Binding characteristics of [3H] guanfacine to rat brain α-adrenoceptors: Comparison with [3H]clonidine

The tritium-labeled α-adrenoceptor agonist and antihypertensive drug guanfacine, N-amidino-2-(2,6-dichlorophenyl)-acetamide (sp. act. 24.2 $\text{Ci}\text{mmole}$) was employed for a direct identification and characterization of α-adrenoceptors in rat brain membranes. Its usefulness as a radioligand was studied in comparison with [³H]clonidine (sp. act. 26.7 $\text{Ci}\text{mmole}$). The nonspecific binding of [³H]guanfacine to rat cerebral membranes was considerably more pronounced than that observed for [³H]clonidine. The specific binding of [³H] guanfacine (0.1–20 nM) and [³H]clonidine (0.1–20 nM) as defined as the excess over blanks containing (?)-norepinephrine (10μM) was saturable. Scatchard analyses of these binding data indicated single populations of binding sites for both ligands. K_D values of 3.9 ([³H]guanfacine) and 3.7 nM ([³H]clonidine) were calculated. Maximal number of specific binding sites amounted to 220 and 195 $\text{fmole}\text{mg}$ protein for [³H]guanfacine and [³H]clonidine, respectively. In case unlabeled guanfacine (1 μM) was used to characterize the specific binding of [³H] guanfacine, K_d value and maximal number of binding sites were about twice as high as determined in the presence of excess (?)-norepinephrine. The rate of association of both radioligands was rapid. Binding reached equilibrium by about 10–15 min of incubation. Half-maximal binding was attained at approximately 1–2 min. The rates of dissociation were biphasic. A rapid and a slow component were identified. The specific binding sites of [³H] guanfacine in rat brain possess the general characteristics of α₂-adrenoceptors. Selective antagonists of α₂-adrenoceptors, like yohimbine and rauwolscine strongly interfered with this binding. However, preferential blocking agents of α₁-adrenoceptors, such as prazosin and corynanthine, were weak competitors. The relative potency of agonists and antagonists in displacing [³H]guanfacine was identical to their effectiveness in competing for [³H]clonidine specific binding sites. It is concluded that [³H]guanfacine labels the same α₂-adrenoceptor population in rat brain as [³H]clonidine. However, [³H]guanfacine seems not as suitable as [³H]clonidine for routine use in the direct identification of α₂-adrenoceptors in view of its relatively high nonspecific binding.     

Binding of adrenergic ligands ([3H]clonidine and [3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [³H]ligands, we studied the binding of [³H]clonidine and [³H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [³H]clonidine (with K_D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [³H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [³H]WB-4101 were also detected in the human frontal cortex (with K_D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [³H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [³H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [³H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [³H]WB-4101 sites was highest in the hippocampus.     

Characterization of non-adrenergic [3H]clonidine binding sites in rat stomach: high affinity of imidazolines,guanidines and sigma ligands

We have identified and characterized non-adrenergic [³H]clonidine binding sites in rat stomach. The binding of [³H]clonidine was rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 68% specific binding at [³H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [³H]clonidine to rat stomach membranes was concentration-dependently inhibited by various imidazolines and guanidines including the sigma site ligand 1,2-di-(2-tolyl)guanidine (DTG), by the butyrophenone derivative haloperidol and by the piperidine derivative (+)-3-PPP[(R)-3-(3-hydroxyphenyl)-N-propylpiperidine]; the latter two compounds are also known to exhibit affinity for sigma sites. In contrast, rauwolscine, histamine, ranitidine and the non-hydrolysable GTP-analogue Gpp(NH)p (5 guanylylimidodiphosphate) did not, or with negligible affinity, inhibit [³H]clonidine binding. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases for a single detectable site) was as follows: cirazoline>idazoxanDTG>(+)-3-PPP> clonidine>guanabenz>haloperidol. This rank order is not compatible with the pharmacological properties of either I₁- or I₂-imidazoline binding sites. However, the ability of haloperidol, (+)-3-PPP and DTG to displace [³H]clonidine (the latter two with high affinity) suggests that the [³H] clonidine binding sites in rat stomach may be related to sigma-like sites.     

Interaction of dihydroxy-2-aminotetralin derivatives at sites labelled with [3H]clonidine, [3H]prazosin and [3H]spiperone in rat brain membranes

The interactions of 5,6- and 6,7-dihydroxy derivatives of 2-aminotetralin with [³H]clonidine and [³H]clonidine and [³H]prazosin as well as with [³H]spiperone binding sites in rat cerebral cortex membrane preparations were investigated. The hydroxy derivatives of 2-aminotetralin tested showed significant interaction with [³H]clonidine as well as with [³H]spiperone binding sites while for [³H]prazosin binding site these agents appeared virtually inactive. For interaction with [³H]clonidine binding site 6,7-dihydroxy substitutions impart greater potency that 5,6-dihydroxy substitutions and N-alkyl substitutions either make no difference or reduce the affinity of these compounds. N-alkyl substitutions, however, markedly enhance the affinity of 5,6-dihydroxy derivatives for interactions with [³H]spiperone binding site. The results suggest that some hydroxy derivatives of aminotetralin have significant interaction with both central α₂-adrenoceptor and D₂-dopamine receptor systems.     

Human brain imidazoline receptors: further characterization with [3H]clonidine

The aim of the present study was to further characterize [³H]clonidine binding in the ventrolateral medulla of the human brainstem, the region involved in the vasodepressor effect of imidazoline drugs of the clonidine type. Under basal conditions, [³H]clonidine can bind both to the imidazoline receptors and to the α-adrenoceptors. The latter represent only a small part of the total [³H]clonidine binding with a B_max of 61 ± 13 fmol/mg proteins and a K_D of 4.9 ± 2.2 nM. Most of the binding was associated with imidazoline receptors with a K_D of 67 ± 13 nM and a B_max of 677 ± 136 fmol/mg protein. α-Adrenoceptor binding of [³H]clonidine could be completely prevented when membranes were either treated during preparation with the aIkylating agent phenoxybenzamine or incubated in the presence of 30 μM (−)-noradrenaline or in the presence of the non-hydrolysable analogue of GTP, guanylyl imidodiphosphate (Gpp(NH)p). When the α-adrenoceptors binding was prevented, we demonstrated the insensitivity of [³H]clonidine binding to Gpp(NH)p and showed that the competition between clonidine and idazoxan for imidazoline receptors was insensitive to Gpp(NH)p suggesting that imidazoline receptors are not G protein coupled receptors. The specificity of [³H]cloniding binding to imidazoline receptors in the human ventrolateral medulla indicates that these receptors are different from imidazole receptors as defined with p-aminoclonidine in the bovine brainstem.     

Binding of [3H]clonidine to I1-imidazoline sites in bovine adrenal medullary membranes

Summary Imidazolines bind with high affinity not only to -adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [³H]clonidine or [³H]idazoxan and termed I₁- and I₂-IBS, respectively. Since bovine adrenal chromaffin cells lack ₂-adrenoceptors, we investigated the pharmacological characteristics of [³H]clonidine binding sites in the bovine adrenal medulla. The binding of [³H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [³H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [³H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (–)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline >- BDF 7579 (4-chloro-2-isoindolinyl guanidine) >-clonidine>- cirazoline >_ BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazolin-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I₁-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5guanylylimidodiphosphate; 100 mol/l) inhibited specific [³H]clonidine binding by about 50%. Equilibrium [³H]clonidine binding was also significantly reduced by K⁺ and Mg²⁺ In conclusion, [³H]clonidine labels non-adrenergic high-affinity sites in plasma membranes of the bovine adrenal medulla; these sites exhibit the pharmacological properties of I₁-IBS, but not of I₂-IBS. Furthermore, the IBS in the adrenal medulla appear to be coupled to a G-protein.Correspondence to G. J. Molderings at the above address     

Effect of treatment by clonidine on the synthesis of catecholamines in the heart, submaxillary and adrenal glands in the rat

The synthesis of catecholamines has been studied in the heart, sub-maxillary glands and adrenal of the rat by intravenous injection of [³H]tyrosine and evaluation, 30 min later, of the newly formed [³H]catecholamines. The synthesis rates of catecholamines have been estimated by calculation of the ratio: amount of [³H]catecholamine/ specific activity of [³H]tyrosine in the tissue. Clonidine (50 $\text{μg}\text{kg}$ i.p.) was administered 1 hr before the injection of [3H]tyrosine. Such treatment did not change significantly the endogenous levels of tyrosine or catecholamines in the organs studied. In clonidine treated rats, the synthesis rate of noradrenaline was reduced by 38 per cent in the heart and by 40 per cent in the sub-maxillary glands. In the adrenal, a 45 per cent reduction of the dopamine synthesis and a 60 per cent reduction of the adrenaline + noradrenaline synthesis were observed after clonidine treatment. The mechanisms by which clonidine reduces catecholamines synthesis are discussed.     

Apomorphine‐Induced Aggressive Behaviour in para‐Chlorophenylalanine‐Treated Male Rats: Implications to Brain [3H]Ketanserin Binding and Monoamine Content

Abstract: The aim of this study was to investigate the role of serotoninergic neurotransmission and the effect of acute para‐chlorophenylalanine (350 mg/kg, intraperitoneally) treatment on apomorphine‐induced aggressive behaviour in adult male Wistar rats. In addition, [³H]ketanserin binding and monoamine content were studied. Repeated administration of apomorphine (1.0 mg/kg, subcutaneously, once daily for two weeks) gradually induced aggressive behaviour. Acute p‐chlorophenylalanine treatment down‐regulated the [³H]ketanserin binding and reduced over 90 per cent the content of serotonin and 5‐hydroxyindolacetic acid. In a half of the p‐chlorophenylalanine‐treated animals, the aggressive behaviour was suppressed, while there was no difference in [³H]ketanserin binding or monoamine content between the p‐chlorophenylalanine treated aggressive and non‐aggressive animals. In conclusion, the acute p‐chlorophenylalanine treatment attenuates the aggressiveness only in half of the animals, while the latter phenomenon is independent on the CNS monoamine content or [³H]ketanserin binding.     

Interaction of opioid drugs with human platelet alpha 2-adrenoceptors.

Morphine, naltrexone and naloxone inhibited the binding of [3H]clonidine to the alpha 2-adrenoceptors in human platelet membranes, provided that Mg2+ was present in the medium. In the presence of 5'-guanylyl imidophosphate (Gpp(NH)p) or in the absence of Mg2+ morphine did not modify the binding of [3H]clonidine. Neither [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAGO), nor [D-Pen2,D-Pen5]enkephalin (DPDPE), nor dynorphin-(1-17) affected the [3H]clonidine binding. The presence of 1 mM naloxone did not alter the affinity of either [3H]clonidine or [3H]yohimbine, but reduced the number of binding sites of [3H]clonidine, having no effect on [3H]yohimbine. Naloxone inhibited the binding of adrenaline to high- but not low-affinity sites. It is concluded that morphine and semisynthetic antagonist derivatives interact with alpha 2-adrenoceptors only in the high-affinity state.     

An increase in cardiac alpha1-adrenoceptors following chronic clonidine treatment

Summary Chronic treatment (22 days) of rats with clonidine (0.5 mg/kg s.c. twice a day followed by 20 h of withdrawal) resulted in a significant increase in the specific [³H]WB4101 binding to ventricular and intraventricular septal ₁-adrenoceptors but no alteration of the atrial ₁-adrenoceptors. Scatchard analysis indicated that the increase in the [³H]WB4101 binding to the clonidine-treated cardiac tissues was due to an enhancement of the ₁-adrenoceptor density since there was a significant increase in the B _max value for the [³H]WB4101 binding to the treated ventricles without a change in the K _d value. The specific [³H]WB4101 binding to cardiac ₁-adrenoceptors was not altered by the acute (1 day) or 7 days treatment with clonidine. Chronic treatment with clonidine had no significant effect on the specific [³H](–)DHA binding to the atrial and ventricular -adrenoceptors. The noradrenaline (NA) concentrations in the clonidine-treated ventricles and intraventricular septae were decreased by 16–20%. These data provide biochemical evidence compatible with a significant reduction of sympathetic outflow to the ventricular myocardium by clonidine.     

Characterization and quantitation of alpha-adrenergic receptor subtypes in rat hypothalamus

The binding of [³H]dihydroergocryptine ([³H]-DHE) to rat hypothalmic membranes was examined. Displacement of [³H]-DHE by 100 nM phentolamine, prazosin and clonidine can be used to assay for total α-, α₁- and α₂-adrenergic receptor sites respectively. The α₁-adrenergic receptor content of the hypothalmus is 4.1 pmoles/g tissue whereas the α₂-level is 6.5 pmoles/g tissue. [³H]-WB 4101 (α₁ selective) and [³H]clonidine (α₂ selective) binding yielded similar levels of 3.2 and 5.8 pmoles/g tissue. It is concluded that [³H]-DHE is a suitable ligand for the assay of α-adrenergic receptor subtypes under the conditions determined in this study.     

Depression by chronic electroconvulsive treatment of clonidine hypotherma and [3H]clonidine binding to rat cortical membranes

The effects of chronic electroconvulsive treatment (ECT) on the binding of [³H]clonidine and on clonidine-induced hypothermia were studied in the rat. After 10 consecutive daily treatments we observed a reduction in the hypothermic action of clonidine and a loss of the high affinity binding sites of clonidine, without changes of the low affinity binding sites. The present results are indicative of the down-regulation of α₂-adrenoceptors during chronic antidepressive treatment.     

EFFECT OF THYROID STATUS ON β-ADRENORECEPTORS AND MUSCARINIC RECEPTORS IN THE RAT LUNG

• 1 The effects of thyroid status on the specific binding of the muscarinic ligand (–)-[³H] quinuclidinyl benzilate (QNB) and of the β-adrenoreceptor ligand (–)-[³H] dihydroalprenolol (DHA) in the adult rat lung were investigated.
• 2 The specific binding of (–)-[³H] quinuclidinyl benzilate (QNB) to lung membranes was saturable and the equilibrium dissociation constant (K_D) determined from Scatchard analysis was 54 pM. Kinetic analysis of the binding of [³H] QNB yielded a K_D of 42 pM. [³H] QNB binding was inhibited by muscarinic agonists and antagonists, the order of their potency was l-hyoscyamine>atropine>scopolamine>oxotremorine>carbachol. These data were consistent with [³H] QNB binding to the muscarinic receptor.
• 3 Adult male rats treated for 2 weeks with the antithyroid agent 3-amino-1,2,4-triazole (ATZ) showed a 52% and 80% reduction in the serum concentration of triiodothyronine (T₃) and thyroxine (T₄) respectively. These hypothyroid rats also had a 39% decrease in the concentration of lung β-adrenoreceptors and a 37% decrease in the concentration of lung muscarinic receptors as compared to euthyroid controls. Concurrent treatment of rats with ATZ and T₄ for 2 weeks resulted in a reduction of 15% and 20% in the concentration of lung β-adrenoreceptors and muscarinic receptors respectively. The K_D values for [³H] DHA and [³H] QNB binding did not change with the ATZ or ATZ + T₄ treated groups.
• 4 Administration of T₄ (500 μg/kg/day) to male rats for 12 days did not result in any significant change in the concentration of either β-adrenoreceptors or muscarinic receptors compared to euthyroid controls. No change in the K_K values for [³H] DHA or [³H] QNB binding were detected.
• 5 The results show that hypothyroid rats have a reduced lung concentration of both β-adrenoreceptors and muscarinic receptors whereas in hyperthyroid rats these receptors do not significantly change from euthyroid controls.

     

Identification and regulation of alpha 2-adrenergic receptors in rabbit ileal mucosa

The alpha 2-adrenergic receptors in rabbit ileal mucosal membranes can be identified by using [3H]clonidine. [3H]Clonidine bound to a homogeneous population of sites (30-120 fmoles/mg protein) with a KD of 2.2 nM at 25 degrees. Alpha-adrenergic agonists and antagonists competed with [3H] clonidine for the binding sites with an order of potency typical for alpha 2-receptors. Mg2+, Ca2+, or Mn2+ (2.4 mM) markedly increased the binding of [3H]clonidine. At the maximally effective concentration, Mg2+ increased both the binding affinity of [3H]clonidine and the number of receptor sites. Both NaCl and GppNHp, the guanyl nucleotide, inhibited [3H]clonidine binding. NaCl decreased the binding affinity of [3H]clonidine, with no appreciable effect on the number of receptor sites. These findings indicate that ileal mucosal alpha 2-receptors can exist in multiple affinity states, which can be regulated by divalent cations, NaCl, and guanyl nucleotides. It appears that NaCl and GppNHp regulate alpha 2-receptors in ileal mucosa by different mechanisms.     

Role of N-methyl-d-aspartic acid and cholecystokinin receptors in apomorphine-induced aggressive behaviour in rats

We studied the aggressive behaviour induced by repeated treatment with apomorphine, a dopamine agonist (0.5 mg/kg s.c. twice daily, 10 days), in rats. The first signs of defensive aggressiveness appeared on the third day of apomorphine treatment and were generally seen on the 7th day. Aggressiveness induced by a challenge dose of apomorphine (0.5 mg/kg s.c.) on the 11th day was antagonized by haloperidol (0.05 and 0.1 mg/kg i.p.) and clozapine (10 mg/kg i.p.). An antagonist of N-methyl-D-aspartate (NMDA)-gated channels, dizocilpine (MK-801), also blocked the aggressive behaviour at 0.25 and 0.5 mg/kg i.p. but caused ataxia. When dizocilpine (0.25 mg/kg i.p.) and apomorphine were coadministered for 10 days, aggressive behaviour did not develop. At 0.025 mg/kg i.p., dizocilpine even accelerated the appearance of apomorphine-induced aggressive behaviour, which manifested on the 3rd day in all rats. In a separate study, a 7-day treatment with dizocilpine (0.25–1 mg/kg i.p.) of rats, sensitized by a prior 10-day apomorphine treatment, did not reverse the established aggressive behaviour. The coadministration of apomorphine and cholecystokinin (CCK)-A or -B antagonists, devazepide or L-365,260 (0.01–2.5 mg/kg i.p.) respectively, neither affected development of apomorphine-induced aggressive behaviour nor intensity of aggressiveness in the sensitized rats.In binding studies neither density nor affinity of striatal dopamine D₂ receptors was changed by acute or chronic apomorphine treatment. The number of [³H]pCCK-8 binding sites in the frontal cortex increased already after a single injection of apomorphine. After 10-day administration of apomorphine, a significant upregulation of [³H]pCCK-8 binding sites occurred in the frontal cortex and striatum, but a downregulation was observed in the hippocampus. A challenge dose of apomorphine (0.5 mg/kg s.c.) on the 11th day of experiment, normalized the upregulated CCK receptors in the frontal cortex and striatum. Acute apomorphine did not change [³H]-MK-801 binding in the rat brain. However, in rats treated for 10 days with apomorphine, the number of NMDA-gated channels in open state was increased in the frontal cortex and hippocampus. In these rats, a challenge dose of apomorphine (0.5 mg/kg s.c.) normalized also the in reased number of [³H]-MK-801 binding sites in the frontal cortex.In conclusion, repeated treatment with apomorphine seems to modify the function of dopamine D₂ receptors without affecting their number or affinity. The increased number of NMDA-gated channels in open state appears to be related to this alteration of dopamine D₂ receptors. The increased density of [³H]pCCK-8 binding sites in the frontal cortex may reflect anxiety and fear due to chronic exposure of rats to apomorphine.     

可乐定镇痛与中枢Ca2+的关系

用大鼠甩尾法和放射配基结合实验,探讨了可乐定镇痛与中枢Ca²⁺的关系。CaCl₂(1μmol/rat,icv)和EGTA(0.2μmol/rat,icv)分别拮抗和增强可乐定(1mg/kg,sc)的镇痛。戊脉安(0.1μmol/rat,icy)对可乐定(1 mg/kg,sc)镇痛无明显影响,但可部分翻转CaCl₂对可乐定镇痛的拮抗。CaCl₂(1×10^-3mol)对[³H]-可乐定结合无明显抑制。结果表明可乐定镇痛与脑室周围组织中Ca²⁺浓度变化密切相关,Ca²⁺至少部分需经对戊脉安敏感的钙通道进入细胞内方可拮抗可乐定镇痛。推沦:可乐定镇痛与神经元内Ca²⁺有关。     

Involvement of sulfhydryl groups in cerebral cortical [3H]clonidine binding in developing rats

The involvement of essential sulfhydryl groups in alpha 2-adrenoceptor function was investigated in the cerebral cortex of 7 and 70 day old rats. N-Ethylmaleimide (NEM) inhibited specific [3H]clonidine binding to cerebral cortical membranes in a concentration-dependent and biphasic manner in both infant and adult rats. The inhibitory effect of NEM was attenuated by simultaneous addition of dithiothreitol 30 microM and 1 mM, though dithiothreitol up to 1 mM did not affect the binding. p-Chloromercuriphenylsulfonic acid also caused a significant reduction in [3H]clonidine binding at both stages. Scatchard analysis of [3H]clonidine binding showed that NEM 10 and 100 microM caused a significant decrease in Bmax of high affinity binding sites without changing KD. Neither Bmax nor KD values were changed by NEM 10 and 100 microM in low affinity sites. The treatment with NEM 10 and 100 microM significantly reduced the increase of binding induced by Mn2+ 10 and 100 microM which was observed on day 70 but not on day 7. It is suggested that two distinct categories of essential sulfhydryl groups are involved in cerebral cortical alpha 2-adrenoceptors and are functionally mature by day 7. Furthermore, sulfhydryl groups are involved in the Mn2+-induced increase of alpha 2-receptors in adult rats.     

Glutamate and benzodiazepine receptor autoradiography in rat brain after repetition of alcohol dependence

During repeated alcohol withdrawal, convulsive withdrawal behaviour has been shown to be increased in a kindling-like manner in both clinical and experimental studies. In the present experiment, quantitative autoradiography was used to investigate binding of tritiated ligands to glutamate receptor subtypes and the benzodiazepine/GABA (BZ/GABA) receptor complex in rats exposed to 14 episodes of alcohol withdrawal. Seizures were detected in 25% of the animals during withdrawal episode 10–13. Repeated alcohol withdrawal resulted in a decrease in the number of [³H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]-AMPA) binding sites in striatum and subregions of the entorhinal cortex, the cerebellum and the hippocampus, while the [³H]-flunitrazepam binding was down-regulated in the frontal cortex. There was no differences between the controls and the multiple withdrawal animals regarding the [³H]-dizocilpine ([³H]-MK801) binding and the [³H]-kainic acid binding. However, within the latter group, those animals in which withdrawal seizures were observed had increased [³H]-MK801 binding sites in focal regions of entorhinal cortex and hippocampus, compared to those in which seizures were not observed. The decreased AMPA binding suggested impaired glutamate neurotransmission. As such, this receptor probably did not contribute to alcohol withdrawal kindling, but rather was involved in seizure protective mechanisms during this process.     

In vitro and in vivo effects of lead,methyl mercury and mercury on inositol 1,4,5-trisphosphate and 1,3,4,5-tetrakisphosphate receptor bindings in rat brain

In vitro and in vivo effects of mercury (Hg), methyl mercury (MM) and lead (Pb) on [³H]inositol 1,4,5-trisphosphate (IP₃) and [³H]linositol 1,3,4,5-tetrakisphosphate (IP₄) receptor binding in the Sprague-Dawley rat brain cerebellar membranes were studied. In vitro studies indicate that binding of [³H]IP₃ and [³H]IP₄ to cerebellar membranes was inhibited by Hg while they were stimulated by MM or Pb in a concentration-ependent manner. MM was more potent (EC₅₀ 3,4 μM) than Pb (EC₅₀ 18.2 μM) in stimulating the [³H]IP₃ receptor binding activity whereas Pb (IC₅₀ 30 μM) was more potent than MM (IC₅₀ 133 μM) in stimulating the [³H]IP₄, receptor binding. When the rats were treated (i.p) with Hg (5 mg/kg body wt.) or MM (5 mg/kg body wt.) or Pb (25 mg/kg body wt.) for 3 or 24 h, no significant alterations in[³H]IP₃ receptor binding were observed in cerebellum and cerebral cortex. But the above treatment of Pb or MM for 3 or 24 h to rats resulted in an increase of [³H]IP₄ receptor binding in the membranes of cerebral cortex. However, the rats treated with Hg (1 mg/kg body wt./day) or Pb (25 mg/kg body wt./day) for 7 days did not show any alteration in binding of [³H]IP₃ to its receptors in cerebellar membranes but an increase in this receptor binding was noticed with the treatment of MM (2.5 mg/kg body wt./day) for 7 days. The cerebellum and cerebral cortex of rats with the above treatment of MM or Pb for 7 days exhibited an increase in [³H]IP₄ receptor binding. These in vitro and in vivo data suggest that alterations in inositol polyphosphate receptor binding by metals could result in alterations in intracellular calcium levels which may influence neuronal activity.     

Further characterization of [3H]ifenprodil binding in rat brain

The present study was undertaken to characterize [³H]ifenprodil binding in rat brain. [³H]ifenprodil showed saturable, high-affinity binding at 4°C. Specific binding, defined with 10 μM ifenprodil as a competitor, was inhibited biphasically by the s receptor ligands, GBR 12909, 1,3-di-o-tolylguanidine (DTG), and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP). At 4°C, 3 μM GBR 12909, which inhibited about 50% of specific binding of [³H]ifenprodil, was used to mask σ receptors. Under these conditions, specific binding of [³H]ifenprodil was inhibited potently by ifenprodil, SL 82.0715, poly(l-arginine), poly(l-lysine), neomycin, ruthenium red, spermine, arcaine and spermidine. In the presence of 3 μM GBR 12909, Zn²⁺ and Mg²⁺ partially inhibited specific binding of [³H]ifenprodil at 4°C. In contrast, in the absence of GBR 12909, at 37°C specific binding of [³H]ifenprodil was partially inhibited by Zn²⁺, but not by Mg²⁺. The anatomical distribution of [³H]ifenprodil binding at 4°C (GBR 12909 included) in rat brain closely paralleled that of [³H]MK-801 (dizocilpine) binding (r = 0.971, P < 0.005). Without GBR 12909, specific [³H]ifenprodil binding at 37°C was inhibited potently by σ ligands. In the presence of 3 μM GBR 12909, [³H]ifenprodil binding at 4°C was highest in synaptosomal and myelin fractions; however, without GBR 12909, [³H]ifenprodil binding at 37°C was highest in microsomal and myelin fractions, consistent with the subcellular distribution of σ receptors. The results suggest that, in the presence of 3 μM GBR 12909, at 4°C, [³H]ifenprodil binds to sites that are sensitive to polyamines and related compounds; and that without GBR 12909, at 37°C, [³H]ifenprodil interacts with σ receptors in rat brain.