Much evidence has shown that neurally localized peptides such as opioid peptides may be involved in the regulation of blood pressure and other cardiovascular functions (1,2). Recently, important changes have been described in the opioid system in hypertension. It was reported that the content of adrenal gland leucine-enkephalin was lower in spontaneously hypertensive rats-stroke prone strain (SHR-SP) than that in normotensive controls, while β-endorphin in the neuro-intermediate lobe of the pituitary was higher in SHR-SP (3). It was also shown that naloxone, an opioid receptor antagonist, reversed the blood pressure reduction induced by clonidine in hypertensive rats (4). In a clinical study, Kraft et al. observed that plasma β-endorphin concentration was lower in patients with essential hypertension both at rest and during exercise (5). These previous findings strongly suggest that altered opioid activities could be a factor in the pathogenesis of hypertension. However, functional significance of central β-endorphin as regards hypertension is not fully understood. We now describe the effects of β-endorphin on the electrically evoked release of [3H]norepinephrine (NE) from rat medulla oblongata. In addition, in order to elucidate a possible role of β-endorphin in the regulation of adrenergic transmission in hypertension, we examined whether the effect of β-endorphin on NE release might be altered in the medulla oblongata of SHR.
MATERIALS AND METHODS
Male Sprague-Dawley rats (bodyweight, 200-250 g; Taconic Farm, Germantown, NY, U.S.A.) were used for the investigation of the effects of β-endorphin in rat medulla oblongata. Male SHR (9-10 weeks of age; systolic blood pressure measured by the tail-cuff method, 177.3 ± 3.5 mmHg; n = 6; Taconic Farm) was studied and compared with age-matched Wistar-Kyoto (WKY) rats (systolic blood pressure, 114.7 ± 6.2 mmHg; n = 6; Taconic Farm). The rats were decapitated and the medulla oblongata was rapidly dissected on ice according to the method described previously (6-8). The tissues were sliced at 0.3 mm thickness using a Brinkman tissue chopper (Brinkman, Westbury, NY, U.S.A.). The sliced tissues were washed three times with 2 ml Krebs-Ringer bicarbonate buffer (NaCl, 118.0 mmol/l; KCl, 4.80 mmol/l; CaCl2, 1.20 mmol/l; KH2PO4, 1.15 mmol/l; MgSO4, 1.20 mmol/l; NaHCO3, 25.0 mmol/l; glucose, 11.1 mmol/l; ascorbic acid, 0.11 mmol/l; and disodium ethylenediamine tetra-acetic acid, 0.04 mmol/l; saturated with a 95% O2 and 5% CO2 mixture at 37°C; pH 7.4). The slices were incubated with 3 ml fresh buffer containing 0.1 μmol/l [3H]NE (specific activity, 1.51 × 1012 Bq/mmol; New England Nuclear Research Products, Boston, MA, U.S.A.) for 20 min at 37°C. After rinsing with fresh buffer, the slices (5-7 mg) were transferred to a superfusion chamber (volume, 200 μl) jacketed with 37°C water and were suspended between two platinum electrodes (25 mm apart, 2.0 mm long). The slices were superfused at a rate of 0.7 ml/min with Krebs-Ringer bicarbonate buffer. Sample collection began after 60 min of superfusion when basal outflow of tritium had stabilized to a constant level. Samples of superfusates were collected at 7-min intervals until the end of the experiment (at 130 min). The first period of electrical stimulation (S1) was applied at 67 min, and the second period of electrical stimulation (S2) was applied at 116 min after the beginning of superfusion. Electrical stimulation was delivered from a Grass stimulator (Model S4K; Grass, Quincy, MA, U.S.A.) and consisted of trains of unipolar, rectangular pulses (1 Hz, 20 mA, and 2 ms duration for 2 min). At the end of the experiment, the slices were sonicated for 20 s. The radioactivity in collected samples and tissue slices was determined by liquid scintillation counting (Packard Tricarb Liquid Scintillation Spectrometer 3255; Packard, Sterling, VA, U.S.A.). The amount of radioactivity in each sample was calculated by dividing the total tritium collected in each sample by the total tritium present in the tissue at the time of sample collection (percent fractional release). The basal outflow during the two pre-stimulation periods was calculated from the tritium in the two 7-min samples just before S1 and S2. The stimulation-evoked release was calculated by subtracting the basal outflow during the 7-min pre-stimulation period from the values in samples collected after 2 and 5 min of electrical stimulation. The effects of β-endorphin were evaluated only in S2, with S1 serving as an internal control. Superfusion with β-endorphin was initiated 14 min before S2 and continued until the end of the experiment. The effects of β-endorphin on the stimulation-evoked release of [3H]NE were determined by comparing the S2/S1 ratios obtained in control slices with the values for slices treated with β-endorphin.
β-Endorphin was purchased from the Protein Institute (Osaka, Japan). All other drugs were standard laboratory reagents of analytical grade.
The data are presented as means ± SEM. The differences between the means of drug treatment and their corresponding controls were tested with a one-way analysis of variance (ANOVA). The means of various groups were compared in statistical analyses using the Mann-Whitney U test. The differences between SHR and WKY rats were analyzed with the two-way ANOVA. p < 0.05 was accepted as the level of significance.
Effects of β-endorphin on the stimulation-evoked release of [3H]NE in medulla oblongata of Sprague-Dawley rats
In the control experiments, the stimulation-evoked [3H]NE release from medulla oblongata in S1 and S2 did not differ significantly (S2/S1 ratio, 0.937 ± 0.043; n = 6). Figure 1 shows the effects of β-endorphin on the stimulation-evoked [3H]NE release from medulla oblongata of Sprague-Dawley rats. β-Endorphin (1 × 10−7 to 1 × 10−6 mol/l) reduced the stimulation-evoked [3H]NE release in a dose-related fashion.
Effects of β-endorphin on the stimulation-evoked [3H]NE release from medulla oblongata of SHR and WKY rats
The inhibitory effect of β-endorphin on the stimulation-evoked [3H]NE release was significantly more attenuated in SHR than in WKY rats [S2/S1 ratio: control: SHR, 0.913 ± 0.022 (n = 6); WKY rats, 0.992 ± 0.025 (n = 6); in the presence of 1 × 10−7 mol/l β-endorphin: SHR, 0.819 ± 0.023 (n = 6); WKY rats, 0.685 ± 0.038 (n = 6); p < 0.05].
This study demonstrated that β-endorphin decreased the stimulation-evoked NE release from rat medulla oblongata. This finding, coupled with previous reports showing that opioid peptides inhibited NE release from rat mesenteric arteries and in other tissues (9-11), suggests that they may act as sympatho-depressors both in the central and peripheral nervous systems. The opioid system is complex because of the multiple species of opioid peptides and multiple forms of receptor subtypes (12,13). Pfeiffer et al. reported that mu-receptors might mediate opioid cardiovascular effects at rat anterior hypothalamic region (14), whereas opioid peptides might inhibit neurotransmitter release in the rabbit ear artery by acting on delta- and kappa-receptors (15). Additional studies should be conducted more thoroughly to assess the mechanisms underlying the inhibitory effects of β-endorphin on NE release in the central nervous system.
In the present study, the inhibitory effect of β-endorphin on the stimulation-evoked NE release was smaller in the medulla oblongata of SHR than in the medulla oblongata of WKY rats. Gaida et al. showed that β-endorphin in the neuro-intermediate lobe of the pituitary was significantly higher in SHR-SP than in the normotensive controls (3). Zamir et al. examined the opiate receptor-binding in the brain and reported that hypertensive rats were accompanied by a low specific [3H]naloxone-binding in the dorsal horn of the spinal cord and in the hippocampus as compared with the normotensive controls (16). It might be possible that the less inhibitory effect of β-endorphin in SHR could be due to a downregulation of the opiate receptors. On the other hand, Bucher et al. showed that the plasma concentration of β-endorphin did not differ in SHR and WKY rats (17). Further studies are necessary to determine the alterations in the opioidergic activity and their contribution to the pathophysiology of SHR.
In the clinical studies, it has also been implicated that the opioid system might be involved in the blood pressure control in essential hypertension (5,18-21). Kraft et al. (18) have reported that plasma concentration of β-endorphin was lower in patients with essential hypertension than in normotensive controls. In contrast, they noted that the plasma norepinephrine concentration was higher in hypertensives, and proposed that reduced opioidergic activity might induce the increased sympathetic nervous activity in hypertension, although the precise relationship between the opioid system and sympathetic tone in hypertension is still uncertain.
In summary, the present study showed that β-endorphin inhibited the stimulation-evoked NE release in a dose-dependent manner in rat medulla oblongata. In the medulla oblongata of SHR, the inhibitory effect of β-endorphin on the stimulation-evoked NE release was significantly smaller than in the medulla oblongata of WKY rats. These results suggest that an insufficient modulation of NE release by β-endorphin might have a crucial role in the regulation of sympathetic nerve activity in hypertension.
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The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.