Comparison of Neuroleptic Binding Characteristics in Rat Striatum and Renal Cortex

Abstract

The binding characteristics of the dopaminergic ligand, ³H- spiperone, were compared in renal cortical and striatal membrane homogenates of the rat. This ligand labelled a single class of high affinity binding sites in striatum with an apparent dissociation constant (Kd) of 0.13 nM and a maximal number of binding sites (Bmax) of 890 fmol/mg protein representing D-2 receptors. In the renal cortex, ³H-spiperone identified a population of binding sites with a Bmax and a Kd of 310 fmol/mg protein and 5.1 nM, respectively. The antagonist displacing profile suggests the dopaminergic nature of the renal binding site. The affinities of dopamine antagonists for the peripheral ³H-spiperone binding site were in general in the micromolar range while the affinities of D-2 or D-2/D-1 dopamine antagonists in striatum were in the nanomolar range. Moreover, these sites showed differential stereoselectivity for (+)- and (-)-isomers of sulpiride. In conclusion, the presence of a D-2/DA-2 dopamine receptor population in renal cortex could not be confirmed. The pharmacological properties of the peripheral ³H-spiperone binding site are also different from the DA-1 receptor but seem to resemble those previously reported for dopamine receptors in sympathetic ganglia and adrenal medulla.     

Interaction of non-steroidal antiestrogens with dopamine receptor binding

The ability of various estrogen antagonists and agonists to compete with [3H]spiroperidol, [3H]domperidone, [3H]dihydroalprenolol, [3H]dihydroergocryptine, [3H]dopamine or [3H]5-hydroxytryptamine for binding to membrane preparations from rat brain tissue was tested. The non-steroidal triphenylethylene-type antiestrogens with an amine side chain--enclomiphene, nitromifene, tamoxifen and zuclomiphene--were found to be competitive inhibitors of [3H]spiroperidol (Kd = 0.12 nM; Bmax = 101 fmol/mg protein) and [3H]domperidone (Kd = 0.62 nM; Bmax = 86 fmol/mg protein) binding to striatal membranes. The Ki values ranged from 4-12 microM. Estradiol-17 beta (Ki = 480 microM) or diethylstilbestrol (Ki = 63 microM) were much less effective inhibitors exhibiting noncompetitive interaction with the in vitro binding of [3H]spiroperidol. The pharmacological relevance of the antiestrogen interactions with dopamine receptor binding is discussed with respect to adverse effects of the in vivo administered compounds such as nausea and vomiting.     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stereospecific binding of a new benzazepine, [3H]SCH23390, in cortex and neostriatum

The binding of the D1 antagonist SCH23390 to membrane preparations from rat cerebral cortex was examined using enantiomers of dopamine agonists and antagonists to compete with the bound [3H]SCH23390 at its Kd value. The competition curves were compared with those obtained with preparations from the neostriatum. The results demonstrate that specific [3H]SCH23390 binding in the cerebral cortex has the same pharmacological profile as in the neostriatum, so that this radioligand can be used to label dopamine D1 receptors in brain regions with a sparse dopaminergic innervation.     

Evidence for the presence of dopaminergic receptors in vas deferens

Specific dopaminergic recognition sites were identified in membranes prepared from rat vas deferens with the ligand (³H)-haloperidol. Specific binding, defined as the difference of (³H)-haloperidol binding in the presence or absence of an excess of unlabelled haloperidol (100 μM), was saturable and a Scatchard analysis of the data revealed a Kd = 21 nM and a Bmax = 74 fmol/mg prot. (+)-Butaclamol was several times more active in displacing (³H)-haloperidol from binding sites than its pharmacologically inactive enantiomer, (?)-butaclamol, demonstrating stereospecificity of binding. Dopamine displaced 50% of (³H)-haloperidol binding at a concentration of approximately 10 μM, while norepinephrine, epinephrine and serotonin were practically ineffective at this concentration. Our results support the notion that there are dopaminergic receptors in the rat vas deferens. We speculate that some of the known effects of dopamine and dopaminergic drugs on sexual behavior may be mediated peripherally and not solely via the CNS as is usually assumed.     

Pregnancy related changes of opiate receptors identified in rat uterine membranes by 3H-naloxone binding

3H-Naloxone was used to demonstrate the presence of specific opiate binding sites in uterine membrane preparations of rats. 3H-Naloxone binding (0.41-27 nM) was found to be rapid, saturable and reversible showing two populations of binding sites with the characteristic of high (KD 2.2 nM; Bmax 46.6 fmol/mg prot.) and low (KD 18.1 nM; Bmax 143.7 fmol/mg prot.) affinity. The number and affinity of the binding sites labelled by 3H-naloxone in the uterus were measured in the rat at mid (14 days), late (21 days) pregnancy and at parturition. The high and low affinity recognition sites labelled by 3H-naloxone showed a consistent reduction during pregnancy and at parturition without changes in the affinity constant. We concluded that pregnancy and parturition are associated with significant changes in the number of the opiate receptors bound in the uterus by 3H-naloxone. This phenomenon which seems to be linked with the several pregnancy-related changes in the levels of endogenous peptides and hormones could be relevant to further explain the pregnancy related changes in pain perception and maternal behavior.     

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.     

Nitrobenzylthioinosine binding in brain: an interspecies study

The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ( [3H]NBI) to cerebral cortical membrane preparations from human, dog, guinea pig, rat, and mouse was investigated. Reversible, specific, saturable, high affinity binding was found in all five species with similar kinetic parameters. (Kd = 0.16-0.44 nM; Bmax = 128-196 fmol/mg prot.). Dilazep, hexobendine, and dipyridamole were all high potency inhibitors of [3H]NBI binding in human, dog, guinea pig and mouse preparations but not in rat. These compounds showed a competitive inhibition of [3H]NBI binding indicating that they are acting at the same site. Discrepancies seen in the rat appear to be a unique, species related anomaly. The dihydropyridine calcium antagonists also inhibited binding with lower potency than the adenosine uptake blockers. This inhibition was most potent in dog and human and suggests a relationship between the calcium channel and the adenosine uptake site.     

Selective labeling of pre-synaptic receptors by 3H-dopamine, 3H-apomorphine and 3H-clonidine; labeling of post-synaptic sites by 3H-neuroleptics.

To test the hypothesis that nM concentrations of ³H-dopamine, ³H-apomorphine and ³H-clonidine prefer pre-synaptic sites, while ³H-neuroleptics and ³H-dihydroergocryptine prefer post-synaptic sites, we tested catecholaminergic agonists and antagonists on the binding of these radio-ligands to calf caudate tissue. 1) Dopamine agonists (apomorphine, NPA and bromocryptine) inhibited ³H-spiperone binding, but not ³H-dopamine binding, in direct correlation to their clinical potencies. 2) Dopamine agonists inhibited ³H-apomorphine binding at concentrations identical to those causing pre-synaptic cardio-inhibition. 3) The IC₅₀ values for ³H-dihydroergocryptine binding of alpha-adrenoceptor drugs did not correlate with the pre-synaptic IC₅₀ values for affecting noradrenaline release; those for ³H-clonidine did. The 3 findings are compatible with the working hypothesis.     

3H-spiroperidol binding sites in blood platelets

3H-spiroperidol, an antagonist of dopamine receptors in brain (striatum), was found to bind to human and rat platelet membrane preparations. The binding was rapid, reversible, saturable and specific. Unlabelled haloperidol displaced the specifically bound 3H-spiroperidol. Binding equilibrium was attained in 15 min at pH 7.4 and 37 degrees C. Scatchard analysis of 3H-spiroperidol binding revealed a single population of binding site with Kd of 7.6 nM in rat platelet membrane and Kd of 15 nM in human platelet membrane. Unlabelled 5-hydroxytryptamine produced no significant effect on 3H-spiroperidol binding to rat or human blood platelet membranes in the presence or absence of haloperidol. Some dopaminergic agents, known to inhibit spiroperidol binding in corpus striatum, also inhibited the same in rat and human blood platelet membranes under in vitro conditions. This study suggests the presence of specific 3H-spiroperidol binding sites in blood platelets.     

Identification of a high-affinity receptor for interleukin-1 beta in rat brain

A single type of high-affinity binding sites for IL-1 beta was identified in the rat hypothalamus (Kd = 1.0 +/- 0.2 nM) and cerebral cortex (Kd = 1.3 +/- 0.2 nM), but not in the pituitary. The maximum binding capacity (Bmax) in the hypothalamus (Bmax = 75.4 +/- 10.8 fmol/mg protein) was 4 times greater than in the cerebral cortex (Bmax = 17.2 +/- 1.5 fmol/mg protein). Neither various neuropeptides nor IL-2 appeared to influence the binding of [125I]IL-1 beta to the hypothalamic membrane preparations. The potency of unlabeled IL-1 alpha to replace the binding of [125I]IL-1 beta to the hypothalamic membrane preparations was considerably less than that of unlabeled IL-1 beta. These findings indicate that IL-1 beta receptors are heterogeneously distributed in the central nervous system and that IL-1 alpha does not bind with IL-1 beta receptors in the brain.     

125I-Spiperone: a novel ligand for D2 dopamine receptors

125I-Spiperone binds with high affinity (KD 0.3 nM) to a single specific site (Bmax 34 pmol/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of 125I-spiperone binding (Ki's 1-10 nM). Selective dopamine antagonists such as sulpiride (Ki 50 nM) and dopamine agonists such as apomorphine (Ki 200 nM) are also potent inhibitors. The drug specificity of 125I-spiperone binding correlates well with that of 3H-spiperone binding, providing good evidence that 125I-spiperone labels D2 dopamine receptors in striatal membranes. 125I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D2 dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.     

Characterization of kinin binding sites: identity of B2 receptors in the epithelium and the smooth muscle of the guinea pig ileum.

Binding of [125I-Tyr8]bradykinin (BK) was measured in homogenates of epithelial and smooth muscle layers of the guinea pig ileum. Binding assays were performed at 4 degrees C for 40 min (smooth muscle) or 90 min (epithelium) in 25 mM PIPES buffer at pH 6.8 in the presence of 1 mM 1,10-phenanthroline, 140 micrograms/mL bacitracin, 1 mM captopril, 1 mM dithiothreitol, and 0.1% bovine serum albumin. Specific binding of [125I-Tyr8]BK (0.32 nM) to epithelial and smooth muscle cell membranes was linearly related to protein concentration between 0.05 and 0.5 mg/mL. Equilibrium experiments showed that specific binding of [125I-Tyr8]BK was saturable and Scatchard analysis indicated the presence of a high affinity site with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg of protein in the epithelial cell membranes. In smooth muscle membranes, Kd was 1.8 nM and the maximum number of binding sites was 58 fmol/mg of protein. Unlabelled peptides, namely bradykinin, [Tyr8]BK, [Hyp3]BK, D-Arg[Hyp3]BK, [Hyp3,Tyr(Me8)]BK, and kallidin displaced [125I-Tyr8]BK binding while other peptides, angiotensin II and substance P, had no effect. A series of B2-receptor antagonists displaced [125I-Tyr8]BK from specific binding sites with IC50 values ranging from 16 to 152 nM on epithelial cell membranes; similar values were obtained from smooth muscle cell membranes. These findings suggest that the binding sites in both preparations are of the B2 type. B1-receptor agonists and antagonists were found to be inactive at concentrations up to 10(-4) M. Results obtained in the two preparations were compared and a positive highly significant correlation was demonstrated between the two sets of data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization and autoradiographic localization of substance P receptors in guinea pig brain

[3H]Substance P ([3H]SP) was used to characterize substance P (SP) receptor binding sites in guinea pig brain using membrane preparations and in vitro receptor autoradiography. Curvilinear Scatchard analysis shows that [3H]SP binds to a high affinity site (Kd = 0.5 nM) with a Bmax of 16.4 fmol/mg protein and a low affinity site (Kd = 29.6 nM) with a Bmax of 189.1 fmol/mg protein. Monovalent cations generally inhibit [3H]SP binding while divalent cations substantially increased it. The ligand selectivity pattern is generally similar to the one observed in rat brain membrane preparation with SP being more potent than SP fragments and other tachykinins. However, the potency of various nucleotides is different with GMP-PNP greater than GDP greater than GTP. The autoradiographic distribution of [3H]SP binding sites shows that high amounts of sites are present in the hippocampus, striatum, olfactory bulb, central nucleus of the amygdala, certain thalamic nuclei and superior colliculus. The cortex is moderately enriched in [3H]SP binding sites while the substantia nigra contains only very low amounts of sites. Thus, the autoradiographic distribution of SP binding sites is fairly similar in both rat and guinea pig brain.     

Evidence for a single class of somatostatin receptors in ground squirrel cerebral cortex

In the present study we characterized high-affinity somatostatin (SRIF) binding sites (Kd = 2.06 +/- 0.32 nM and Bmax = 295 +/- 28 fmol/mg protein) in cerebral cortex membrane preparations of European ground squirrel using 125I-[Tyr0-D-Trp8]-SRIF14 as a radioligand. The inhibition of radioligand specific binding by SRIF14, as well as by its agonists (SRIF28, Tyr0-D-Trp8-SRIF14, SMS 201 995) was complete and monophasic, thus revealing a single population of somatostatinergic binding sites. Radioautographic analysis of 125I-[Tyr0-D-Trp8]-SRIF14 labeled brain sections confirmed the results of our biochemical study. The homogeneity of SRIF binding sites in the ground squirrel neocortex was not dependent on the animal's life-cycle phase.     

Specific vasopressin binding to rat adrenal glomerulosa cells. Relationship to inositol lipid breakdown.

Cells from the zona glomerulosa of rat adrenals were isolated and maintained for 3 days in primary culture. Specific vasopressin binding was determined by using [3H]vasopressin. [3H]Vasopressin binding was time-dependent (half-time of about 2 min for 6 nM free ligand) and reversible on addition of unlabelled vasopressin (80% dissociation within 30 min). Dose-dependent [3H]vasopressin binding at equilibrium indicated that vasopressin interacted with two populations of sites: high-affinity sites (dissociation constant, Kd = 1.8 nM; maximal binding capacity = 10 fmol/10(6) cells) and low-affinity sites. Vasopressin increased the cellular content of labelled inositol mono-, bis- and tris-phosphate in cells prelabelled with myo-[3H]inositol. The vasopressin concentration eliciting half-maximal inositol phosphate accumulation was very close to the Kd value for vasopressin binding to high-affinity sites. Competition experiments using agonists and antagonists with enhanced selectivity for previously characterized vasopressin receptors indicated that vasopressin receptors from rat glomerulosa cells are V1 receptors of the vascular or hepatic subtype. The detected specific vasopressin-binding sites might represent the specific receptors mediating the mitogenic and steroidogenic effects of vasopressin on glomerulosa cells from rat adrenals.     

Identification and characterization of (3H)-rauwolscine binding to alpha 2-adrenoceptors in the canine saphenous vein

The biochemical exploration of the alpha 2-adrenergic receptors was investigated in the canine saphenous vein using the highly selective alpha 2-adrenergic antagonist rauwolscine as a tritiated ligand. Following an enzymatic digestive pretreatment, we isolated a purified smooth muscle cell membrane fraction from saphenous veins in quantity sufficient to permit us to study the venous alpha 2-adrenoceptor content. The binding of tritiated rauwolscine was rapid, specific, saturable and reversible. The presence of high affinity binding sites (Kd = 1.53 +/- 0.71 nM) with a density of binding Bmax of 125.2 +/- 43.1 fmol/mg protein was demonstrated on a unique class of non interacting sites (nHill = 1.001 +/- 0.06). The kinetically derived Kd was 1.28 nM, in good agreement with the value obtained from saturation isotherms. The pharmacological profile of these sites was assessed by the comparison of the potency of alpha-adrenergic agonists and antagonists to inhibit 1 nM (3H)-rauwolscine. Their efficacy was respectively: rauwolscine greater than phentolamine greater than RX 781094 greater than clonidine much greater than prazosin greater than (-)-phenylephrine greater than (-)-noradrenaline. The results showed that (3H)-rauwolscine bound specifically to sites in our membranal preparation, which had the pharmacological characteristics of the alpha 2-adrenoceptors. The correlation between biochemical and pharmacological data revealed the usefulness of binding methods in the further study of adrenergic mechanisms in the canine saphenous vein.     

Irreversible interaction of beta-haloalkylamine derivatives with dopamine D1 and D2 receptors

The interaction of beta-haloalkylamine derivatives of dopamine agonists and antagonists with 3H-spiperone binding (D2 sites) and 3H-flupenthixol binding (D1 sites) was studied. N-chloroethyl derivatives of phenothiazines and thioxanthenes were potent inhibitors of the binding of both ligands. The in vitro inhibition of binding produced by these compounds was irreversible. The drugs were however only weakly active in vivo. The results suggest that beta-haloalkylamine derivatives of neuroleptics may be useful compounds for studying dopamine receptors in vitro.     

Binding of vasoactive intestinal polypeptide (VIP) by human blood monocytes: demonstration of specific binding sites

Vasoactive intestinal polypeptide (VIP) interaction with a 94% pure preparation of monocytes isolated from human peripheral blood was studied by direct binding technique using 3-[125I]tyrosyl-VIP as a tracer ligand. Scatchard analysis of binding data was compatible with two classes of binding sites, one with Kd = 0.25 nM and maximal binding capacity of 16 fmol/10(6) cells, and another one with Kd = 25 nM and maximal binding capacity of 180 fmol/10(6) cells. The binding was time-, temperature-, and pH-dependent and was saturable, reversible, and specific. This study has demonstrated that human monocytes have high affinity/low capacity as well as low affinity/high capacity binding sites for VIP. No specific VIP binding was found in pure preparations of human granulocytes, platelets or erythrocytes.     

The bindings of 3H-prazosin and 3H-yohimbine to alpha adrenoceptors in the guinea-pig stomach

Alpha adrenoceptor subtypes have been investigated by radioligand binding study in guinea-pig stomach using 3H-prazosin and 3H-yohimbine. The specific 3H-prazosin binding to guinea-pig stomach was saturable and of high affinity (KD = 1.4 nM) with a Bmax of 33 fmol/mg protein. Specific 3H-yohimbine binding to the tissue was also saturable and of high affinity (KD = 25.5 nM) with a Bmax of 150 fmol/mg protein. Adrenergic drugs competed for 3H-prazosin binding in order of prazosin greater than phentolamine greater than methoxamine greater than norepinephrine greater than clonidine greater than epinephrine greater than yohimbine. These drugs competed for 3H-yohimbine binding in order of yohimbine greater than phentolamine greater than clonidine greater than epinephrine greater than norepinephrine greater than prazosin greater than greater than prazosin greater than methoxamine. We also examined whether dopamine receptors exist in guinea-pig stomach, using radioligand binding study. Specific binding of 3H-spiperone, 3H-apomorphine, 3H-dopamine and 3H-domperidone was not detectable in the stomach. Dopaminergic drugs such as dopamine, haloperidol, domperidone and sulpiride competed for 3H-prazosin binding in order of haloperidol greater than domperidone greater than dopamine greater than sulpiride. Metoclopramide, sulpiride and dopamine competed for 3H-yohimbine binding in order of metoclopramide greater than sulpiride greater than dopamine. These results suggest that guinea-pig stomach has alpha 1 and alpha 2 adrenoceptors and has no specific dopamine receptors. It is also suggested that some dopamine receptor antagonists such as domperidone, haloperidol, sulpiride and metoclopramide have antagonistic actions on alpha adrenoceptors.