C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

Localization of α1-adrenoceptors in rat and human hearts by immunocytochemistry

The localization of the ai adrenoceptors (₁-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human ₁-AR (AS sequence 192–218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, ₁-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of ₁-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the -adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.     

Distribution of the α2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues

Summary The hepatic ₁-macroglobulin receptor (₂MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses ₂-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of ₂MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed ₂MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocytederived cells displayed marked ₂MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of ₂MR/LRP in certain cell types of most organs suggests two main routes for ₂MR/LRP ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.     

G proteins,adenylyl cyclase and related phosphoproteins in the developing rat heart

The postnatal alterations of the composition of a subunit isoforms (G_ic, G _i3 G_o, and _Gq of G proteins, the adenylyl cyclase activity as well as of cAMP-regulated phosphoproteins e.g. troponin and phospholamban were investigated in the ventricular tissue of 1, 7, 30 days old rats. Quantitative immunodetection revealed a 5.7-fold decrease in G_i3 at 30th postnatal day compared with the postnatal day 1 and up to 15-fold at 4 months. The amounts of G_q and G as well as the G subunits were found to be higher in the earlier life period compared to the adult. In contrast, the content of G_s was uneffected by the developmental state. Basal adenylyl cyclase activity (pmoles cAMP/min × mg protein) increased from 30.9 ± 5.0, 36.8 ± 5.0 to 63.9 ± 5.9 at 1st, 7th and 30th postnatal day, respectively. Isoprenaline (100 M) enhanced the activity of adenylyl cyclase from day 1, 7–30 from 46.2 ± 7.0, 79.1 ± 9.2 to 120.5 ± 7.2, respectively. The effects of forskolin and NaF on adenylyl cyclase activity was found to be not influenced within the first postnatal month. Furthermore, a developmentally controlled expression of cardiac troponin I was observed (6-fold from the first to the 28th postnatal day) whereas the level of phospholamban was found to be age-independent.In conclusion, there is an increase in the efficiency of the -adrenergic signal transfer mainly caused by a reduction of the inhibitiory G proteins and a dominance of the G_s-linked pathway in the postnatal rat heart. Furthermore the developmentally controlled expression of troponin I might be of functional importance in the cAMP-supported relaxation. Additionally, altered G_q, G_o and G pattern of the developing rat ventricle may play a role in the observed change of -adrenerg-mediated heart contractility as well as in cardiac differentiation and growth processes.     

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

Phospholipase C activation in rat pituitary adenoma (GH) cells

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G_q and/or G₁₁ has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G_q and/or G₁₁ confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G_q/11-sera coprecipitated PL-C activity. In essence, only G_q/11 (but neither G_i2, G_i3 nor G_o) seems to mediate the TRH-sensitive PL-C activity, while G_o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH₃ pituitary cell line PL-C activity. Finally, the steady state levels of G_q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP).     

GABA A -receptors: Structural requirements and sites of gene expression in mammalian brain

GABA_A-receptors, the major synaptic targets for the neutotransmitter GABA, are gated chloride channels. By their allosteric drug-induced modulation they serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension and epileptiform activity can be regulated. Despite their functional prominence, the structural requirements of fully functional GABA_A-receptors are still elusive. Expression of cDNAs coding for the ₁- and ₁-subunits of rat brain yielded GABA-gated chloride channels which were modulated by barbiturates but displayed only agonistic responses to ligands of the benzodiazepine receptor. GABA_A-receptors with fully functional benzodiazepine receptor sites were formed when the ₁- and ₁-subunits were coexpressed with the ₂-subunit of rat brain. These receptors, however, failed to show cooperativity of GABA in gating the channel. In order to determine the subunit repertoire available for receptor assembly in different neuronal populations in vivo, the sites of subunit gene expression were (₁, ₂, ₃, ₅, ₆, ₁, ₂, ₃, ₂) mapped by in situ hybridization histochemistry in brain sections. The mRNAs of the ₁-, ₁- and ₂-subunits were co-localized e.g. in mitral cells of olfactory bulb, pyramidal cells of hippocampus as well as granule cells of dentate gyrus and cerebellum. The lack of colocalization in various other brain areas points to an extensive receptor heterogeneity. The presence of multiple GABA_A-receptors in brain may contribute to synaptic plasticity, differential responsiveness of neurons to GABA and to variations in drug profiles.Special issue dedicated to Dr. Erminio Costa     

The alpha2-adrenergic receptor system in the hypothalamus of the Pekin duck

Summary In the present study, we have employed the monoradioiodinated ₂-agonist clonidine ([¹²⁵I]-CLO) to characterize duck hypothalamic ₂-adrenoceptors and to localize ₂-specific binding sites in the duck brain. To validate the ₂-specificity of [¹²⁵I]-CLO using an enriched duck hypothalamic membrane fraction, a radioreceptor assay was established by altering the membrane protein concentration, time, temperature and ionic milieu of incubation, and in the presence or absence of protease inhibitors. Competitive displacement studies revealed the following sequence of potency to displace [¹²⁵I]-CLO: yohimbine>(-)-epinephrine>clonidine> (-)-norepinephrine>phentolamine>(-)-phenylephrine>(-)-isoproterenol>prazosin. The non-hydrolyzable guanosine 5-triphosphate analog guanylylimidodiphosphate markedly inhibited [¹²⁵I]-CLO binding suggestive of G-protein involvement. With regard to the histological distribution, diencephalic structures, such as the habenula and the nucleus reticularis of the thalamus, were densely labeled by [¹²⁵I]-CLO. In the hypothalamus, ₂-adrenoceptors were detected in the antidiuretic hormone-synthesizing nucleus paraventricularis, the nucleus praeopticus medialis, the nucleus anterior medialis hypothalami, the nucleus magnocellularis praeopticus, the nucleus commissurae pallii, the nucleus inferior hypothalami and the regio lateralis hypothalami. Circumventricular organs, such as the plexus choroidei, organum subfornicale, organum paraventriculare and the corpus pineale, were endowed with ₂-specific binding sites, as were the cell layers of the tectum opticum. In addition, telencephalic structures revealed high receptor densities. The presence of well characterized ₂-specific adrenoceptors in hypothalamic structures of the duck brain including associated telencephalic regions supports physiological investigations with regard to functional ₂-adrenergic modulation of antidiuretic hormone release in the duck.     

Selective down-regulation of the Cqα/G11α G-protein family in tumour necrosis factor-α induced cell death

Investigations into the regulation of heterotrimeric GTP-binding protein a-subunits in models of tumour necrosis factor- (TNF)-induced cell death, revealed the selective down-regulation of the G_q/G₁₁ family of G-proteins. The human HeLa and murine L929 cells treated with recombinant human TNF for up to 24 h displayed down-regulated G_q/G₁₁ family protein levels, but not G_s, G_i and G_o protein levels as determined by Western analyses. This effect of TNF was observed in a concentration - and time-dependent manner, consistent with the profiles of TNF-induced cell death observed. Moreover, the functioning of G_q/G₁₁ family proteins were found to be impaired in TNF-treated cells, as measured by agonist-induced [Ca²⁺],_i release. In contrast, G_s activity was unaltered by TNF-treatment, determined by measurement of agonist-induced intracellular cyclic AMP generation. These findings in TNF-induced cytotoxic models, indicate a novel 'cross-talk' mechanism by which TNF alters Ca²⁺-signalling mechanisms, which may contribute towards the apoptotic and necrotic cell death.     

Distribution and quantification of alpha1-integrin subunit in rat organs

Summary The ₁₁-integrin is known to be a receptor for collagen and laminin mediating cell-matrix interactions. A monoclonal antibody, 33.4, which specifically inhibits the ₁-integrin-mediated in vitro cell-collagen binding of rat hepatocytes and hepatoma-derived A-cells (Löster et al., 1994), was used to purify by immunoaffinity chromatography the ₁-integrin subunit from rat liver in large quantities for inducing a polyclonal antiserum. In immunoblot analysis on membrane extracts of several rat organs this polyclonal antiserum recognized only a 190 kDa-band, suggesting that it is highly specific for the ₁-integrin subunit. A sandwich-ELISA with monoclonal antibody 33.4 and the polyclonal antiserum against the ₁-integrin subunit, respectively, enabled the quantitative expression pattern of the ₁-integrin subunit to be studied in different rat organs. With the exceptions of brain (not detectable) and muscle (low concentration), the ₁-integrin subunit was detectable in almost all organs of the digestive, respiratory and urogenital system as well as in lymphatic organs. The highest relative concentrations of ₁-integrin subunit were found in uterus, lung and spleen, whereas in seminal vesicle, stomach, parotid gland, epididymis, kidney and liver only modest concentrations were evident. The organ distribution and localization of ₁-integrin subunit were studied by immunohistochemistry with monoclonal and polyclonal antibodies. Immunoreactivity was present in the plasma membranes of all smooth muscle cells, vascular endothelial cells of many organs and fibrocyte-fibroblast sheaths in the heart and kidney. Since these cells are in close contact with collagen-containing basal membranes as well as reticular fibrils, strong evidence exists that in rat tissues the ₁-integrin subunit is expressed at sites where collagen is present and might be involved in vivo in cell—ollagen binding.Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

Functional Coupling of G Proteins to Endothelin Receptors Is Ligand and Receptor Subtype Specific

1. The aims of the present study were (a) to determine the identity of the G proteins with which the endothelin receptor interacts and whether this interaction is subtype specific and (b) to determine whether agonist exposure can result in specific coupling between the endothelin receptor and G proteins.2. Coupling between endothelin A (ET_A) or endothelin B (ET_B) receptors and G proteins was assessed in two fibroblast cell lines, each expressing one receptor subtype. Four ligands, ET-1, ET-3, SRTXb, and SRTXc, were used for receptor stimulation. The G protein -subunit coupled to the receptor was identified by immunoprecipitation with an antibody against the endothelin receptor and immunoblotting with specific antibodies against different G protein -subunits.3. Unstimulated ET_A and ET_B receptors (ET_AR and ET_BR, respectively) were barely coupled to G_o. The unstimulated ET_AR coimmunoprecipitated with G_i3, whereas the unstimulated ET_BR was much less strongly coupled to G_i3. The coupling of ET_BR to G_i1G_i2 -subunits was much stronger than the coupling of ET_AR to these -subunits. Stimulation with the different ET agonists also resulted in differential coupling of G proteins to the receptor subtypes. All four ligands caused a strong increase in coupling of the ET_BR to G_i3, whereas coupling of the ET_AR to this subunit was not affected by ET-1 and was even decreased by SRTXc. On the other hand, all four ligands caused a much greater increase in the coupling of ET_AR to G_q/G₁₁ than in the coupling of ET_BR to these -subunits. Ligand-induced coupling between the receptors and the G_i1 and G_i2 -subunits is similar for the two receptor subtypes. The same was true for ligand-induced coupling of the receptors to G_o, except that ET-3 increased the coupling of this -subunit to ET_BR and decreased the coupling to ET_AR. Taken together, the results of this study show that coupling between ET receptors and G proteins is ligand and receptor subtype specific.4. It remains to be established whether this diversity of receptor–G protein coupling is of relevance for the various endothelin signaling pathways and/or pathological states.     

Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: Possible involvement of tyrosine kinase and protein kinase C

Monocytic U937 cells were differentiated into mature macrophages in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h at 37°C. We investigated the alterations in the expression of GTP-binding proteins that take place during differentiation of these cells. A 40 KDa -subunit of the inhibitory G-protein was identified by specific antibodies to G_i-1/2 and G_i-3 on Western blots and also by ADP-ribosylation catalyzed by pertussis toxin. The expression of the 40 KDa G_i subunit was increased 3.4 fold in differentiated cells. The expression of a 43 kDa G_s subunit identified by Western blotting using specific antibody to G_s and by ADP-ribosylation in the presence of cholera toxin was increased approximately 2 fold in differentiated cells. A faintly recognizable 46 KDa G_s subunit was also increased but to a lesser extent (1.3 fold). Small molecular weight GTP-binding proteins identified by [³⁵S]GTPS binding on nitrocellulose blots were also increased significantly. The PMA-induced expression of G_i-1/2 and G_s subunits was blocked to control level by both genistein and staurosporine, inhibitors of protein tyrosine kinase and protein kinase C, respectively. However, staurosporine was unable to block the PMA-induced expression of G_i-3; this was blocked only by genistein. These data suggest a role for tyrosine kinase and protein kinase C in the expression of G-proteins during differentiation of U937 cells.     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups

The triated adrenergic antagonists Prazosin ([³H]PRZ) and Idazoxan ([³H]IDA, or RX-781094) bind specifically and with high affinity to ₁ and ₂-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (K _d) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (–SS–) bridges and sulfhydryl (–SH) groups in the specific combination of [³H]PRZ and [³H]IDA to the ₁ and ₂-adrenoceptors. Pretreatment of the membranes with the –SS– reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [³H]IDA binding (₂-sites) was more sensitive to both DTT and NEM than the [³H]PRZ sites (₂-adrenoceptors), and the initial changes induced by alkylation of the ₂-site were due to an important decrease in the affinity for [³H]IDA, as judged by the increase in theK _d. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the ₂-site. The results provide evidence for the participation of –SS– and –SH groups in the binding site of ₁ and ₂-adrenoceptors in the cerebral cortex.