Norepinephrine kinetics in freely moving rats

Emmanuelle Maignan, Monique Legrand, Ilham Aboulfath, Michel Safar, and Jean-Louis Cuche

Catecholamine Biology Research Laboratory, Institut National de la Santé et de la Recherche Médicale, Broussais Faculty of Medicine, 75 270 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Norepinephrine (NE) kinetics were investigated in freely moving (FM) and minimally stressed (MS) rats with the isotope dilution technique. 1) The mean NE spillover rate (NE-SOR) was 79 ± 6 ng · kg-1 · min-1, and the mean NE metabolic clearance rate (NE-MCR) 179 ± 9 ml · kg-1 · min-1 (n = 31). Thus the NE kinetics in FM and MS rats are much faster than in human beings, probably related to a higher sympathetic drive. 2) Whether the magnitude of NE-MCR is related to the level of plasma NE concentration was investigated. No significant correlation was calculated between plasma NE concentration and NE-MCR in 31 control rats. When plasma NE concentration was varied during either acute or chronic infusion of exogenous NE, NE-MCR remained unchanged as long as animal hemodynamics were not altered. When plasma NE concentration was high enough to increase mean arterial pressure (MAP), NE-MCR was decreased. However, when MAP was increased within comparable magnitude, NE-MCR was decreased during NE and increased during epinephrine (Epi) infusion. Thus the existence of an alpha -/beta -adrenergic mechanism involved in the regulation of NE-MCR independent of known hemodynamic mechanisms is suggested. 3) The "epinephrine hypothesis" was revisited in FM and MS rats. At variance with humans, very high plasma Epi concentrations have to be induced to increase NE-SOR in resting rats. Furthermore, NE-MCR was also increased, accounting for the nonsignificant increase of plasma NE concentration. Within the range of Epi concentrations with no effect on NE-SOR, an increase of NE release was revealed when the presynaptic alpha 2-adrenoreceptors were partially inhibited by yohimbine. This suggests the existence of a second epinephrine hypothesis.

norepinephrine spillover rate; norepinephrine metabolic clearance rate; epinephrine; dopamine; epinephrine hypothesis; yohimbine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RECENTLY, Grassi and Esler (18) stated that assessment of the adrenergic neuronal function remains one of the major fields in cardiovascular research. Reviewing available techniques, they observed that radiotracer methods, allowing assessment of norepinephrine (NE) kinetics, and microneurography, allowing direct recording of sympathetic nerve traffic, have supplanted others. The radiotracer technique provides the investigator with an estimate of what comes in plasma, NE spillover rate (NE-SOR), and what comes out, metabolic clearance rate (NE-MCR), information of a broader physiological meaning than plasma NE concentration alone. Also, it allows a more refined understanding of sympathetic activity through estimates of regional contributions, at least in humans. Thus the radioisotope dilution technique has strongly improved our understanding of sympathetic activity in humans.

The aim of the present study was to investigate the relationship between plasma catecholamine and the kinetics of NE in normotensive, freely moving rats. A three-step study was designed. 1) The physiology of NE kinetics in the rat remains poorly known; previous studies have dealt with NE kinetics in controls or in response to antihypertensive drugs in spontaneously hypertensive rats (10, 21, 22). Our working hypothesis was to consider that NE kinetics should be in agreement with a high sympathetic drive in the rodent (28, 34, 36). 2) Whether the capacity of the rat organism to clear NE, as judged with the NE-MCR, adapts itself to plasma NE concentration was investigated in rats given various infusion rates of exogenous NE during short (90 min) or long-lasting (7 days) periods of time. Special attention was paid to investigating the NE kinetics in the range of plasma catecholamine concentrations apparently devoid of hemodynamic effect. 3) Epinephrine (Epi) was proposed ("epinephrine hypothesis") to participate in the presynaptic regulation of the release of NE, with conflicting conclusions (1, 3, 16, 23, 24, 26, 29, 30, 33). It was revisited in freely moving rats kept in apparently quiet conditions and given increasing doses of exogenous Epi. The effect on NE-SOR appears to be dose dependent; furthermore, on the basis of previous reports (30, 31, 33, 35), we hypothesized a presynaptic regulation of NE release under the control of a delicate alpha 2-inhibition/beta 2-activation balance. The Epi-induced beta 2-activation could become more efficient when the alpha 2-inhibition was partially canceled by yohimbine. Thus the rat appears to be a mammal with a high sympathetic drive and with a tight control of presynaptic regulation of NE release.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Wistar rats (n = 154; Elevage Janvier, Le Genest Saint Isles, France) were housed in individual cages and fed ad libitum 24 h/day; lights went off at 1800 and on at 0600. Previous work has shown that anesthesia can create misleading information on sympathetic activity, making inappropriate any investigation carried out on anesthetized animals (25). All were studied in freely moving and apparently undisturbed conditions, equipped according to the following procedure.

Implantation of Catheters

All animals were anesthetized with pentobarbital sodium (60 mg/kg ip) and surgically implanted with femoral arterial and venous catheters for measurement of mean arterial pressure (MAP) and heart rate (HR) and for administration of tritiated NE, as well as other physiological or pharmacological compounds according to further-described experimental protocols. These catheters were routed subcutaneously to the back of the neck, exteriorized, and flushed regularly with sterile saline. A full week was allowed for recovery from anesthesia. At the end of the experiment, the animal was killed with an extraphysiological dose of pentobarbital sodium.

Blood Pressure and HR Recordings

The femoral artery catheter was connected to a Gould pressure processor via a pressure transducer (Gould P231 D). MAP and HR were electrically integrated on the signal of instantaneous pressure by means of a Gould DC amplifier and a Gould Biotach, respectively. Between 20 and 30 min were allowed for equilibration of the preparation.

Measurements of NE Kinetics

The isotope dilution technique described by Esler et al. (13) was used. Briefly, tritium-labeled norepinephrine ([3H]NE; [levo-ring-2,5,6-3H]NE, 40-60 Ci/mmol, 1 mCi/ml, New England Nuclear, Boston, MA) was diluted with 500 µl of 0.2 M acetic acid plus 50 µl of sodium sulfite (100 mg/ml), 350 µl of reduced glutathione (6 mg/ml) qsp 10 ml with 0.9% sodium chloride. The [3H]NE solution was infused at 27 µl/min intravenously i.e., 0.06 µCi · kg-1 · min-1 with the Harvard pump for 90 min. Arterial blood samples were obtained to measure alumina-extractable 3H and endogenous NE. In an Eppendorf tube, 150 µl of plasma were added to 100 µl of Tris buffer (1 M Tris, 54 mM Na2EDTA · 2H2O, pH 8.6) and 10 mg of alumina, activated according to the Anton and Sayre procedure (2). Vortexing, centrifugation, and aspiration of supernatant were performed. Alumina was washed twice with water. [3H]NE was eluted with phosphoric acid and acetic acid, and radioactivity was counted in the supernatant. The recovery of [3H]NE was calculated for each series of measurements; mean recovery was 87.7 ± 0.6%. Alumina-extractable 3H was measured after various periods of [3H]NE infusion. An apparent plateau was observed between 60 and 110 min (unpublished data); 90 min was chosen as the optimal period of [3H]NE infusion.

Whether alumina-extractable tritium measured after 90 min of [3H]NE infusion is appropriate to evaluate the kinetics of NE deserves comment. Although infused at tracer doses, [3H]NE is actively removed from the circulating volume by neuronal uptake, is translocated into storage vesicles, and enters the metabolic pathways of NE with production of 3H-labeled metabolites (9, 10, 19). Thus alumina-extractable 3H represents a mixture of various 3H-labeled compounds, in which [3H]NE accounts for a fraction only, a fraction that varies according to the time of [3H]NE infusion and the sites of blood sampling. Therefore, it appeared important to separate [3H]NE from other 3H-labeled metabolites, i.e., [3H]dihydroxyphenyl glycol ([3H]DHPG) and [3H]dihydroxymandelic acid ([3H]DOMA), and to evaluate the respective contribution of each fraction within the limits of our experimental protocols. Because the level of [3H]DOMA in plasma was shown to be very low, and to some extent negligible (10), special attention was paid to the plasma concentration of [3H]DHPG, having in mind that the [3H]DHPG/[3H]NE ratio increases (10) when plasma NE concentration is experimentally increased during infusion of exogenous NE.

HPLC procedure is the appropriate method (9) to estimate plasma concentrations of both [3H]NE and [3H]DHPG during infusion of [3H]NE. Thus a pilot study was designed to validate our data. Three groups of conscious rats were given either sodium chloride (control) or exogenous NE infusion (500 or 3,000 ng · kg-1 · min-1). The arterial plasma sample obtained at the end of each experiment was divided into two parts: one fraction was run according to procedures routinely used in our laboratory (Paris), whereas the other was sent to the Clinical Neuroscience Branch, National Institutes of Health (NIH), Bethesda, MD (11, 27). One of us (E. Maignan) was kindly welcomed to run the series of samples with the HPLC procedure at NIH.

The mobile phase [50 µM Na2PO4 · H2O, 130 µM EDTA, 3.6% acetonitrile, and octanesulfonic acid (48 mg/ml), adjusted to pH 3.1 with phosphoric acid] was filtered (0.22-µm membrane, Millipore, Bedford, MA), degassed, and pumped through the system at 1 ml/min. It was changed daily because of the presence of tritium.

Between 0.3 and 0.8 ml of plasma samples and 20 µl of infusate were mixed for 30 min with 5 mg of alumina (acid washed and stored at 100°C before use), DHBA as internal standard (2 ng), and 500 µl of Tris (pH 8.6). After brief centrifugation, the supernatant was discarded, and alumina was washed twice with milli-Q water. One hundred microliters of an acid solution [0.2 M phosphoric acid-0.2 M acetic acid (5-10%)] were added to the alumina, and the mixture was vortexed for 5 min to elute the catecholamines. A second elution was performed with 50 µl of the same acid solution to increase the recovery. After centrifugation, the supernatant (145 µl) was transferred into microsample vials, ready for injection by the autosampler.

The collected tritiated fraction was mixed with scintillation cocktail (Ecoscint A, National Diagnostic, Atlanta, GA), and tritium content was determined using a liquid scintillation analyzer (LS3801, Beckman). A part of the infusate was counted without extraction.

The mean (±SE) recovery of the DHBA internal standard was 77.7 ± 4.4%. Plasma concentrations of labeled and endogenous catecholamines were corrected according to the recovery of DHBA. Sensitivity of the assay was within the range of 1 and 4 pg of catecholamines measurable per milliliter of plasma. Interassay coefficients of variation, obtained using a quality control, were 4.3% for DHPG, 4.7% for NE, and 16.4% for epinephrine.

Endogenous catecholamines were measured according to a radioenzymatic assay routinely used in our laboratory (4). NE-SOR and NE-MCR were calculated according to Esler's procedure (12).

Experimental Protocols

Intravenous infusion of tritiated NE. Data obtained in freely moving rats given sodium chloride with or without tritiated NE are shown Table 2. No statistically significant difference was observed. In plasma, NE was slightly raised (+15%) but not significantly higher in rats given tritiated NE.

Study of NE clearance: infusions of exogenous NE. In given experimental conditions, one may wonder whether the capacity of the rat's organism to clear NE is dependent on the level of NE released and circulating in plasma. Previous reports came to differing conclusions. In humans, plasma NE concentration and NE clearance were significantly negatively correlated for Gordon et al. (17), whereas infusion of pressor doses of NE failed to alter NE clearance for Ziegler et al. (37); in the rat, an apparently nonsignificant 22% increase was reported in rats given pressor doses of exogenous NE (10). Our working hypothesis was to consider that the capacity of rat's organism to clear NE could increase as long as the hemodynamic state remained unaltered. Beyond a still undefined range of plasma NE concentrations, when MAP increases, the NE-MCR may be expected to decrease through various mechanisms where cardiac output and blood flows are likely to have a major role (12), whereas other mechanisms should not be excluded (6). Intravenous infusion rates of exogenous NE were empirically chosen to mimic various rates of NE delivered in plasma. The first infusion rate (50 ng · kg-1 · min-1) was chosen to increase plasma NE concentration with no measurable effect on rat hemodynamics (MAP and HR). A second group of animals was given NE at 500 ng · kg-1 · min-1. MAP was not changed, and HR was slower. In a third group of rats, exogenous NE was infused at 3,000 ng · kg-1 · min-1 to induce a moderate hypertensive effect. Rats entering that series of experiments were randomly assigned to four groups and given either sodium chloride or exogenous NE infusion together with tritiated NE for a period of 90 min.

Because adaptation of NE-MCR might be time dependent, a second series of experiments was carried out in rats given either sodium chloride or exogenous NE (50 or 500 ng · kg-1 · min-1) infusion for a period of 7 days.

Effects of Exogenous Epi Infusion on NE Kinetics

Several years ago, it was hypothesized that Epi incorporated into nerve terminal stores could act to facilitate sympathetic neurotransmission via activation of presynaptic beta 2-adrenoreceptors (24). Several reports have confirmed an Epi-dependent release of NE in humans (3, 15, 23, 26). In the study of Kjeldsen et al. (23), intra-arterial infusion of Epi in the human forearm was shown to increase both plasma NE concentration and forearm blood flow, suggesting a disassociation between metabolic and hemodynamic effects induced by Epi. Nonetheless, the "epinephrine hypothesis" was not always confirmed (16). In animals, reported conclusions are conflicting. Although a pronounced facilitation of endogenous NE release by presynaptic beta 2-adrenoceptors was reported in freely moving rats (30), and to a lesser extent in the dog heart (29), Epi or isoprenaline failed to facilitate stimulus-induced NE overflow in the isolated perfused rat kidney or in the rabbit isolated ear artery, respectively (1, 33). Thus NE kinetics were investigated in freely moving (FM) and mildly stressed (MS) rats given exogenous Epi infusion at various rates that were chosen 1) to have no hemodynamic as well as no metabolic effect (50 ng · kg-1 · min-1), as judged by the lack of effect on MAP and HR as well as on plasma glucose concentration (32); 2) to incrementally increase plasma Epi concentration in the neighborhood of its upper physiological limit (500 ng · kg-1 · min-1) as judged by plasma Epi concentrations in exercising rats (31) while MAP was not altered; and 3) to induce hemodynamic effects as judged by an increased MAP (3,000 ng · kg-1 · min-1). Rats entering that series of experiments were randomly assigned to four groups and given either sodium chloride or exogenous Epi infusion, together with tritiated NE, during 90 min.

Effect of alpha 2-Antagonist Yohimbine on beta 2-Facilitation of NE Release Induced by Exogenous Epi Infusion

Several groups (30, 31, 33) have suggested the existence of a pronounced counterregulatory role of presynaptic alpha 2-adrenoceptors in the rat; alpha 2-adrenoceptors tonically restrain NE synthesis, release, and turnover in sympathetic nerves (35). Further injection of the presynaptic alpha 2-adrenoreceptor antagonist yohimbine in combination with infusion of the beta 2-selective agonist fenoterol into adrenalectomized rats caused an enormous increase in plasma NE (31). Thus we assumed that Epi could increase the release of NE if the alpha 2-dependent inhibition was canceled, at least partially, by yohimbine. FM rats entered that series of experiments in randomized order. Three rats were given Epi (500 ng · kg-1 · min-1) alone; because results were not different from those obtained in the previous series, the two groups (8 + 3) of experiments were combined. Eleven rats were given intravenous infusion of yohimbine at 5,000 ng · kg-1 · min-1; the yohimbine infusion rate was empirically defined to be the highest possible, not affecting MAP. Twelve rats were given a combined infusion of exogenous Epi (500 ng · kg-1 · min-1) + yohimbine (5,000 ng · kg-1 · min-1).

Statistical Analysis

Data were expressed as means ± SE. NE-SOR and NE-MCR were normalized according to body weight. Differences were tested by either paired t-test or nonparametric Mann-Whitney test. P < 0.05 was accepted as the minimal level of significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Methodology: Pilot Study

Because alumina-extractable tritium represents a mixture of several radioactive compounds, a mixture that makes NE kinetics data unclear, a pilot study was carried out to compare data obtained according to the procedure used in "Paris" (alumina-extractable tritium and radioenzymatic method when needed) to that used in the Clinical Neuroscience Branch (NIH), which combines an HPLC analysis of the radioactivity eluted from alumina ([3H]NE and [3H]DHPG) with HPLC measurement of catecholamines (later on defined as "NIH"). Data obtained are shown in Table 1.

                              
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Table 1.   Comparative study of two biochemical procedures: Paris vs. NIH

The second column of Table 1 reports data obtained when the radioactivity, expressed in disintegrations per minute, of given [3H]NE infusates was measured by direct scintillation counting in Paris and NIH. The two series should be identical; however, tritium infusion rates turned out to be 25% higher in Paris than in NIH. Whether Paris countings were contaminated by a degradation product was ruled out by data obtained in NIH: there was no difference in the radioactivity of infusates after alumina extraction compared with direct counting. Thus we have to consider that the difference was related to a difference in quench correction by the two scintillation counters. Scintillation counting carried out in Paris was used, when needed, to obtain reported data.

A statistically significant positive correlation was computed between tritium infusion rate and tritium concentration in plasma: y = 516 + 0.005x, P < 0.001, in 31 control rats. Thus variations in tritium infusion rates account for ~29.5% in variation of tritium concentration in plasma. As expected, tritium concentration measured in Paris (679 ± 77 dpm/ml) was significantly higher than [3H]NE measured in NIH (337 ± 39 dmp/ml). Three factors accounted for that difference: 1) higher infusion rate measured in Paris, likely to account for ~29.5%; 2) [3H]DHPG measured in NIH (20 ± 5 dpm/ml), which accounts for 5.6% of [3H]NE + [3H]DHPG in control animals; 3) an unknown fraction that accounted for ~18% of the radioactivity eluted in Paris.

NE kinetics were substantially different according to the methodology used. NE-SOR was 136 ± 18 ng · kg-1 · min-1 and NE-MCR 298 ± 32 ml · kg-1 · min-1 in NIH, values significantly higher (P < 0.01) than the values measured in Paris: 82 ± 14 and 201 ± 28 ml · kg-1 · min-1, respectively. Thus NE kinetics, according to the procedure in Paris, were underestimated by 39.7% for NE-SOR and 32.5% for NE-MCR.

Because the ratio of DHPG to NE was shown to increase when exogenous NE was infused (10), the present pilot study was also run in rats given either 500 or 3,000 ng · kg-1 · min-1 of exogenous NE. Indeed, the percentage of radioactivity associated with [3H]DHPG increased up to 10.1% during 500 and 18.9% during 3,000 ng · kg-1 · min-1 infusion rate of exogenous NE. Thus the radioactivity associated with [3H]DHPG that could be considered as small in control rats (5.6%) became substantial in rats given exogenous NE.

NE Kinetics in Resting Rats

Data obtained in a group of 31 FM and MS animals are shown in Table 2. The mean NE-SOR was 79 ± 6 ng · kg-1 · min-1, whereas the mean NE-MCR was 179 ± 9 ml · kg-1 · min-1. Figure 1 shows a lack of statistical correlation between plasma NE and NE-MCR.

                              
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Table 2.   Lack of effect of [3H]norepinephrine iv infusion in freely moving rats



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Fig. 1.   Lack of correlation between plasma norepinephrine (NE) concentration and NE metabolic clearance rate in 31 freely moving and minimally stressed Wistar rats.

Effects of Exogenous NE Infusions

In rats given infusion of exogenous NE, NE-SOR are not reported, because their physiological meaning is difficult to assess in such experimental conditions.

In rats given acute infusion of exogenous NE (Table 3), the NE-MCR remained unchanged within a range of NE concentration from control to about a sixfold increase. The upper limit of that stability appears to be indicated by the slowing of HR. Beyond about six times the control level of NE concentration, an increased MAP was observed, associated with a significant decrease of NE-MCR. The statistically significant increase of plasma dopamine concentration during intravenous infusion of NE at 50 ng · kg-1 · min-1 was unexpected; its physiological meaning remains unclear.

                              
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Table 3.   Effects of exogenous NE infusion on NE-MCR on freely moving rats

In rats given chronic infusion (7 days) of exogenous NE, an apparent increase of the animal's general metabolism is suggested by the statistically significant decrease in body weight of those that were given either 50 or 500 ng · kg-1 · min-1 of exogenous NE. NE-MCR remained unchanged (Table 4).

                              
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Table 4.   Increased plasma NE concentration has no significant effect on NE-MCR during chronic (7 days) infusion of exogenous NE

Effects of Exogenous Epi Infusions

Data obtained are shown in Table 5. When plasma Epi concentration was increased up to ~3,200 pg/ml, plasma catecholamine concentrations as well as NE kinetics were not changed. When plasma Epi concentration was in the neighborhood of 20,000 pg/ml, MAP was significantly increased as expected, whereas HR was not. Both NE-SOR and NE-MCR were significantly increased, accounting for the nonsignificant increase of plasma NE concentration. Plasma dopamine concentration was significantly increased for a still-unknown reason.

                              
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Table 5.   Effects of exogenous epinephrine infusion on NE kinetics in freely moving rats

Effects of Exogenous Epi Infusion in Association With Yohimbine

Results are shown in Table 6. Data obtained during yohimbine (5,000 ng · kg-1 · min-1) infusion alone have to be compared with those of controls (Table 2). As expected, plasma NE and NE-SOR were slightly but nonsignificantly increased. Compared with preinfusion levels, MAP was not changed, whereas HR was significantly increased (+32 ± 6 beats/min, P < 0.05).

                              
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Table 6.   Effects of epinephrine infusion in the presence of yohimbine on NE kinetics

In rats given combined infusions of Epi with yohimbine, plasma Epi levels were not different from Epi levels measured in rats given Epi infusion alone. However, their plasma NE concentration and NE-SOR were significantly increased when compared with animals receiving either Epi or yohimbine infusion alone. NE-SOR was about three times higher in rats given Epi + yohimbine compared with those given Epi alone. Changes in the NE-SOR accounted for changes in plasma NE concentration, as NE-MCR was not changed.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was carried out to investigate the relationship between the level of circulating catecholamines and the kinetics of NE in normotensive FM and MS rats. The isotope dilution technique was adapted to small-sized animals, and alumina-extractable tritium was used.

Methodologies and NE Kinetics in Control Rats

Measurement of catecholamines and their metabolites remains a challenge for the investigator. Three points deserve comment. 1) Although the radioenzymatic procedure runs the sample of plasma in its original physiological equilibrium (there is no need for protein precipitation with strong acid), the HPLC procedure was developed up to the point where it has become a standard procedure. Paired comparisons of the two procedures have reported conflicting conclusions. Although plasma NE concentration was reported to be 40% higher (P < 0.001) with radioenzymatic assay (8), it is shown to be 15% lower (P < 0.05) in the present study when compared with HPLC (Table 1). That contradiction remains unexplained at the moment. 2) A second difference could have a potential role in discussing data obtained in the present study. The radioenzymatic procedure reports plasma concentration of DOMA in the range of 4,000 pg/ml (7); although DOMA is clearly identified by thin-layer chromatography, a nonspecific contamination cannot be ruled out. On the other hand, HPLC indicates a plasma DOMA concentration below 10 pg/ml (10); in such a low range of plasma concentration, it is acceptable to consider that plasma DOMA is negligible from a physiological point of view. 3) Adsorption on alumina is a well-known procedure to extract catecholamines and their metabolites (2); it is commonly accepted that only diphenolic structures are adsorbed on alumina. Elution plus HPLC provides the investigator with two well-identified compounds ([3H]NE and [3H]DHPG); unfortunately, the whole quantity of radioactivity eluted from alumina was not available. Thus, after elution of alumina-extractable tritium, one has to deal with a mixture of [3H]NE + [3H]DHPG + a still-unknown fraction; that unknown fraction was tentatively evaluated as representing 18% of total radioactivity. Whether that unknown fraction represents [3H]DOMA cannot be established at the moment. Because alumina-extractable tritium is made of [3H]NE + [3H]DHPG (which contributes minimally in control animals) + an unknown fraction, it is understood that NE kinetics data obtained in Paris are 39.7 and 32.5% lower for NE-SOR and NE-MCR, respectively, than data obtained in NIH. Thus data on NE kinetics reported in the present study will be discussed in light of these methodological limitations.

As shown in Table 2, intravenous infusion of radiolabeled NE had no significant effect on either circulating catecholamines or systemic hemodynamics. These data obtained in FM and MS rats (n = 31) indicate that their mean NE-SOR was 79 ± 6 ng · kg-1 · min-1 and their mean NE-MCR was 179 ± 9 ml · kg-1 · min-1. These data, confirmed with those obtained in NIH, demonstrate a high NE turnover in the rat, with high NE-SOR and high NE-MCR, whereas plasma NE concentration is not very much different in the rat compared with humans. In healthy humans, Esler et al. (14) have reviewed several studies and concluded that venous NE-SOR ranges between 0.6 and 1.6 µg/min (i.e., between 8 and 23 ng · kg-1 · min-1 for 70 kg body wt) and the venous NE-MCR between 2.3 and 4.8 l/min (i.e., between 32 and 68 ml · kg-1 · min-1 for 70 kg body wt). Our data on NE kinetics obtained in FM and MS rats suggest a high sympathetic drive in the rodent, as already suggested (28, 34, 36).

Clearance of NE

The concept of clearance was developed by renal physiologists in the early 1940s to define the volume of plasma cleared from a given compound by a unit of time. It was extended by endocrinologists to cover the whole series of mechanisms that participate in the clearing process of a given hormonal agent. The concept of metabolic clearance has been applied to circulating NE (10, 12, 14). Besides neuronal reuptake, known to have a predominant role in NE catabolism, several other mechanisms (as distinct from urinary excretion, protein binding, platelet conjugation, etc.) are assumed to have a role in the MCR of NE. Whether that metabolic process can be activated by an increased level of plasma NE is not clear. In humans, plasma NE concentration and MCR were reported to be negatively correlated (17), although this was not confirmed (37), whereas in the rat, NE clearance tended to increase during infusion of a pressor dose of exogenous NE (10). Our working hypothesis was to consider that the capacity of the rat organism to clear NE could increase as long as the hemodynamic state remained unaltered. Three series of data are reported. Figure 1 shows a lack of significant correlation between plasma NE and NE-MCR in 31 FM and MS rats; it suggests that the whole capacity of Wistar rats to clear NE is independent of plasma NE level, at least in resting conditions. A second series of experiments was carried out in rats given exogenous NE infusion to set up plasma NE concentrations at various levels. In acute conditions (Table 3), the NE-MCR remained unchanged when plasma NE was increased up to ~2,500 pg/ml, concentrations measured in the rat during swimming exercise (31). This is in agreement with previously reported work. Careful examination of the data of Eisenhofer et al. (10) reveals no apparent change in either DHPG or MHPG concentrations in rats given exogenous NE infusion when plasma NE remained <2,000-3,000 pg/ml. A step further, we suspected that activation of the NE-MCR could be time dependent, and exogenous NE was chronically infused for 7 days, with no effect on NE-MCR (Table 4). Thus the whole capacity of the rat organism to clear NE is not increased when plasma NE is experimentally increased as long as the hemodynamic state is not altered. Nonetheless, we previously reported a statistically significant correlation between arterial concentration and urinary excretion of NE in the dog (5). Whether the isotope dilution technique is not sensitive enough to identify small variations of NE-MCR should be considered. In the third series of experiments, plasma NE was increased in such a way that MAP was increased (Table 3), and NE-MCR was shown to be significantly reduced. A first interpretation is to consider that hemodynamic changes account for the decreased NE-MCR: either a decrease of cardiac output (12), a reduction of peripheral blood flow (14), or both. However, it deserves further comment. When plasma NE is experimentally increased, especially when it counterbalances the NE synaptic-plasma concentration gradient, NE stimulates both postsynaptic mechanisms, leading to an increase of MAP and presynaptic neuronal uptake, leading to a greater activation of the deamination pathway to form DHPG. Table 1 shows a significant increase of DHPG concentration in rats given exogenous NE at 3,000 ng · kg-1 · min-1, with a significant increase of [3H]DHPG, and a 36.7% increase (P < 0.06) of alumina-extractable tritium. Thus the neuronal component of the NE-MCR, at least, was stimulated, although its effect on the whole capacity of rat's organism to clear NE, as measured by the isotope dilution technique, was apparently overwhelmed by NE-induced hemodynamic effects.

Epinephrine Hypothesis Revisited

Several years ago, Majewski (24) hypothesized that Epi incorporated into sympathetic neuronal stores could act to facilitate sympathetic neurotransmission via activation of presynaptic beta 2-adrenoceptors. Although several reports in humans demonstrated that experimentally increased plasma Epi concentration tends to increase the release of NE (3, 23, 26), it was not always confirmed (16). Data obtained in animals are even less clear (1, 29, 30, 33). The epinephrine hypothesis was revisited in FM rats assumed to be in resting conditions and with special attention being paid to the level of circulating Epi. Our results (Table 5) show that NE-SOR remained unchanged when plasma Epi concentration was increased to ~3,200 pg/ml. When plasma Epi reached a very high level of concentration (~20,000 pg/ml), two effects were observed. 1) The NE-SOR was significantly increased, suggesting that the epinephrine hypothesis is concentration dependent in the rat, revealed by the upper range of plasma Epi levels only. From a physiological point of view, one may wonder whether the increased MAP induced by high Epi concentrations represents a direct activation of adrenergic receptors leading to vasoconstriction of various vascular beds (20), an indirect activation of NE release through presynaptic beta 2-receptors, or both. 2) High Epi concentration triggered an increase of NE-MCR, accounting for the nonsignificant increase of plasma NE concentration. That effect cannot be explained at the moment; nonetheless, it leads to extending one's view on the regulation of NE-MCR. So far, hemodynamic modifications (cardiac output or vascular blood flows) have been thought to have a predominant role in the regulation of the NE-MCR (14). Accordingly, the increase of MAP induced by the high infusion rate of exogenous NE could have accounted for the decrease of NE-MCR (Table 3). However, the comparable increase in MAP induced by the high infusion rate of exogenous Epi is shown to be associated with an increase of NE-MCR (Table 5). Thus the hemodynamic factors involved in the regulation of NE-MCR in the rat can be overwhelmed by mechanisms likely to be of beta -adrenergic type. Thus an alpha -/beta -adrenergic equilibrium seems to have a role in the regulation of NE clearance in the rat, as already suggested in human beings (6).

The epinephrine hypothesis was further investigated in the lower range of plasma Epi concentrations not having, by themselves, an effect on NE-SOR (Table 5). The presynaptic membrane presents, apparently, structural features sensitive to beta 2-adrenergic stimuli, leading to a facilitation of the release of NE and alpha 2-adrenoceptors that have a counterregulatory role, a counterregulation that appears to be predominant in the rat (29, 30, 32, 34). We have speculated on the existence of a delicate presynaptic regulation of the release of NE on the basis of an alpha -/beta -adrenergic equilibrium. Such an equilibrium could be species dependent; the alpha -component could be much more predominant in the rat, accounting for a much higher Epi concentration (Table 5) than in humans (22), to trigger an increase of NE release. If so, it could be swung toward a predominant beta -component by architectural reordering of the presynaptic membrane with an alpha 2-adrenoreceptor inhibitor, yohimbine, for example. Our results support such a working hypothesis. A 500 ng · kg-1 · min-1 infusion rate of Epi, which had no effect by itself on the NE-SOR (Table 6), increased it when infused in association with yohimbine (Fig. 2). Thus, besides a faster NE kinetics, the rat presents a quantitatively different regulation of the presynaptic component of the NE release.


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Fig. 2.   Epinephrine (Epi) increased the NE spillover rate (NE-SOR) when infused in the presence of yohimbine. Intravenous infusions of either Epi or yohimbine alone are shown to have no significant effect on NE-SOR compared with data obtained in 31 control rats. Horizontal lines represent mean values for each group.

In summary, the present work was carried out to study the relationship between plasma catecholamine and the NE kinetics in the rat. 1) In freely moving rats, likely to be at rest, mean NE-SOR and NE-MCR were both much higher values than were previously reported in humans, in agreement with a strong sympathetic activity in the rodent. 2) Several lines of results tend to demonstrate that the NE-MCR is independent of plasma NE concentration as long as the hemodynamic state is not altered. When MAP is increased during infusion of exogenous NE, NE-MCR was decreased, suggesting a predominant role for hemodynamic factors in the control of NE-MCR. 3) Nonetheless, besides the hemodynamic, another mechanism should be considered. The NE-MCR appears also under the control of an alpha -/beta -adrenergic equilibrium, because for a comparable hypertensive effect, NE-MCR was decreased during NE and increased during Epi infusion. 4) The epinephrine hypothesis was revisited and was shown to be concentration dependent, revealed only at very high plasma Epi concentration. 5) A second epinephrine hypothesis is proposed: Epi can reveal a stimulating effect on NE-SOR, even at low plasma concentration, when presynaptic alpha 2-adrenoceptors are partially inhibited by yohimbine.


    ACKNOWLEDGEMENTS

We thank Dr. Graeme Eisenhofer (Clinical Neurochemistry Laboratory, National Institutes of Health) for professional support and hospitality. In addition, we would like to thank all the staff of the Clinical Neurochemistry Laboratory for kind and efficient support.


    FOOTNOTES

This work was supported by a grant-in-aid from the Centre National d'Etudes Spatiales.

Address for reprint requests and other correspondence: J.-L. Cuche, 104 rue de la République, 50 600 Saint Hilaire du Harcouët, France (E-mail: Jean-Louis.Cuche{at}wanadoo.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 March 2001; accepted in final form 29 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 281(4):E726-E735
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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