Effects of morphine in rats treated chronically with U-50,488 H, a kappa opioid receptor agonist

The pharmacological effects of morphine, namely analgesic, hyperthermic and cataleptic effects, were assessed in rats rendered tolerant to U-50,488H, a kappa opioid receptor agonist. Male Sprague-Dawley rats were injected intraperitoneally with U-50,488H (25 mg/kg) twice a day for four days. The rats which served as controls were injected similarly with the vehicle. Chronic administration of U-50,488H resulted in the development of tolerance to its analgesic and hypothermic effects, but not to its diuretic effect. The development of tolerance to the pharmacological effects of U-50,488H was associated with decreased binding of [3H]ethylketocyclazocine [( 3H]EKC) to brain and spinal cord membranes. The decreased binding of [3H]EKC in U-50,488H-treated rats was due to changes in the Bmax value; the Kd values remained unaltered. Intraperitoneal administration of morphine (8 mg/kg) to rats produced analgesia (as determined by the tail-flick test) and hyperthermia. A dose of 50 mg/kg of morphine produced cataleptic response. The intensity of analgesic, hyperthermic and cataleptic effects of morphine were unaltered in rats tolerant to U-50,488H. The development of tolerance to analgesic and hypothermic effects of U-50,488H were associated with down-regulation of brain and spinal cord kappa opioid receptors. Finally, U-50,488H does not confer cross-tolerance to morphine, a predominantly mu opioid receptor agonist.     

Down-regulation of central receptors for thyrotropin-releasing hormone in kappa opiate agonist-induced abstinence in the rat.

The effect of U-50,488H, a selective kappa opiate agonist, on tolerance-dependence and abstinence on the TRH receptors of the spinal cord and discrete regions of the brain of male Sprague-Dawley rats was determined. Rats were injected intraperitoneally twice daily with 25 mg/kg of U-50,488H for 4 days. Rats serving as controls were injected with the vehicle. On day 5, rats which were labeled as tolerant to U-50,488H were injected with U-50,488H (25 mg/kg) and sacrificed 1 hr later, whereas those labeled as abstinent were sacrificed without any injection. The above procedure has been previously shown to produce a high degree of tolerance to the analgesic and hypothermic effects of U-50,488H. The spinal cord and regions of the brain (hippocampus, cortex, midbrain, hypothalamus, corpus striatum, pons and medulla, and amygdala) were isolated for binding studies. The ligand [3H]MeTRH was used for TRH receptors. The binding constants, Bmax and Kd values, of [3H]MeTRH to bind to membranes prepared from various regions of the brain and spinal cord of rats tolerant-dependent on U-50,488H were unaffected. However, in rats abstinent to U-50,488H, the binding of [3H]MeTRH to membranes of the hypothalamus, and pons and medulla, was decreased. The decreased binding of [3H]MeTRH to hypothalamic membranes was due to changes in Bmax value, while in pons and medulla it was due to an increase in the Kd value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding characteristics of [3H]SCH 23390 in spinal cord and discrete brain regions of kappa-opiate tolerant-dependent and abstinent rats.

The effects of chronic administration of U-50,488H, a highly selective kappa-opiate receptor agonist, and its subsequent withdrawal were determined. The binding characteristics of [3H]SCH 23390 to dopamine D1 receptors were investigated on membranes of rat spinal cord and in discrete brain regions. Male Sprague-Dawley rats received U-50,488H (25 mg/kg) intraperitoneally twice a day for 4 days. Animals serving as controls received only vehicle. Two different treatment schedules were used. In one, rats were injected with U-40,588H on day 5 and were sacrificed 1 h later (tolerant-dependent rats). In another, the rats were sacrificed 16 h after the last injection of U-50,488H (abstinent rats). Chronic administration of U-50,488H resulted in the development of tolerance to its analgesic effect, as demonstrated by the decreased response to a challenge dose of U-50,488H compared to vehicle-injected rats. The binding characteristics (Bmax and Kd values) of [3H]SCH 23390 were unaffected in spinal cord and other regions of brain of tolerant-dependent rats. In U-50,488H-abstinent rats, Bmax values of [3H]SCH 23390 were increased in hypothalamic and striatal membranes, but Kd values were unaffected. These results suggest that, in U-50,488H-abstinent rats, dopamine D1 receptors are up-regulated in hypothalamus and striatum.     

Effect of repeated administration of U-50,488H, a kappa opioid receptor agonist, on central 5-HT1 and 5-HT2 receptors in the rat

The effect of repeated administration of U-50,488H, a kappa-opioid receptor agonist, on the development of tolerance to its analgesic effect and on the 5-HT1 and 5-HT2 receptors in the cerebral cortex and spinal cord of the rat were determined. Male Sprague-Dawley rats were injected twice daily with U-50,488H, (25 mg/kg, i.p.) or its vehicle for 4 days. The assessment of tolerance to the analgesic effect and biochemical determinations were made on day 5. Repeated administration of U-50,488H resulted in the development of tolerance to its analgesic effect. The 5-HT1 and 5-HT2 receptors were characterized by using 3H-5-hydroxytryptamine (3H-5-HT) and 3H-spiperone as the ligands and unlabeled 5-hydroxytryptamine (5-HT) and ketanserin, respectively, to determine the nonspecific binding. In the spinal cord 3H-5-HT bound to 5-HT1 receptors at a single high-affinity site with a Bmax value of 41.3 +/- 9.6 fmol/mg protein and a Kd value of 22.6 +/- 7.0 nmol/l. 3H-Spiperone bound to 5-HT2 receptors in the spinal cord with a Bmax value of 16.1 +/- 3.8 fmol/mg protein and a Kd value of 0.36 +/- 0.15 nmol/l. Repeated administration of U-50,488H to rats did not affect spinal cord 5-HT1 and 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of endothelin receptors in the central nervous system of spontaneously hypertensive rats.

The binding of [125I]sarafotoxin 6b (SRT 6b) and [125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared, from regions of the brain and spinal cord of 8 week-old, spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. Spontaneously hypertensive rats had significantly higher blood pressure as compared to WKY rats. Heart rate was similar in SHR and WKY rats. [125I]SRT 6b and [125I]ET-1 bound to the membranes of the cerebral cortex, hypothalamus, ventrolateral medulla, dorsomedial medulla and spinal cord at a single, high affinity site. The Kd and Bmax values of the binding of [125I]SRT 6b were found to be similar to binding of [125I]ET-1 in all the regions. The concentration-dependent inhibition of binding of [125I]ET-1 by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 2.66 +/- 0.59 nM and a Ki value of 2.35 +/- 0.52 nM in WKY rats and an IC50 value of 2.82 +/- 0.76 nM and a Ki value of 2.43 +/- 0.70 nM in SHR rats. On the other hand, the concentration-dependent inhibition of the binding of [125I]SRT 6b by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 10.31 +/- 1.82 pM and a Ki value of 8.44 +/- 1.41 pM in WKY rats, while SHR rats showed an IC50 value of 10.28 +/- 1.94 pM and a Ki value of 8.89 +/- 2.00 pM. The binding of [125I]SRT 6b and [125I]ET-1 in the cerebral cortex, dorsomedial medulla and spinal cord membranes was found to be similar in SHR and WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS) [corrected]     

Effect of age and of hypertrophy on cardiac Ca2+ antagonist binding sites

We explored the effect of age and of hypertrophy on Ca2+ antagonist binding site density (Bmax), affinity (Kd), and selectivity in cardiac membranes harvested from the hearts of young adult (9-week-old) and older (25-week-old) Sprague Dawley (SD) rats, Wistar Kyoto rats (WKY), and spontaneously hypertensive rats (SHR). Radioligand binding studies with (+)[3H]PN200-110 failed to show a significant difference between the Bmax obtained for the cardiac membranes of 9-week-old SD, WKY, or SHR. Similarly, at 25 weeks, the Bmax values were the same for each group, but in each group the Bmax tended to increase with age. The Kd and selectivity were unchanged. For (-)[3H]D888 binding, the Kd values changed with age, but there was no hypertension or hypertrophy-linked increase in Bmax. In the SD and SHR series, but not in the WKY, there was a tendency for the Bmax to increase with age. We interpreted these results to mean that age may contribute to the different Kd and Bmax values described for cardiac membranes from 25-week-old WKY and SHR.     

Abstinence from U-50,488H, a kappa-opiate receptor agonist, decreases the binding of [3H]DPAT to 5-HT1A receptors in the hypothalamus of the rat.

The effect of trans-( ± )-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzene-acetamide methane sulfonate (U-50,488H), a κ-opiate agonist-induced tolerance and abstinence on 5-HT_1A receptors was determined in regions of the brain and spinal cord of the rat. The administration of U-50,488H (25 mg/kg, i.p., twice daily) to male Sprague-Dawley rats for 4 days resulted in the development of almost complete tolerance to its analgesic and hypothermic effects. On day 5, the animals were divided into tolerant and abstinent groups and sacrificed. The brain and spinal cord were excised from all groups of rats and the brain was dissected into 6 regions, namely, amygdala, hypothalamus, striatum, midbrain, pons + medulla and cortex. The 5-HT_1A receptors were characterized by using [³H]8-hydroxy-2-(di-n-propylamino)tetralin ([³H]DPAT) as the ligand. The binding constants (B_max and K_dvalues) of [³H]DPAT in regions of the brain and spinal cord of rats tolerant to U-50,488H and vehicle did not differ. However, the B_max value of [³H]DPAT in the hypothalamus of U-50,488H-abstinent rats was decreased but the K_d value did not change. In the other regions of the brain and spinal cord of U-50,488H-abstinent rats, the B_max and K_d values of [³H]DPAT were unaffected. Subcutaneous administration of DPAT produced hypothermic response in vehicle- and U-50,488H-treated rats. The intensity of this effect was more marked in U-50,488H-abstinent group. It is concluded that 5-HT_1A receptors are down-regulated in the hypothalamus of U-50,488H-abstinent rats but the hypothermic response to 5-HT_1A agonist is intensified.     

Characteristics of endothelin binding sites in the spinal cord of spontaneously hypertensive rats.

The binding of [125I]sarafotoxin 6b (SRT 6b) and [125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and spinal cord of 8-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) was determined. SHR had significantly higher blood pressure as compared to WKY. Heart rate was similar in SHR and WKY. [125I]SRT 6b and [125I] ET-1 bound to the membranes of cerebral cortex and spinal cord at a single, high affinity site. The binding of [125I]SRT 6b and [125I]ET-1 in the cerebral cortex and spinal cord membranes was found to be similar in SHR and WKY. Concentration-dependent inhibition of [125I]ET-1 binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 was performed. The Ki values were found to be 2.35 +/- 0.68, 0.29 +/- 0.08 and 24.10 +/- 5.90 nM for ET-1, ET-2 and ET-3, respectively in WKY. The Ki values of ET-1 and ET-2 were found to be similar in WKY and SHR. However, the Ki value of ET-3 were found to be significantly lower (P less than 0.004) in SHR as compared to WKY. Concentration-dependent inhibition of [125I]SRT 6b binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 were also performed. The Ki values were found to be 9.50 +/- 2.10 pM, 0.17 +/- 0.04 nM and 31.20 +/- 6.00 nM for ET-1, ET-2 and ET-3, respectively in WKY. The Ki values of ET-1 and ET-2 were found to be similar in WKY and SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acceleration by chronic treatment with clorgyline of the turnover of brain alpha 2-adrenoceptors in normotensive but not in spontaneously hypertensive rats.

1. The aim of this study was to quantitate and compare the turnover of alpha 2-adrenoceptors in the cerebral cortex of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, and its modulation during chronic treatment with the monoamine oxidase (MAO) inhibitor, clorgyline. 2. In SHR, the specific binding of the agonist [3H]-UK 14304 and of the antagonist [3H]-RX 821002 was significantly reduced in the brain (Bmax 15-19% lower) as compared to that in sex- and age-matched WKY rats. In contrast, no significant changes in the Kd values for both radioligands were found between WKY and SHR rats. Therefore, SHR rats offer a genetic model with a lower density of alpha 2-adrenoceptors in the brain. 3. Chronic treatment (21-35 days) with clorgyline (1 mg kg-1, i.p.) markedly decreased the density of brain alpha 2-adrenoceptors ([3H]-UK 14304 binding) in Sprague-Dawley (Bmax reduced by 50%) and in WKY (Bmax reduced by 30%) rats without any apparent change in the affinity of the radioligand. In contrast, the density of brain alpha 2-adrenoceptors in SHR was not down-regulated by chronic clorgyline treatment. 4. The recovery of [3H]-UK 14304 binding after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; 1.6 mg kg-1) (an alkylating agent for the alpha 2-adrenoceptor) was assessed in control and clorgyline-treated (1 mg kg-1; i.p. for 7-21 days) WKY and SHR rats to study the process of alpha 2-adrenoceptor repopulation and to calculate receptor turnover parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of two kappa-opiate agonists, U-50,488H and U-69,593, on the binding of 3H-(3-MeHis2) thyrotropin-releasing hormone to rat spinal cord and amygdala membranes.

The effect of delta- and kappa-opiate receptor agonists on the binding of 3H-(3-MeHis2) thyrotropin-releasing hormone (3H-MeTRH) to membranes of the spinal cord and amygdala of male Sprague-Dawley rats was determined in an effort to further understand interactions between opiates and TRH receptors. The agonists used were D-Ala2-MePhe4-Gly-ol5-enkephalin (DAMGO, mu-receptor), cyclic D-penicillamine2-D-penicillamine5-enkephalin (DPDPE, delta-receptor), cyclic D-penicillamine2-L-penicillamine5-enkephalin (DPLPE, delta-receptor), D-Ala2-D-Leu5-enkephalin (delta-receptor), U-50,488H and U-69,593 (kappa-receptor). 3H-MeTRH bound to amygdala and spinal cord membranes at a single site with Bmax values of 35.7 +/- 5.4 and 15.8 +/- 2.6 fmol/mg protein, and Kd values of 6.3 +/- 1.1 and 5.2 +/- 0.7 nmol/l, respectively. The competition experiments were carried out at a concentration of 2 nmol/l 3H-MeTRH. The concentration of opiate ranged from 10(-9) to 10(-4) mol/l. DAMGO, DPDPE and DPLPE had no effect on the binding of 3H-MeTRH to amygdala or spinal cord membranes. The two highly selective kappa-agonists differed in their interaction with TRH receptors. Whereas U-69,593 did not modify the binding of 3H-MeTRH, U-50,488H significantly inhibited the binding of 3H-MeTRH to both spinal cord and amygdala membranes. U-50,488H has been found to be 10 times more potent than U-69,593 at the central kappa-opiate receptors and may explain their differential action at the TRH receptors. It is concluded that mu- and delta-opiate agonists do not interact with brain and spinal cord TRH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of 3H-(3-MeHis2) thyrotropin-releasing hormone to spinal cord membranes of spontaneously hypertensive and Wistar-Kyoto normotensive rats

Previous studies from this laboratory had indicated that in spontaneously hypertensive (SHR) rats the development of hypertension paralleled increases in brain receptors for thyrotropin-releasing hormone (TRH). The increase appeared to be confined to hypothalamus and striatum. The present studies were undertaken to determine the binding characteristics of TRH receptors in the peripheral tissues of SHR and normotensive Wistar-Kyoto (WKY) rats. TRH receptors were labeled with 3H-MeTRH. The binding of 3H-MeTRH to membranes prepared from spinal cord, heart, lung, kidney and adrenal gland of SHR and WKY rats was determined. In WKY rats, the binding of 3H-MeTRH to various tissue membranes was in the following order: spinal cord greater than kidney greater than lung greater than heart = adrenals. The binding of 3H-MeTRH to spinal cord membranes of SHR rats was significantly higher in comparison to WKY rat tissues, whereas the binding of 3H-MeTRH in kidney, heart, lung and adrenals of the two strains of rats did not differ. The increase in the binding of 3H-MeTRH to spinal cord of SHR rats was due to increases in the Bmax values and not in the Kd values. The results suggest that in addition to the brain, TRH receptors in the spinal cord of SHR rats are also up-regulated and may also play an important role in the regulation of blood pressure.     

Decreased density of alpha 2-adrenoceptors in medulla oblongata of spontaneously hypertensive rats

To study the role of medullary alpha-adrenoceptors in hypertension, we compared specific binding of [3H]prazosin and [3H]clonidine in different brain regions of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and normotensive Wistar-Kyoto rats (WKY). As compared with age-matched WKY, Bmax values for specific [3H]clonidine binding in the medulla oblongata were significantly lower in SHR and SHRSP at 16-24 weeks of age. In the SHRSP medulla oblongata, the decrease was more prominent in dorsomedial and ventrolateral regions than in the ventromedial region. Density of alpha 2-adrenoceptor binding sites was also decreased in the medulla oblongata of young (4-5-week-old) SHRSP. In contrast, there was no difference in Kd and Bmax values for medullary [3H]prazosin binding between WKY and SHRSP. The dorsomedial and ventrolateral regions of the SHRSP medulla oblongata showed significantly lower levels of norepinephrine (NE). Thus, the present study demonstrates that there is a specific loss of alpha 2-adrenoceptors in the medulla oblongata of SHR and SHRSP that may be partly involved in the pathogenesis of spontaneous hypertension.     

Binding of [3H]nitrendipine to cardiac and cerebral membranes from normotensive and renal, deoxycorticosterone/NaCl and spontaneously hypertensive rats

The properties of [3H]nitrendipine binding to cardiac and cerebral membranes from normotensive Wistar-Kyoto (WKY) and renal (RHR), deoxycorticosterone/NaCl (DOCA-HR) and spontaneously hypertensive (SHR) rats were investigated. The maximal numbers of binding sites (Bmax) in the striatum, thalamus and hippocampus for SHR increased by 21.4-40.0, 28.1-40.4 and 21.4-34.1% of the numbers in WKY, but the apparent dissociation constants (KD) in the cerebral membranes differed very little between WKY and SHR. In the cardiac membranes, KD and Bmax values differed very little between WKY and SHR. In the RHR and DOCA-HR, the Bmax values in the striatum, thalamus and hippocampus were similar to those of WKY. These findings suggest that the increase in Bmax of [3H]nitrendipine in the striatum, thalamus and hippocampus of SHR may play a part in the development and maintenance of high blood pressure in SHR.     

Modulation of mu, kappa and delta opioid receptors in the rat brain by isoflurane and enflurane.

This study was done to investigate whether inhalational anesthetics modulated the binding of specific ligands to opioid receptors in the brain of the rat. The effect of isoflurane and enflurane on the binding of specific ligands to various subtypes of opioid receptors in vitro was studied. Isoflurane inhibited the binding of [3H]naloxone to opioid receptors by 50% in the spinal cord, midbrain and cortex at 22, 49 and 50 mM, respectively. Enflurane was more potent than isoflurane in inhibiting the binding of [3H]naloxone. Scatchard analysis of the binding of [3H]naloxone, done in the presence of therapeutic level (5 mM) of isoflurane, suggested that it did not affect the KD (1.3 nM) but decreased the Bmax by 41% in the cortex. Isoflurane and enflurane, at large doses (30-50 mM), inhibited the binding of [3H]ethylketo-cyclazocine (EKC) to kappa receptors in midbrain, cortex and spinal cord. At a smaller dose (5 mM), they increased the binding of EKC in spinal cord. The binding of the analogs of enkephalin [3H]DSTLE(Tyr-D-Ser-Gly-Phe-Leu-Thr-enkephalin) to delta receptors and [3H]DAGO (Tyr-D-Ala-Gly-Methyl-Phe-Glyol-enkephalin) to mu receptors in the midbrain and cortex was inhibited by isoflurane at a significantly smaller concentration than the binding of [3H]naloxone, indicating that the binding of peptides was more susceptible to the inhibition by inhalational anesthetics than the binding of alkaloids, such as naloxone or EKC. These results suggest that the modulation of opioid receptors by inhalational anesthetics is a function of both the nature of the ligand and the tissue used for the receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative studies of muscarinic and dopamine receptors in three strains of rat

Cholinesterase activities and characteristics of muscarinic and dopamine receptors from 9 week old male Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were studied. Plasma cholinesterase activity in WKY was significantly lower (50%) than activity in the other strains. In studies of muscarinic receptors, the number of [3H]QNB binding sites in striata from SD rats was lower (18%) than those from WKY and SHR. However, muscarinic receptor properties (Kd and Bmax) were the same in hypothalami. Studies of dopamine receptors revealed that the densities of both D-1 and D-2 receptors in both striata and hypothalami were significantly higher in SHR than in other strains. However, there were no differences in the affinity constant (Kd). The higher densities in hypothalami from SHR were mainly due to the high population of D-1 and D-2 receptors in the posterior hypothalamus. In the anterior hypothalamus, there was no difference in the population of D-2 receptors. These results provide a substantive basis, i.e. demonstration of alterations in drug metabolizing enzymes and receptor populations, on which to build an understanding of the genetic predisposition to the actions of xenobiotic agents.     

Differences in the binding of [3H][D-Ser2,Thr6]leucine-enkephalin and [3H][D-Pen2,D-Pen5]enkephalin to brain membranes of morphine tolerant-dependent rats.

The effect of morphine tolerance-dependence and abstinence on the characteristics of delta-opiate receptors was determined in male Sprague-Dawley rats. Two ligands used for characterizing the receptors were [3H][D-Ser2,Thr6]leucine-enkephalin ([3H]DSTLE) and [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE). Rats were implanted s.c. under light ether anesthesia with six morphine pellets (each containing 75 mg of morphine free base). Rats which served as controls were implanted similarly with placebo pellets. Two sets of rats were used. In one group of rats, the pellets were left intact (tolerant-dependent) at the time of sacrificing and in the other the pellets had been removed 18 h earlier (abstinent). The spinal cord and brain regions (amygdala, hippocampus, hypothalamus, corpus striatum, mid-brain, pons and medulla and cortex) were dissected for binding studies. The binding of [3H]DSTLE to membranes of cerebral cortex of morphine-tolerant-dependent rats was decreased in comparison to control rats, and was due to a decrease in Bmax rather than Kd value. The binding of [3H]DSTLE to other brain regions or spinal cord of morphine-tolerant-dependent and abstinent rats did not differ from their respective controls. On the other hand, the binding of [3H]DPDPE was unaffected in any brain region or the spinal cord of morphine-tolerant-dependent and abstinent rats when compared to their controls. The decrease in binding of [3H]DSTLE to cortical membranes of morphine-tolerant-dependent rats amounted to 15%. Since DSTLE also binds to mu-opiate receptors, which have earlier been shown to be decreased in cortex of morphine-tolerant-dependent rats, and the binding of a more selective delta-opiate ligand [3H]DPDPE was unaffected, it is concluded that central delta-opiate receptors do not play a role in the development of morphine-induced tolerance-dependence or abstinence processes in the rat.     

Down-regulation of hypothalamic 5-HT1A receptors in morphine-abstinent rats

The effects of morphine tolerance-dependence and abstinence on 5-HT1A receptors in brain regions and spinal cord of the rat were determined. Tolerance to and physical dependence on morphine was induced in male Sprague-Dawley rats by implanting six morphine pellets (each containing 75 mg of morphine free base) during a seven day period. Two groups of rats were used for binding studies. In one group the pellets were left intact (tolerant-dependent) and in the other they were removed (abstinent). Rats were killed, and spinal cords and brains were excised. Brain was dissected into seven regions (amygdala, hippocampus, hypothalamus, striatum, midbrain, pons + medulla and cortex). 5-HT1A receptors were characterized by using [3H]8-hydroxy-di-n-propylaminotetralin [( 3H]DPAT) as the ligand and unlabelled 5-HT to determine the non-specific binding. In morphine and placebo tolerant-dependent rats the binding of [3H]DPAT to 5-HT1A receptors in brain regions and spinal cord did not differ. The Bmax value of [3H]DPAT in the hypothalamus of morphine abstinent rats was decreased by 61.9%. No change in Bmax value was observed in other brain regions and spinal cord. The Kd values were unaffected. Subcutaneous administration of DPAT produced hypothermia in rats from which pellets had been removed. The intensity of DPAT-induced hypothermic response was greater in morphine abstinent rats as compared to placebo abstinent rats. Since DPAT is believed to have a major action on the presynaptic 5-HT neurons, it is concluded that in morphine abstinent rats 5-HT1A receptors are down-regulated in hypothalamus, but in morphine tolerant-dependent rats they are unaffected.     

Characterization of specific [3H]nociceptin binding in rat brain and spinal cord

The present study was undertaken to characterize simultaneously [3H]nociceptin binding to opioid receptor-like 1 (ORL1) receptors in the rat brain and spinal cord. Specific binding of [3H]nociceptin to crude membranes from the rat brain and spinal cord at 25 degrees C was saturable, reversible and of high affinity, and it also exhibited a pharmacological specificity involving the ORL1 receptor. The Kd and Bmax values for [3H]nociceptin in the spinal cord were significantly lower than those in the brain. At 4 degrees C, there was a significant increase in the dissociation constant (Kd) for [3H]nociceptin in the brain and spinal cord with little change in the maximal number of binding sites (Bmax) compared with that at 25 degrees C. Nociceptin and its analogue, [Phe1 psi(CH2-NH)-Gly2]nociceptin(1-13)NH2 were found to be potent inhibitors of [3H]nociceptin binding to crude membranes from the brain and spinal cord, while opioid ligands such as naloxone-benzoylhydrazone, naltrindole and nor-binaltorphimine, exhibited an inhibitory effect only at high concentrations. The Ki values for nociceptin, its analogue and opioid ligands in the spinal cord were significantly lower than those in the brain. There were regional variations in the specific [3H]nociceptin binding to crude membranes from the rat brain: a relatively high density of [3H]nociceptin binding in the cerebral cortex, hippocampus, thalamus and midbrain, moderately dense binding in the corpus striatum and pons/medulla oblongata, and the lowest density of binding in the cerebellum. In conclusion, the present study has shown that [3H]nociceptin binds selectively to ORL1 receptors in the rat brain and spinal cord.     

Increased 45Ca influx in response to alpha 1-adrenoceptor stimulation in spontaneously hypertensive rat caudal artery

The isometric tension development and 45Ca influx in response to norepinephrine (NE) and methoxamine stimulation were investigated in caudal arteries of spontaneously hypertensive rats (SHR) and Wistar Kyoto normotensive rats (WKY). The maximum isometric tension developed as well as 45Ca influx in response to NE and methoxamine stimulation were significantly increased (p less than 0.05) in SHR caudal arteries as compared with WKY. On the other hand, neither the isometric tension developed nor the 45Ca influx in response to K+ depolarization were different between WKY and SHR caudal arteries. Estimation of [3H]prazosin binding to the membranes isolated from caudal artery of WKY and SHR showed a single class of high-affinity binding sites with Kd values for SHR 128 +/- 14 pM and for WKY 141 +/- 19 pM, and Bmax values for SHR 108 +/- 14 fmol/mg protein and for WKY 113 +/- 21 fmol/mg protein. From these results, we conclude: (a) Increased contractile response of SHR caudal artery rings to alpha 1-adrenoceptor stimulation appears at least in part to be due to an increased Ca2+ influx through receptor-operated Ca2+ channels; (b) the affinity or density of alpha 1-adrenoceptors estimated by [3H]prazosin binding is not altered in the SHR caudal artery.     

Autoradiographic localization of muscarinic acetylcholine receptors in the rat pulmonary vascular tree.

The pharmacological characteristics and the anatomical localization of muscarinic receptors in the pulmonary vascular tree were investigated in lung sections of Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [3H]Quinuclidinyl benzylate [( 3H]QNB) was bound by sections of rat lung in a manner consistent with the labeling of muscarinic acetylcholine receptors, with a dissociation constant value (Kd) of 0.41 +/- 0.3 nM in WKY rats and of 0.37 +/- 0.2 nM in SHR. The density of muscarinic acetylcholine receptors was higher in sections of lung of WKY rats than of SHR. In the pulmonary vasculature these sites were associated with the smooth muscle of the medial layer of different size branches of the pulmonary artery and vein. No [3H]QNB binding sites were found within the endothelium in the blood vessels of either WKY rats or SHR. The density of [3H]QNB binding sites was significantly lower in the smooth muscle of pulmonary vein and its branches in SHR. There were no significant hypertension-dependent changes in the density and pattern of muscarinic receptors of pulmonary artery smooth muscle.