Decrease In Bmax Value Research Articles

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes.

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. In the presence of 1-10 μM AEA, suppression of both Na(+) and L-type Ca(2+) channels was observed. Inhibition of Na(+) channels was voltage and Pertussis toxin (PTX) - independent. Radioligand-binding studies indicated that specific binding of [(3) H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd . Further studies on L-type Ca(2+) channels indicated that AEA potently inhibited these channels (IC50 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba(2+) currents. MetAEA also inhibited Na(+) and L-type Ca(2+) currents. Radioligand studies indicated that specific binding of [(3) H]isradipine, was inhibited significantly by metAEA. (10 μM), changing Bmax but not Kd . Results indicate that AEA inhibited the function of voltage-dependent Na(+) and L-type Ca(2+) channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation.

Down-regulation of 3H-lofentanil binding to opiate receptors in different cultured neuronal cells

There was stereospecific binding of 3H-lofentanil (KD value = 1.53 nM) to membranes of neuroblastoma-glioma NG 108-15 cells which are known to bear high affinity binding sites for enkephalin derivatives (delta-opiate receptor subtype). There was no high affinity specific binding of the mu-opiate specific ligand 3H-sufentanil. The specific binding of 3H-lofentanil to delta-opiate receptor subtype was down-regulated (decrease in Bmax value without change in the KD value) after prolonged incubation of the cells in the presence of leu- and met- enkephalin (0.1 microM). There was no down-regulation of the opiate receptors (3H-lofentanil and 3H-D-ala-D-leu-enkephalin specific binding) after incubation of NG 108-15 cells with drugs from the fentanyl series (alfentanil or sufentanil). In cultured neurones from rat forebrain (15 day old embryos), the 3H-lofentanil binding was specific with high affinity (KD: 0.048 nM) and a slow dissociation rate similar to that in adult rat cortex. Drugs of the fentanyl series (4-anilino-piperidines) were potent displacers whereas agonists of the delta- (enkephalin derivatives), sigma- (phencyclidine, haloperidol, 3-hydroxyphenyl-propylpiperidine) or K- (U 50488) opiate sites had a low affinity (Ki greater than 0.5 microM) for 3H-lofentanil specific binding sites. Since there was also specific binding of 3H-sufentanil, the opiate receptors in cultured neurones seem to be mainly of the mu-subtype and this is consistent with the ontogeny of opiate receptors subtypes. These receptors were down-regulated after incubation in the presence of etorphine, sufentanil and alfentanil but not enkephalin derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Ex vivo occupancy by tamsulosin of α1-adrenoceptors in rat tissues in relation to the plasma concentration

At 0.5–12 h after oral administration of tamsulosin (2.3 μmol/kg) in rats, there was a significant decrease in specific [ 3H]prazosin binding in the prostate as compared to the control value. The greater decrease occurred in the submaxillary gland. The effect of tamsulosin was mainly due to a marked reduction of [ 3H]prazosin binding sites (Bmax) rather than to an increase in the dissociation constant (Kd). In contrast, there was only a slight decrease or no change in the [ 3H]prazosin binding in the spleen, heart, and cerebral cortex of tamsulosin-administered rats at 0.5–12 h. Oral administration of terazosin (21.7 μmol/kg) significantly increased Kd values for [ 3H]prazosin binding with little effect on Bmax values in the rat prostate at 3 and 6 h. The greater increases in Kd values were observed in the submaxillary gland, spleen and heart at 0.5–12 h. Terazosin had a slight effect on Kd values for the cerebral cortical [ 3H]prazosin binding. Tamsulosin was absorbed rapidly after oral administration at a dose of 2.3 μmol/kg in rats, and at 6 h, plasma concentration decreased markedly to approximately one-twentieth of the 0.5 h peak level. α 1-Adrenoceptor occupancy was estimated as a percentage of decrease in Bmax values for [ 3H]prazosin binding in tissues of tamsulosin-treated rats compared with control rats. The α 1-adrenpceptor occupancy by tamsulosin in the prostate and submaxillary gland occurred rapidly in parallel with the rise in plasma concentration of tamsulosin, and lasted for over 12 h despite the marked decrease in plasma concentration. Consequently, it is suggested that tamsulosin produces more selective and sustained occupancy in vivo of α 1-adrenoceptors in the submaxillary gland and prostate of rats than in other tissues.

Plasticity of 5‐HT2A Serotonin Receptor in Patients With Analgesic‐Induced Transformed Migraine

Chronic drug exposure can induce a significant change in neurotransmitter receptor systems and is possibly involved in the pathogenesis of drug-induced neurological disorders. Abuse of analgesics is known to induce deterioration in headache status in patients with primary headaches, especially migraine. To assess the possibility of 5-HT2A serotonin receptor plasticity in this condition, we investigated receptor binding on the platelet membrane in patients with analgesic-induced transformed migraine, patients with migraine, and nonheadache controls. Various concentrations of [3H]-spiperone (0.4 to 12 nmol) was used as a radioligand, and ketanserin was used to determine nonspecific binding. A lower maximal number of receptors (Bmax) was observed in patients with migraine as compared to patients with transformed migraine, and controls (467 +/- 58, 708 +/- 36, and 786 +/- 64 fmol/mg protein, respectively, P<0.01); whereas the value of the dissociation equilibrium constant (Kd) remained unchanged (1.72 +/- 0.16, 1.41 +/- 0.13, and 1.25 +/- 0.21 nmol for patients with migraine, patients with transformed migraine, and nonheadache controls, respectively). A significant decrease in Bmax value was observed in patients with transformed migraine after 4 weeks of analgesic withdrawal (770 +/- 25 and 345 +/- 31 fmol/mg protein, P<0.001), whilst no significant change in Kd value was observed (1.95 +/- 0.12 and 2.47 +/- 0.30 nmol, respectively). These findings indicate that 5-HT2A serotonin receptor system is altered in patients with transformed migraine with analgesic overuse. Such receptor plasticity may be an important step in the pathogenic mechanism of transformed migraine.

Effects of exposure to cypermethrin on saxitoxin binding in susceptible and pyrethroid-resistant houseflies.

Saxitoxin (STX) binding was measured in susceptible (SBO) and pyrethroid-resistant (KDR) female houseflies having only target site insensitivity as a resistance mechanism. In KDR flies, there was a quantitative decrease in STX binding capacity (Bmax) relative to SBO flies coupled with an increase in binding affinity (Kd). Treatment of SBO flies with sublethal doses of cypermethrin resulted in a large decrease in the number of STX binding sites and an increase in STX binding affinity. In KDR flies, identical treatments had the opposite effects. Treatment of both strains with higher doses of cypermethrin resulted in smaller decreases in Bmax values coupled with decreases in binding affinities. The results show that physiological changes in STX binding occur upon exposure to extremely low doses of cypermethrin. The data suggest that the kdr resistant gene may be expressed as changes in STX binding kinetics and that measurements of STX binding in pyrethroid-treated insects may be a useful approach for studying pyrethroid's mode of action and resistance.

Alteration of D1 and D2 dopaminergic receptor kinetics in specific rat brain regions following repeated administration of opiates.

The present study was conducted to investigate the effects of repeated administration of opiates on the binding characteristics of D1 and D2 dopamine receptors in specific rat brain regions. Male Sprague-Dawley rats (150-175 g) were adapted for 1 week under controlled conditions (22 +/- 1 degree C, 40% relative humidity, 12 h light:12 h dark illumination cycle). After the adaptation period, rats were randomly assigned into six groups (n = 15/group) for two sets of experiments; one for D1 and the other for D2 receptor binding evaluation. In each set, group 1 served as saline control, group 2 was treated with morphine (1 mg/kg/day for 7 days) and group 3 was treated with naloxone (2 mg/kg/day for 7 days). Following the third day of morphine injection, rats showed restlessness, hyperactivity, increased urination, diarrhea, lacrimation, irritability and squealing on touch, head and body shakes and salivation just prior to morphine dosage. These observed signs are typical for morphine withdrawal. One hour after the last injection, rats were sacrificed by decapitation, brains were removed and kept at -70 degrees C. The frozen brains were further dissected into cortex (CTX), hypothalamus (HYPO), hippocampus (HIPP) and midbrain (MID), pooled (5/pool), homogenized and used for radioreceptor assays. For D1 binding study, 3H-SCH-23390 was used as ligand and for D2 receptor binding 3H-YM-09151-2 was employed. Following repeated morphine or naloxone treatment, Bmax values for 3H-SCH-23390 binding to membranes of HYPO and MID were increased and decreased, respectively, whereas Kd values were significantly decreased in both HYPO and MID of morphine and naloxone-treated animals. In rat brain CTX, both morphine and naloxone decreased Kd and Bmax values of 3H-SCH-23390 binding, however the decrease in Bmax values noted after morphine administration was not statistically significant. Decrease in both Bmax and Kd values of dopamine D1 receptors were observed after naloxone but not morphine treatment in HIPP. Morphine administration increased the density of D2 receptors in HIPP and MID and decreased the affinity of 3H-YM-09151-2 binding in CTX, HIPP and MID. Naloxone treatment resulted in increased number of 3H-YM-09151-2 binding sites in CTX, HYPO and HIPP. While naloxone treatment increased Kd values of 3H-YM-09151-2 in HYPO and HIPP, it decreased these values in CTX and MID. It is concluded that repeated intermittent treatment with opiates induces alterations in D1 and D2 dopamine receptors binding properties and that these changes are regionally specific.(ABSTRACT TRUNCATED AT 400 WORDS)

Nicotinic acetylcholine receptor subtypes in human frontal cortex: Changes in Alzheimer's disease

Molecular genetic and pharmacological studies have suggested that several subtypes of nicotinic acetylcholine receptors exist in the mammalian and avian brain. Combining 3H-(-)-nicotine, 125I-alpha-bungarotoxin, and 125I-kappa-bungarotoxin as ligands, we report here the first evidence for the existence in human frontal cortex of at least three different subtypes of nicotinic receptors. Autoradiographic analysis shows that specific 125I-kappa-bungarotoxin binding sites are concentrated mainly in several cortical layers. We also show that kappa-bungarotoxin, but not alpha-bungarotoxin decreases the evoked release of 3H-acetylcholine in rat cortical slices, indicating a likely presynaptic localization for some of the alpha-bungarotoxin-insensitive kappa-bungarotoxin sites in mammalian brain. The brains of patients with Alzheimer's disease show marked decreases in Bmax values for low-affinity 125I-kappa-bungarotoxin sites and both high- and low-affinity 3H-nicotine sites, whereas 125I-alpha-bungarotoxin sites are not significantly different in number from age-matched control brains. We conclude that Alzheimer's disease does not affect all subtypes of nicotinic receptors in the frontal cortex to the same extent.

Decrease in μ-opioid receptor binding capacity in rat brain after chronic PL017 treatment

In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or d-Ala 2, d-Leu 5-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases, μ-opioid receptor binding capacity was attenuated together with the δ-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both μ and δ receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the μ-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5–20 μg/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the AD 50 of the peptide to elicit the antinociceptive response and about 14-fold increase in the ED 50 of the peptide to elicit the catatonic effect. When μ- and δ-binding was determined using [ 3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20–30%) in the μ-opioid receptor binding but not in δ-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [ 3H]DAMGO saturation binding data revealed a decrease inB max values and no change in theK d values. Hence, μ-opioid receptors can be specifically regulated by ligand in the brain as δ-receptors are in neuroblastoma × glioma NG 108-15 cells. Chronic activation of the μ-opioid receptor in the brain would result in the down-regulation of the binding sites.

Alloimmunization-induced changes in β-adrenoceptor expression and cAMP on B lymphocytes

In this report we have examined the effect of alloimmunization on β-adrenergic expression in lymphocytes. We have observed a variation in the number of β-adrenoceptors (Bmax) according to the degree of immunization without modifications in their affinity (Kd). This phenomenon was accompanied with parallel variations of intracellular cAMP levels. A decrease in Bmax values was observed during the first and second immunizations. Then the Bmax began to increase, exceeding control values up to the fourth and fifth immunizations, and remaining constant at the sixth immunizations. Only B cell-enriched populations showed variation in Bmax values of β-adrenoceptors with alloimmunization. In contrast, the Bmax values of T cell-enriched populations did not change. Kd values were similar in all cell types tested. The number of binding sites was not dependent on the animal's age. Modifications in cAMP levels of B cell-enriched populations were correlated with changes in β-adrenoceptor expression. These results suggest that β-adrenoceptor expression and cAMP intracellular levels in B cell-enriched populations vary with the number of alloimmunizations. In addition, the antibody synthesis induced by allogenic stimulus was inversely proportional to the number of β-adrenoceptors expressed on B cells. Perhaps these findings are evidence of a control mechanism that regulates antibody synthesis during the immune response.

Changes in 3H-Spiperone, 3H-WB 4101 and 3H-Dihydroalprenolol Bindings to Brain Membranes Produced by Postnatal Pretreatment with Chlorpromazine in Adult Rats

In order to elucidate possible mechanisms of the learning deficit produced by postnatal pretreatment with chlorpromazine (CPZ), changes in catecholamine receptors in the rat brain were investigated. Male neonates of Wistar strain rats were given s.c. 2 mg/kg/day of CPZ for 7 successive days from days 6 to 12 after birth. Effect of the postnatal pretreatment with CPZ on saturation constants for specific bindings of 3H-spiperone, 3H-WB 4101 and 3H-dihydroalprenolol, respectively, in 8 brain regions was investigated at 60 days after birth. Significant decreases in Bmax values of 3H-WB 4101 binding sites in the cortex, thalamus, hypothalamus, mid brain and medulla oblongata/pons and decreases in Kd values of the binding sites in thalamus, hypothalamus and mid brain were observed in CPZ-pretreated rats when compared with corresponding Bmax and Kd values obtained in saline-pretreated rats. Furthermore, significant decreases in both Bmax and Kd values of 3H-DHA binding sites in the thalamus were detected in CPZ-pretreated rats when compared with those obtained in saline-pretreated rats. However, no alterations in 3H-spiperone binding sites in all brain regions were found between CPZ- and saline-pretreated rats. These results suggest that the learning deficit observed in CPZ-pretreated rats may be produced by a functional disorder of catecholaminergic, in particular alpha 1-noradrenergic neurons in the brain.