Introduction

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Prostanoids modulate the depolarization-evoked norepinephrine release from postganglionic sympathetic neurons in many tissues, including blood vessels (for reviews, see Güllner, 1983; Malik and Sehic, 1990; Rand et al., 1990). Prostaglandin (PG)E₂ reduced the stimulation-evoked norepinephrine release from blood vessels in rat, rabbit, and humans (Molderings et al., 1992, 1994; Jensen and Nedergaard, 1997). PGD₂ enhanced the stimulation-evoked norepinephrine release in vessels from dog (Nakajima and Toda, 1984) and humans (Molderings et al., 1994).

The actions of prostanoids are mediated via specific receptors that have been classified on the basis of their sensitivity to the naturally occurring eicosanoids, PGD₂, PGE₂, PGF₂, prostacyclin, and thromboxane A₂ and named DP, EP, FP, IP, and TP, respectively (Kennedy et al., 1982). A lack of a variety of subtype-selective antagonists and insufficient agonist specificity for the subtypes have so far hampered an unequivocal receptor classification (Fuder and Muscholl, 1995). However, with the use of synthetic prostaglandin analogs, four subtypes of PGE₂ receptors have been characterized and named EP₁, EP₂, EP₃, and EP₄ (Coleman et al., 1994). The aims of this study were to determine whether prejunctional EP, FP, IP, and DP receptors are present on postganglionic sympathetic neurons in rabbit aorta and to identify the subtype (EP₁, EP₂, EP₃, EP₄) of the putative EP receptor. A preliminary account of this investigation was given at the Ninth Meeting on Adrenergic Mechanisms (Jensen and Nedergaard, 1996a) and the Eight International Catecholamine Symposium (Jensen and Nedergaard, 1996b).

	Materials and Methods

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Drugs. AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) was obtained from Glaxo (Stevenage, UK). Butaprost was obtained from Bayer (West Sussex, UK). Cicaprost, iloprost, and sulprostone were obtained from Schering AG (Berlin, Germany). Corticosterone, PGD₂, PGE₁, PGE₂, PGF₂, and 9,11-dideoxy-11, 9-epoxymethano-PGF₂ (U46619) were obtained from Sigma Chemical Co. (St. Louis, MO). ()-Cocaine hydrochloride and fluprostenol were obtained from BIOMOL (Plymouth Meeting, PA). ()-7-[³H](N)-Norepinephrine hydrochloride (specific activity, 10.4-10.5 Ci/mmol) was obtained from New England Nuclear Research Products (Boston, MA). Misoprostol was obtained from G.D. Searle (Skokie, IL). Rauwolscine hydrochloride was obtained from Carl Roth (Karlsruhe, Germany).

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PSS. The composition of the PSS was 1.442 × 10¹ M Na⁺, 4.9 × 10³ M K⁺, 1.2 × 10³ M Ca²⁺, 1.267 × 10¹ M Cl, 2.50 × 10² M HCO₃, 1.2 × 10³ M SO₄², 1.2 × 10³ M H₂PO₄², and 1.11 × 10² M D-(+)-glucose,. The solution also contained calcium disodium EDTA (CaNa₂EDTA; 3 × 10⁵ M) and L-(+)-ascorbic acid (10⁴ M). The solution was maintained at 37.0°C and equilibrated before and during the experiment with O₂ containing 5% (v/v) CO₂ in the tissue bath (pH 7.4).

Release of [³H]Norepinephrine. A modification of the method described by Nedergaard (1980) was used. Albino rabbits (2-3 kg) of either sex were sacrificed by cervical dislocation and exsanguinated. Rings of thoracic aorta were prepared and incubated in 6-ml test tubes containing PSS (2.0 ml). After an equilibration period (20 min), the rings were incubated with [³H]norepinephrine (10⁷ M) for 30 min. They were washed three times for 5 min each with PSS by transferring them to new test tubes. The rings were then mounted in isolated tissue baths, which were automatically emptied and refilled with PSS (2.0 ml) every 5 min for the remainder of the experiment. The fractions (5-min) were collected from 135 min after the outset of washout directly in a counting vial by means of a fraction collector. At the end of each experiment, each ring was treated with Solvable (DuPont de Nemours, Dreieich, Germany) for 16 h at room temperature (18-22°C). The ³H content in each 5-min fraction and tissue was determined by a liquid scintillation spectrometer (Tri-Carb 2100TR; Packard Instrument Company, Meriden, CT). The spectrometer automatically corrected for quenching and determined the counting efficiency by means of an external standard.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Diagrammatic illustration of the protocol used to determine passive 3H outflow and stimulation-evoked 3H overflow in rabbit aorta preloaded with [3H]norepinephrine. Ordinate, 3H outflow/3H overflow expressed as norepinephrine (pmol/g). Abscissa, time (min) after the onset of washout with PSS. Each fraction (F1-F7) is numbered in the columns. A, stimulation (S1-S7)-evoked 3H overflow. The horizontal bars indicate the electrical field stimulation period. S3 was used as an initial control value (100%). B, passive (P3-P7) 3H outflow refers to F3-F5. Stimulation (S3-S7)-evoked 3H overflow (gray area) was calculated by subtracting the estimated passive 3H outflow in F3-F5 from the total tritium in F3-F5. The latter was calculated by using F1-F2 and F6 (see Materials and Methods).

Statistical Analysis. Data are expressed as mean ± S.E. Log concentration-response curves were plotted. pEC₅₀ values, defined as the negative logarithm of the molar concentration (log EC₅₀) required to produce 50% of maximal inhibition of the stimulation (S₃)-evoked ³H overflow, were calculated by linear regression analysis of the results in the 20 to 80% response interval. Differences between mean values were evaluated using an unpaired t test. In the case of unequal variance between the mean values compared (evaluated with a variance ratio test), an unpaired t test for unequal variance was used. When multiple comparisons were made, the t test was combined with a Bonferroni correction. Significance was accepted at the .05 level of probability. Analysis of data was performed with Excel 97 (Microsoft, Redmond, WA).

	Results

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Passive and Stimulation-Evoked ³H Overflow. In rings of aorta preincubated with [³H]norepinephrine, passive (P₄-P₈) ³H outflow under control conditions decreased with time (Table 1). However, when the ³H outflow was corrected for tissue ³H content, the ³H outflow remained essentially unaltered with time (Table 1). Basal ³H outflow was not altered by the prostanoids and other drugs at the concentrations used (results not shown).

Effect of Prostanoids. PGE₁, PGE₂, PGD₂, PGF₂, sulprostone (EP₁/EP₃ receptor agonist), misoprostol (EP₂/EP₃ receptor agonist), and iloprost (EP₁/IP receptor agonist) concentration-dependently reduced the stimulation-evoked ³H overflow (Fig. 2). The potencies (pEC₅₀ values) and maximum inhibitory effects (E_max) are shown in Table 2. Cicaprost (IP receptor agonist) inhibited the stimulation-evoked ³H overflow only at a concentration of >3 × 10⁵ M, whereas fluprostenol (FP receptor agonist) had no effect (Fig. 3). U46619 (thromboxane A₂ analog) slightly reduced the ³H overflow (Fig. 4). Butaprost (EP₂ receptor agonist) had no effect (Fig. 4). AH6809 (EP₁ receptor antagonist; 10⁵ M) did not alter the inhibitory effect of PGE₂ and sulprostone (Fig. 5). AH6809 (10⁵ M) alone had no effect on the stimulation-evoked ³H overflow (results not shown; n = 6).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of prostanoids on the stimulation-evoked 3H overflow from rabbit aorta preincubated with [3H]norepinephrine. Ordinate, mean stimulation-evoked 3H overflow expressed as a percentage of S3 (100%). Abscissa, concentration (log M) of prostanoids. A: , sulprostone; , PGE2; , misoprostol; , PGE1. B: , PGF2; , iloprost; , PGD2. Vertical lines represent ±S.E.M. (n = 5-8). A, all values below 85% were significantly different from the controls (at least p < .01). B, *p < .05; ***p < .001.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of fluprostenol and cicaprost on the stimulation-evoked 3H overflow from rabbit aorta preincubated with [3H]norepinephrine. Ordinate, mean stimulation-evoked 3H overflow expressed as percentages of S3 (100%). Abscissa, concentration (log M) of prostanoids. , fluprostenol; , cicaprost. Vertical lines represent ±S.E.M. (n = 6; **p < .01).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effect of butaprost and U46619 on the stimulation-evoked 3H overflow from rabbit aorta preincubated with [3H]norepinephrine. Ordinate, mean stimulation-evoked 3H overflow expressed as percentages of S3 (100%). Abscissa, concentration (log M) of prostanoids. A: , U46619. B: , butaprost. Vertical lines represent ±S.E.M. (n = 5; **p < .01).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of AH6809 on the action of PGE2 and sulprostone on the stimulation-evoked 3H overflow from rabbit aorta preincubated with [3H]norepinephrine. Ordinate, mean stimulation-evoked 3H overflow expressed as percentages of S3 (100%). Abscissa, concentration (log M) of drugs. A: , PGE2 alone; , PGE2 in the presence of AH6809 (105 M). B: , sulprostone alone; , sulprostone in the presence of AH6809 (105 M). Vertical lines represent ±S.E.M. (n = 6).

	Discussion

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PGE₁, PGE₂, and several other prostanoids inhibited the stimulation-evoked [³H]norepinephrine release from several species (for reviews, see Güllner, 1983; Malik and Sehic, 1990; Rand et al., 1990), including the rabbit aorta (Jensen and Nedergaard, 1997). In the latter tissue, there was an interaction between prejunctional inhibitory ₂-adrenoceptors and EP receptors (Jensen and Nedergaard, 1997). It is therefore optimal to block prejunctional ₂-adrenoceptors when the interaction of prostanoids with their prejunctional EP receptors is examined. In the present study, we therefore blocked prejunctional inhibitory ₂-adrenoceptors with the rather selective ₂-adrenoceptor antagonist rauwolscine. Although neither cocaine nor corticosterone modified the inhibitory effect of PGE₂ (Jensen and Nedergaard, 1997), these inhibitors were used in the present experiments to rule out the theoretical possibility that the effect of prostanoids on the [³H]norepinephrine release was due to an action on the neuronal and extraneuronal uptake mechanisms.

The results suggest that the EP receptor mediating the PGE₁- and PGE₂-evoked reduction in stimulation-evoked [³H]norepinephrine release in rabbit aorta belongs to the EP₃ receptor subtype. This view is based on the following findings: 1) the effect of PGE₁ and PGE₂ was mimicked by the EP₁/EP₃ agonist sulprostone and the EP₂/EP₃ agonist misoprostol (Fig. 2); 2) the rank order (pEC₅₀) were sulprostone = PGE₂ > misoprostol > PGE₁ iloprost > PGF₂ > PGD₂ > U46619 (Table 2) (this rank order of potency is in accordance with the rank order for the prostanoids at the EP₃ receptor; Kennedy et al., 1982); 3) the selective EP₂ receptor agonist butaprost had no effect (Fig. 3) (this excludes the presence of EP₂ receptors); and 4) the selective EP₁ receptor antagonist AH6809 (McKenniff et al., 1988) did not modify the inhibitory effect of PGE₂ or sulprostone (Fig. 5). This rules out the possibility that PGE₂ and sulprostone acted via EP₁ receptors. Our conclusion that PGE₁ and PGE₂ act on prejunctional inhibitory EP₃ receptors is consistent with previous reports for other blood vessels (Molderings et al., 1992, 1994).

The effects of PGF₂ and iloprost were not mimicked by fluprostenol (FP receptor agonist) and cicaprost (IP receptor agonist), respectively. This rules out the possible existence of prejunctional FP or IP receptors.

PGD₂ was a weak partial agonist (pEC₅₀ < 5.0; Table 2) that caused a reduction in the stimulation-evoked ³H overflow (Fig. 2). In contrast, PGD₂ either enhanced the stimulation-evoked [³H]norepinephrine release in canine mesenteric artery (Nakajima and Toda, 1984) and human saphenous vein (Molderings et al., 1994) or had no effect in rat vena cava (Molderings et al., 1994). The discrepancy between these findings and our results could be related to species differences. It is unlikely that the discrepancy is due to the inhibition of prejunctional ₂-adrenoceptors in the present study because rauwolscine also was used in the investigation with rat vena cava and human saphenous vein (Molderings et al., 1992, 1994).

In summary, the results indicate that the postganglionic sympathetic neurons in rabbit aorta are endowed with prejunctional inhibitory EP₃ receptors. FP and IP receptors are not present, and the possible presence of inhibitory DP receptors requires further study.