inactive metabolite. The paucity of unconjugated DA (PDA) in plasma throws doubt on its physiological significance. However, PDA, when measured with a highly sensitive radioenzymatic method, showed quite different features from norepinephrine and epinephrine in some types of clinical hypertension, lower in essential hypertension and higher in primary aldosteron-ism and pheochromocytoma. There was a weak but significant correlation between the values of PDA and DA sulfate measured in the same specimens, but DA sulfate was more susceptible to impaired renal function. Upright posture, high salt diets and an intravenous injection of metoclopramide (MCP, 10 mg), a DA receptor antagonist, induced a slight but significant increase in PDA in normal and hyperten-sive subjects. An intravenous dexamethasone (2 mg) caused a gradual increase in PDA over 150 min af-ter medication, which was completely blocked by concomitant administration of a-methyl p-tyrosine, a tyrosine hydroxylase inhibitor. The responses of PDA to both high salt diets and MCP were blunted in salt-sensitive patients with uncomplicated essential hypertension. The results suggest that DA is not only a precursor of norepinephrine biosynthesis but also plays an inherent role as an active neurotransmitter in the peripheral sympathoadrenal system, and that PDA is a sensitive marker of peripheral dopa-minergic activity, which may operate to modulate the cardiovascular and endocrine functions and par-ticipate in the pathogenesis of some types of hypertension. (Hypertens Res 1995; 18 Suppl. I: S65-S72)

Key Words: plasma free dopamine, primary aldosteronism, essential hypertension, salt-sensitivity, de-xamethasone

Norepinephrine (NE) and epinephrine (E) are well established as principal neurotransmitters of the peripheral sympathoadrenal system and their con-centrations in plasma are widely used as a useful marker of peripheral sympathoadrenal activity (1-3). Although dopamine (DA) is the largest constit-uent of plasma catecholamines (CA), it is mostly conjugated to sulfate or glucuronide, biologically in-active metabolites, and unconjugated (free) DA is said to be as little as 1-3% or less of total plasma DA, throwing doubt on its physiological significance (4-6). The difficulties in studying the significance of plasma free DA (PDA) result from lack of the sensitive method that permits its accurate measure-ment under physiological and pathological condi-tions (6, 7). Fortunately, recent advances in the methodology for PDA assay have disclosed that PDA shows physiological variability in response to various stimuli, suggesting that PDA is a marker of peripheral dopaminergic activity, which may play a role as a physiological regulator (7).

In this paper, we will summarize our recent stud-ies on PDA in various types of clinical hypertension and its responses to physiological and pharmacolog-ical stimuli. These studies have disclosed the possi-bility that peripheral dopaminergic activity operates to modulate the cardiovascular and endocrine func-tions and participates in the pathogenesis of some types of hypertension.

Improvement of PDA Assay

In this study, we used our newly developed proce-dures, which were based on the radioenzymatic method using catechol-0-methyltransferase (COMT) (7-9). The most important improvement was made on the extraction and condensation procedures for free DA from a sufficient amount of plasma. Namely, despite the extremely low concentration of PDA, plasma specimens, when directly subjected to the assay, must be limited to 0.1 ml or less, other-wise some plasma constituents interfere with the

From *'the Department of Informatics on Pathophysiology, Tohoku University Graduate School of Information Science, the Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai Municipal Hospital, and

* 4East Japan Railway Sendai Hospital, Sendai, Japan.

Address for Reprints: Yukio Miura, M.D., The Health Administration Center, the Department of Informatics on

Pathophysiology, Tohoku University Graduate School of Information Science, Kawauti, Kita Campus, Aoba-ku, Sendai

980-77, Japan.

S66 Hypertens Res Vol. 18, Suppl. I (1995)

COMT reaction. As a practical measure, some pre-vious methods used the procedure of acid depro-teinization of plasma, extraction of DA using alumi-na and elution by acidic solutions (10-13). Acid treatment, however, may cause a deconjugation of DA sulfate, the influence of which, even occurring in the least portion of plasma DA sulfate, should be very critical, since plasma contains so large amount of DA sulfate (14). Aluminium ions eluted from alumina may interfere with the COMT reaction (15). Aromatic acid decarboxylase, if any, con-taminated in the COMT preparation may synthesize DA from dihydroxyphenylalanine (DOPA) and falsely raise the PDA concentration (16). To avoid these technical problems, plasma specimens (2-3 ml), mixed with ascorbic acid, were applied on an Amberlite CG50 (Lit ) microcolumn as a substitute for alumina and DA absorbed on the Amberlite column was eluted by magnesium-ethanol solution. After desalination steps, final eluate was condensed to dry residua, which were proved to be free from any plasma inhibitors as well as inhibitory cations. There were also some modifications in the solvent extraction steps of the radioenzymatic procedures reported by Peuler and Johnson, 1977 (17). The sensitivity (blank X 2) was close to 6.0 pg / sample, being equivalent to 3.0 pg / ml of plasma when 2.0 ml of plasma were used. Interassay and intraassay variations were 7.0-9.1% and 10-23%, respectively, for each CA fraction.

PDA Profile in Hypertensive Patients

All patients studied here came to the Tohoku Uni-versity Hospital for evaluation of their hypertensive disorders and gave informed consent for the inves-tigation. Healthy volunteers were also studied as normal controls. All subjects were studied on their daily meals, and all medications, if any, were dis-continued for two weeks or longer. Blood speci-mens were obtained from an indwelling catheter placed in the antecubital vein while subjects were lying for 60 min after an overnight fast and then standing for 15 min.

The baseline levels of plasma CA concentrations in normal and hypertensive subjects are summarized on Table 1 (7-9). In each group of hypertensive pa-tients and normal controls, PDA concentrations were invariably the smallest of the CA fractions. In hypertensive patients except pheochromocytoma, both NE and E concentrations were comparable to those of normal controls, whereas PDA concentra-tions tended to be lower in patients with essential hypertension, and slightly higher in some patients with primary aldosteronism. All types of CA in-creased remarkably in patients with pheochromo-cytoma. Upright posture induced a significant rise in all CA fractions. Although the responses of PDA in each group of subjects were much smaller than those of E and NE, changes in PDA were statisti-cally significant (p < 0.05) (8, 9). Similar findings were also reported by others (18), though their PDA values both at rest and during standing were much larger than ours. The distinctive features of PDA in various types of clinical hypertension and its prompt responsiveness to upright posture indi-cate that peripheral dopaminergic activity is a phys-iological regulator of the cardiovascular functions.

Correlation of PDA to Other Variables

The baseline values of PDA, when data were com-bined in normal and hypertensive subjects, corre-lated negatively with their mean blood pressures (r _ - 0 .301, n = 61, p < 0.05), which were recorded at the time of blood sampling. A weak positive cor-relation was also observed between PDA and plas-ma NE concentrations (r = 0.381, p < 0.01) in nor-mal and hypertensive subjects (Fig. 1) (9). In this figure, it should be noteworthy that there were some hypertensive patients, especially borderline hypertensive patients, who showed a slight increase in both PDA and plasma NE. These findings may support the view that both DA and NE in plasma are concomitantly released from the same sym-pathoadrenal tissues (19, 20), and that hypertensive patients, even borderline hypertensive patients, con-

Table 1. Blood Pressure, Heart Rate, and Plasma Free Catecholamines in Nomal and Hypertensive Subjects

Miura et al: Plasma Free Dopamine in Hypertension S67

sist of a heterogeneous population for sympathetic nerve activity (19, 21, 22). Such a significant correlation between PDA and

plasma NE, however, could not extend to the pa-tients with primary aldosteronism, who showed a slight rise in PDA but not in plasma NE, suggesting that the regulatory mechanisms or the sources of PDA and NE may not be the same.

Relation of PDA to Plasma Conjugated DA

The major component of human plasma DA exists in the form of sulfoconjugated compound (DA sul-fate) (4, 14). It has been proposed that plasma DA sulfate is useful to measure the sympathoadrenal function, so long as dietary influences are controlled (5, 23, 24), since virtually all DA in plasma are rapidly sulfoconjugated upon release and plasma concentration of DA sulfate varies in response to the stimuli (4, 6). It is therefore interesting whether any relationships exist between plasma concentra-tions of free and conjugated DA. When plasma sulfo-

conjugated and free DA were measured in the same specimens, DA sulfate tended to be higher in pa-tients with primary aldosteronism, pheochromo-cytoma and renal hypertension (Table 2). These profiles are well comparable to those of PDA, at least in primary aldosteronism and pheochromo-cytoma, but not in renal hypertensive patients who showed a marked increase in DA sulfate alone without any significant changes in PDA.

It has been proposed that the ratio of DA to NE may be useful to measure the balance between peripheral dopaminergic and noradrenergic activity (19, 25, 26). Our findings on this point were as fol-

lows (Table 3): the ratio of plasma free DA to NE showed significantly larger values in patients with primary aldosteronism and tended to be smaller in patients with pheochromocytoma. The ratios of con-jugated DA to NE were also significantly larger in primary aldosteronism and significantly smaller in pheochromocytoma patients. At the basal condi-tion, NE and DA, either free or conjugated, showed weak but significant correlations (p < 0.05) to each other in normal subjects and in patients with uncomplicated essential hypertension (PDA vs. NE, r=0.464; PDA vs. NE sulfate, r=0.339; PDA vs. DA sulfate, r = 0.340; DA sulfate vs. NE sul-fate, r=0.716, n=37 for each pair). It is necessary, however, to note that conjugated CA is more sus-ceptible to the renal function. Moderately impaired renal function induced a significant increase in con-jugated plasma CA as a result of decreased renal clearance (27, 28) and possibly of decreased renal desulfation (24).

Effects of Dietary Salt Intake on PDA

To evaluate a functional significance of PDA, we studied the effects of dietary salt intake on PDA and its related hormonal factors in hypertensive pa-tients and in age-matched normal controls, while they were receiving ordinary meals (urinary Na, 130-180 mEq daily) followed by high sodium diets (250-300 mEq daily) for a week. Under high salt intake, both plasma NE and E tended to decrease, whereas

Fig.!. The correlation between plasma free dopamine

(PDA) and plasma norepinephrine (PNE), which were measured at the basal condition in normal controls (N) and in patients with borderline hypertension (BH) and essential hypertension (EH).

Table 2. Plasma Free and Sulfoconjugated Catecholamines in Normal and Hypertensive Subjects

S68 Hypertens Res Vol. 18, Suppl. I (1995)

PDA increased slightly in patients with essential hypertension from 7.2 ± 0.8 pg/ml [SE] to 9.3 ± 1.0 and in normal controls from 9.1 ± 1.8 to 11.2 ± 1.3, showing clearly different trends between PDA and other CA (Fig. 2) (29). At the baseline condition, a significant correlation was observed between PDA and NE (r=0.733, pG

0.01, n =16), but this correlation disappeared on high salt intake, because PDA and NE changed in opposite directions. Plasma renin activity (PRA) and plasma aldosterone concentration (PAC) de-creased in all subjects following high salt diets, while plasma prolactin (PRL) remained unchanged. When all data measured before and during high salt intake were combined, PDA showed a weak but significant negative correlation to PAC in normal and hypertensive subjects (r= - 0.351, p < 0.05, n = 32), suggesting that peripheral dopaminergic activity plays a role in the physiological regulation of aldo-sterone secretion (30, 31). Upright posture induced a significant increase in NE, E, DA, PRA and PAC on ordinary meals. The responses of NE and PAC were apparently blunted on high salt diets, while PDA response tended to become greater on high salt diets (29). An intravenous injection of metoclopramide (10 mg, MCP, Fujisawa Pharmaceutical Co., Ltd., Osa-ka, Japan), a DA receptor antagonist, provoked a slight increase in both plasma NE and DA in nor-mal and hypertensive subjects at the basal condition (Fig. 3) (7, 29). The response of PDA to MCP was further enhanced on high salt diets. MCP also in-duced a significant rise in PAC and PRL in all sub-jects studied, while plasma E and PRA did not change.

In summary, the responses of PDA to upright posture and to MCP were similar to those of NE at the basal condition, but there was a sharp contrast in the response to sodium loading between PDA and NE, indicating that different regulatory mechanisms are operating between DA and NE re-lease. These results reinforce the view that peripheral dopaminergic activity is stimulated by high salt intake and participates in the mechanisms responsible for inhibition of aldosterone secretion from the adrenal glands (30, 31) as well as for de-crease in NE release from sympathetic nerve ter-minals observed during high salt intake (19).

Effects of Dexamethasone on PDA

Dexamethasone is known to stimulate peripheral dopaminergic activity, although the precise mecha-nisms remain unclear (32, 33). To extend our under-standing of the dopaminergic modulation in the pathogenesis of hypertension, we examined the effects of dexamethasone on plasma CA and related hormonal factors in normal and hypertensive sub-

Table 3. The Ratios of Plasma Dopamine (DA) to Norepinephrine (NE) in Normal and Hypertensive Subjects

Fig. 2. The baseline values o f plasma norepinephrine (NE), epinephrine (E), dopamine (DA), plasma renin activity (PRA), plasma aldosterone concentration (PAC) and plasma prolactin concentration (PRL) and their re-sponses to upright posture in patients with essential hyper-tension (n = 8), while they were receiving ordinary (nor-mal) hospital diets followed by high sodium diets for a week.

Miura et al: Plasma Free Dopamine in Hypertension S69

jects (34). In the morning after an overnight fast, the subjects rested for 30 min and then dexametha-sone (2 mg) or saline as a placebo was administered intravenously. Blood specimens were obtained be-fore and at every 30 min over 150 min after medica-tion. One week later, a-methyl-p-tyrosine (250 mg, a-MPT, Merck Sharp & Dohme, West Point, Pa, USA), a tyrosine hydroxylase inhibitor, was admin-istered orally at 60 min before dexamethasone in-jection and the same protocol was repeated. Dexamethasone administration provoked a grad-

ual increase in PDA (Fig. 4), reaching the levels as high as 153% in normal control, 206% in patients with primary aldosteronism and 158% in patients with essential hypertension, compared with their baselines at 150 min after medication. Plasma NE remained unchanged and E tended to increase slightly. PAC decreased slightly, but PRL did not change. Dexamethasone-induced increases in PDA and E

were completely blocked by concomitant adminis-tration of a-MPT. These findings suggest the possi-bility that dexamethasone stimulates peripheral dopaminergic activity by enhancing CA biosynthetic enzyme activities, probably tyrosine hydroxylase activity. It has been reported that glucocorticoid stimulates tyrosine hydroxylase activity in the sym-pathetic ganglia (35) and the brain (36). A delayed and gradual increase in PDA may reflect the changes in biosynthesis. However, no significant changes were observed in NE, suggesting an addi-tional possibility that other enzymes, for example, dopamine-R-hydroxylase, may be influenced by dexa-methasone as well.

In addition, it may be also possible that dexa-

methasone stimulates arylsulf atase activity and accelerates a deconjugation of sulfated CA, result-ing a rise in free CA fractions (24, 37), although the physiological significance of conjugation-deconjuga-tion interplay remains controversial (38). In this study, however, any significant changes were not found in free NE fraction and changes in PDA and E disappeared following pretreatment with a-MPT, suggesting that dexamethasone-induced change in PDA is mainly due to the changes in biosynthesis rather than in deconjugation. The response of PDA to dexamethasone tended

to be greater in patients with primary aldosteronism than others. This finding should again reinforce the view that peripheral dopaminergic activity is stimu-

Fig. 3. The responses of plasma norepinephrine (PNE) and dopamine (PDA) to intravenous injection of metoclopramide

(MCP) in normal controls (N, n = 8) and in patients with essential hypertension (EH, n = 8), while they were receiving ordinary hospital diets (control) followed by high sodium diets for a week.

Fig. 4. The responses o f plasma dopamine to intravenous injection of dexamethasone (Dx, 2 mg) with and without concomitant administration of a-methyl-p-tyrosine (a-MPT) in patients with essential hypertension (n=6).

S70 Hypertens Res Vol. 18, Suppl. I (1995)

lated in patients with primary aldosteronism and counteracts the excess mineralocorticoid state (7).

Interestingly, PDA responses to upright posture were completely blocked by concomitant adminis-tration of a-MPT, but the responses of plasma NE and E were not changed. The results may be com-patible with the subcellular process that NE and E are released from their storage particles in the sym-pathoadrenal tissues, which may be unaffected from rapid changes in CA biosynthesis, whereas DA ex-ists mainly in the cytosol outside the storage parti-cles (39, 40). An inhibition of DA biosynthesis may induce rapid depletion of cytosolic DA content, re-sulting in a failure of PDA to respond to upright posture.

PDA and Salt-Sensitivity

From these findings mentioned above, it must be crucial to study whether PDA participates in the pathogenesis of salt-sensitivity in essential hyperten-sion. In our study (41), the salt-sensitivity was iden-tified by blood pressure response to changes in salt intake from 3 g to 20 g daily for a week. Daily salt intake was confirmed by measuring urinary sodium

excretion throughout the study period. Salt-sensitive (SS) patients were defined according to the arbi-trary criteria that the patients showed a rise in mean blood pressure as much as 8% or greater on the 7th day of high salt diets compared with that on the last day of low salt diets. Other patients were classified as the non salt-sensitives (NSS). Plasma CA and re-lated hormonal factors were measured on the 7th day of low and high salt diets. The responses of plasma CA and hormonal factors to an intravenous MCP were also determined on high salt diets. High sodium diets induced a decrease in plasma NE and an increase in PDA (Fig. 5). However, a reduction of NE tended to be smaller in SS patients and a rise in PDA was significantly greater in NSS patients. PDA response to MCP tended to be small-er in SS than in NSS patients (Fig. 6). There were no significant differences in PRA,

PAC and PRL between SS and NSS patients on each dietary condition. However, the ratio of PAC to PRA under high salt diets was significantly (p 0.05) greater in 55 patients (4.98 ± 0.81, n = 6) than

Fig. 5. Changes in plasma norepinephrine (PNE), epinephrine (PE) and dopamine (FDA) in salt-sensitive

(SS, n= 6) and non salt-sensitive (NSS, n = 9) patients with essential hypertension, while they were receiving low salt diets for a week followed by high salt diets for a week. Plasma catecholamines were measured in a supine position after an overnight fast on the 7th day of each diet.

Fig. 6. The responses o f plasma norepinephrine (PNE), epinephrine (PE) and dopamine (FDA) to intravenous in-

jection of metoclopramide (MCP) in salt-sensitive (SS, n= 6) and non salt-sensitive (NSS, n=9) patients with essential hypertension, while they were receiving high salt diets for a week. Experiment was performed on the 7th day of high salt diets.

in NSS patients (2.56 ± 0.80, n = 9), indicating an attenuated inhibition of aldosterone secretion under high salt diets. All of these findings are in line with the previous reports that peripheral dopaminergic activity is re-duced in SS patients (42 -44) and participates in re-duced inhibition of both aldosterone secretion from the adrenals and NE release from sympathetic nerve terminals (19).

In this context, Kuchel OG, and Kuchel GA pro-posed an interesting idea that volume expansion in-duced by high salt intake causes an inhibition of dopamine-j9-hydroxylase activity in the sym-pathoadrenal tissues, leading to increase in DA and decrease in NE biosynthesis (19). This response may be complemented by stimulation of tyrosine hydroxylase activity, which increases plasma DOPA, serving as a precursor of both plasma and urinary DA production. As a result, peripheral sympathoadrenal activity may be functionally in-clined to "dopaminergic" predominance. This idea is well in line with the view that DA plays an in-herent role as an active neurotransmitter, indepen-dently from NE and E, not only in the central but also in the peripheral sympathetic regulation. However, it remains unclear what kinds of the biochemical mechanisms are involved in the mod-ulation of CA biosynthetic enzyme activities when such stimuli as volume expansion or high salt intake are imposed on the living body. Another idea that PDA is derived from the peripheral dopaminergic neurons, which operate in-d

ependently from the noradrenergic neurons, may be possible to explain our present findings (45). Our recent study has demonstrated that a significant amount of renal DA spillover can be detected in conscious rabbits, indicating that DA is actually re-leased into the circulation within the kidney (46). This finding may be a first biochemical evidence to support the existence of dopaminergic innervation in the kidney that has been identified morphologi-cally (47). However, at present, we have no conclu-sive evidences enough to prove the possibility that PDA is mostly derived from the peripheral dopa-minergic neurons.

In conclusion, PDA measured with a sensitive radioenzymatic method, showed quite different fea-tures from NE and E in some types of clinical hypertension. PDA was also proved as a responsive variable to various physiological and pharmacologi-cal stimuli, including upright posture, changes in dietary salt intake, DA receptor blockade, dexa-methasone challenge and an inhibition of CA biosynthesis. The results suggest that DA is not only a precursor of NE biosynthesis but also plays an inherent role as an active neurotransmitter in the peripheral sympathoadrenal system, and that PDA is a sensitive marker of peripheral dopaminergic activity, which may operate to modulate the car-diovascular and endocrine functions and participate in the pathogenesis of some types of hypertension.

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