Prostaglandin E2 binding site distribution and subtype classification in the rabbit iris-ciliary body.

The distribution and characteristics of specific binding sites for tritium labeled prostaglandin E2 (3H-PGE2) were examined in membrane preparations from rabbit iris-sphincter, iris and ciliary body. The majority of 3H-PGE2 specific binding sites were found in the ciliary body (46%) followed by the iris (37%) and the iris-sphincter muscle (5%). Scatchard analysis of saturable 3H-PGE2 binding sites in the ciliary body indicated a single binding site with a Kd of 2.81 nM and Bmax value of 84 fmoles bound/mg protein. Competition by agonists selective for the EP1, EP2 and EP3 receptor subtypes of the EP (PGE2) prostanoid receptor indicated that the majority of rabbit ciliary body 3H-PGE2 binding sites are of the EP2 subtype. Incomplete displacement of labeled 3H-PGE2 from its binding sites by the EP2 selective agonist 11-deoxy PGE1 suggests the presence of additional EP or non-EP binding sites. There was essentially no binding to EP1 receptor sites as defined by the displacement of 3H-PGE2 by 17-phenyl-trinor PGE2. A weak displacement of 3H-PGE2 by the EP3/EP1 specific agonist, sulprostone, may account for the presence of a small number of EP3 specific binding sites in this tissue. The predominant distribution of PGE2 binding sites in the ciliary body and their identification as EP2 selective, supports recent functional studies where topical application of prostanoids with EP2 but not EP1 or EP3 agonist activity resulted in breakdown of the blood-aqueous barrier.     

Effect of pituitary hormones on phosphodiesterase and adenyl cyclase activity of brain tissue in vitro

Effects of seiwhale somatotropin (STH), its biologically active fragment 77--107, porcine corticotropin (ACTH) and seiwhale prolactin on phosphodiesterase and adenylate cyclase activity of glial cells and synaptosomes isolated from the rat brain cortex were investigated. As compared with control, ACTH increased phosphodiesterase activity of glial cells by 392%, of synaptosomes by 123%, while STH by 49 and 77%, respectively, somatotropin fragment by 455 and 74%, and prolactin by 30 and 37%, respectively. Adenylate cyclase activity was significantly changed only by ACTH and only in synaptosomes (a 50% decrease). STH, its fragment and prolactin virtually failed to alter adenylate cyclase activity. The data obtained indicate that some of pituitary hormones, primarily ACTH and STH, may play the role of neuromodulators in some brain structures by decreasing the cyclic AMP level, by activating phosphodiesterase (STH and ACTH) and inhibiting adenylate cyclase (ACTH in synaptosomes).     

Thiopental and ether anaesthesia decrease the prostaglandin receptor density of the rat liver.

Male Sprague-Dawley rats were killed either by decapitation or during anaesthesia with thiopental or diethylether by aortectomy. Livers were removed and liver plasma membranes were prepared using standard techniques. Direct binding experiments with 3H-PGE1 and 3H-iloprost revealed heterogeneity of the binding sites (high and low affinity binding sites), whereas 3H-PGE2 demonstrated only high affinity binding to the liver. The highest binding capacity for all radioligands was found for livers after decapitation. Livers obtained during anaesthesia showed a significantly (p less than 0.05 to p less than 0.001) lower binding capacity and binding affinity for 3H-PGE1, 3H-PGE2 and 3H-iloprost. The reduction in binding activity was more pronounced in livers obtained during inhalation than thiopental anaesthesia. Specific binding amounted to 82.1 +/- 7% for 3H-PGE2, 75.3 +/- 9% for 3H-PGE1 and 78.9 +/- 8% for iloprost in livers obtained after decapitation. In livers obtained during anaesthesia specific prostaglandin binding was significantly (p less than 0.01) decreased, again being more pronounced during inhalation than thiopental anaesthesia. These results suggest that some anaesthetics interfere with prostaglandin receptors of the liver.     

Steroid hormone modulation of 3H-prostaglanding E1 binding to bovine corpus leteum cell membranes.

The specific binding of 3H-prostaglandin E1 (3H-PGE1) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0x10-minus-6M. At concentrations above 2.5 x 10-minus-6M; estrone, 17beta-estradiol (but not 17alpha-estradiol or 17beta-estradiol glucuronide), estroil, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of 3H-PGE1. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced 3H-PGE1 binding. These findings permitted the following correlations between steroid structure and modulation of 3H-PGE1 binding: steroids with a free phenolic ring and a 17beta-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of 3H-PGE1. PGE receptors are heterogeneous with respect to affinity for 3H-PGE1. The steroids that decreased 3H-PGE1 binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased 3H-PGE1 binding caused appearance of new low affinity PGE receptors. Association rate constants for 3H-PGE1 binding were decreased by 17beta-estradiol (61%) and increased by DHT (59%).     

Specific 3H-PGE1 binding sites in rat ovary in estrous cycle and pregnancy.

Stimulation of cAMP synthesis by prostaglandins E series in the rat ovary is consistent with the presence of a prostaglandin receptor in this tissue. Prostaglandin binding sites with specificity for PGE1 in vitro incubation systems have been demonstrated in rat ovary slices and corpora lutea. The binding of 3H-PGE1 was progressively inhibited with increasing amounts of unlabelled PGE1 and PGE2. PGF2alpha inhibitory effect was markedly smaller than that of PGE. 3H-PGE1 binding to the ovary was higher in 3-day-old rats than in 5-day-old and adult animals, when the highest binding was present in estrus. The specific binding of 3H-PGE1 to rat corpora lutea (CL) decreased on days 11 and 13 of pregnancy and then gradually returned to the level found on day 1 during the second half of gestation. This binding of labelled prostaglandin during pregnancy has been studied in relation to the PGE1 stimulation of cAMP synthesis in rat corpora lutea, but no consistent changes were observed in responsiveness.     

Regulation of prostaglandin E2 receptors in vivo by dietary fatty acids in peritoneal macrophages from rats

Groups of rats were pretreated with 4-week diets containing 12.5% corn oil or linseed oil. At the end of this period peritoneal macrophages were elicited and isolated. These cells were used for binding experiments with 3H-PGE2 and for estimation of prostaglandin-stimulated cAMP production. Specific binding of 3H-PGE2 was saturable, reversible, protein-dependent, and correlated with stimulation of cAMP production, indicating that specific binding referred to receptor binding. PGE1 and PGI2 were far less effective than PGE2 in competition of binding with 3H-PGE2, indicating receptor selectivity for PGE2. Scatchard analysis of the specific binding data revealed a high affinity component (Kd 17 nM) and low affinity component. The total number of high- and low-affinity binding sites, respective Kd values, and PG stimulation of cAMP production of cells from rats fed the linseed oil diet were comparable to controls. The corn oil diet, however, resulted in a twofold increase in total number of high- and low-affinity binding sites, while respective Kd values were unchanged. This enhancement of binding capacity could be explained by an increased density of binding sites on the cells, and may itself be responsible for the increased sensitivity of the macrophages in this diet group for PG-stimulated cAMP production. The data suggest a regulatory mechanism at the receptor level and are discussed in terms of possible altered bioavailability of arachidonic acid-derived PGE2.     

Exploring the structural basis of the selective inhibition of monoamine oxidase A by dicarbonitrile aminoheterocycles: Role of Asn181 and Ile335 validated by spectroscopic and computational studies

Since cyanide potentiates the inhibitory activity of several monoamine oxidase (MAO) inhibitors, a series of carbonitrile-containing aminoheterocycles was examined to explore the role of nitriles in determining the inhibitory activity against MAO. Dicarbonitrile aminofurans were found to be potent, selective inhibitors against MAO A. The origin of the MAO A selectivity was identified by combining spectroscopic and computational methods. Spectroscopic changes induced in MAO A by mono- and dicarbonitrile inhibitors were different, providing experimental evidence for distinct binding modes to the enzyme. Similar differences were also found between the binding of dicarbonitrile compounds to MAO A and to MAO B. Stabilization of the flavin anionic semiquinone by monocarbonitrile compounds, but destabilization by dicarbonitriles, provided further support to the distinct binding modes of these compounds and their interaction with the flavin ring. Molecular modeling studies supported the role played by the nitrile and amino groups in anchoring the inhibitor to the binding cavity. In particular, the results highlight the role of Asn181 and Ile335 in assisting the interaction of the nitrile-containing aminofuran ring. The network of interactions afforded by the specific attachment of these functional groups provides useful guidelines for the design of selective, reversible MAO A inhibitors.     

Modulation of 3H-prostaglandin E2 binding sites in the rabbit gastric mucosa

The binding of 3H-prostaglandin E2 (PGE2) to rabbit gastric mucosa was investigated. Binding depended on incubation time, temperature and pH, and was saturable and reversible. Scatchard plot analysis revealed a single class of binding sites with a dissociation constant (Kd) of 5.33 +/- 0.21 nM and a maximum number of binding sites (Bmax) of 138.1 +/- 3.4 fmol/mg protein. PGE1 and 16,16-dimethyl PGE2 potently competed with 3H-PGE2 for the binding sites of gastric mucosa, whereas PGA2, PGF2 alpha, 6-keto PGF1 alpha and thromboxane B2 were less potent. The gastric mucosa prepared from the rabbits given indomethacin (5 mg/kg s.c. three times) showed a lower Kd (2.47 +/- 0.19 nM) for 3H-PGE2 than that from untreated one. Treatment with a PGE1 analog, misoprostol (320 micrograms/kg s.c. three times) lowered the Bmax to 74.1 +/- 2.4 fmol/mg protein without any significant effect on the Kd value. It is concluded that rabbit gastric mucosa has specific binding sites for 3H-PGE2 which may be modulated by the levels of PGs in vivo.     

Steroid regulation of monoamine oxidase activity in the adrenal medulla

Administration of different steroid hormones in vivo has distinct and specific effects on the MAO activity of the adrenal medulla. In an effort to reconstitute these effects in defined cells, we have isolated endothelial cells and chromaffin cells from the bovine adrenal medulla and tested each cell type for sensitivity to these steroids. As in the intact animal, we found that endothelial cell MAO activity was stimulated 1.5- 2.5-fold by 10 microM progesterone, hydrocortisone, and dexamethasone, inhibited by ca. 50% by 17-alpha-estradiol, but unaffected by testosterone. The type of MAO in the endothelial cells was found to be exclusively of the A type. The chromaffin cells had MAO B exclusively and were inert to treatment with dexamethasone. The mode of action of the various steroids on MAO A activity in endothelial cells seemed to be that of affecting the number of MAO molecules, as binding of [3H]pargyline, an MAO inhibitor, changed in proportion to changes in enzyme activity. Consistently, the kinetic parameters for MAO A showed changes in Vmax but not Km under all conditions. The specificity of steroid action on MAO A activity was also supported by the fact that steroid-induced changes in total cell division ([14C]thymidine incorporation) and total protein synthesis ([14C]leucine incorporation) were seen after changes in MAO A. We conclude that the differential effects of steroids on MAO activity in the intact adrenal medulla can be reproduced in cultured adrenal medullary endothelial cells but not in chromaffin cells. Therefore we suggest that the action of these steroid hormones on the intact adrenal medulla may be restricted to the endothelial cell component of this tissue.     

Release and specific binding of prostaglandins in bovine pineal gland

Incubated bovine pineal glands released prostaglandin E-and prostglandin F-like material (304 +/- 20 and 582 +/- 56 pg/mg dry tissue wt/h, respectively) and the release was increased 2.2 2.9-fold by adding 10(-4)-10(-6)M of norepinephrine to the medium. Binding assays revealed the existence of high affinity binding of 3H-prostaglandin E2 (3H-PGE2) and 3H-prostaglandin F1 alpha (3H-PGF2 alpha) in low speed supernatants of pineal homogenates. Binding was increased by increasing Ca++ concentration in medium up to 2 mM, was heat labile and was depressed following incubation with trypsin. In subcellular fractionation studies maximal 3H-PG binding was found in the 27000 x g pellet. Scatchard analysis of 3H-PGE2 binding revealed the presence of a single population of binding sites with a Kd= 1.2 nM and a binding site concentration of 1-2 pmoles/g protein. A single population of binding sites for 3H-PGF2 alpha was also detected with a Kd= 1.7 nM and a similar binding site concentration. Non-radioactive PGE1 and PGE2 were almost equally effective to compete for 3H-PGE1 binding sites (ED50= 5 and 2 nM, respectively). Unlabeled PGF1 was relatively ineffective to compete for 3H-PGE2 binding (ED50 greater than 1000 nM) but displaced effectively 3H-PGF2 alpha binding (ED20=1.2 nM).     

Effect of mercaptoethanol in the radioactive thin layer chromatography assay of NAD+-15-hydroxyprostaglandin in dehydrogenase.

The use of mercaptoethanol in the assay of rat kidney 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to have minimal effect on activity assayed with the spectrophotometric and substrate loss assays. However, mercaptoethanol appeared to inhibit PGDH when assayed by thin-layer chromatography, based upon conversion of 3H-PGE1 to 15-keto-3H-PGE1. Mercaptoethanol reacted with 15-keto-PGE1 to alter its chromatographic mobility and to suppress the U.V. absorption spectrum of 15-keto-PGE1. The implication of the use of ME in radiometric assays is discussed.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS

A high-affinity (K_d= 5.9 nM) specific binding site for [³H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [³H]serotonin ([³H]5-HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra-synaptosomal origin exhibited very similar properties with respect to [³H]harmaline binding characteristics and MAO A activity. Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [₃H]harmaline. Logit-log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [³H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 10²-2 × 10³ times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [³H]harmaline from its specific binding site. K_i and IC₅₀ values for the inhibition of [³H]harmaline binding by MAO I and MAO substrates (tryptamine, 5-HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [³H]5-HT as the substrate. In conclusion, the specific binding site for [³H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [³H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.     

Monoamine oxidase in rat placenta, human placenta, and cultured choriocarcinoma.

The activity of monoamine oxidase (MAO), an enzyme which metabolized catecholamines and indoleamines, was determined in rat placenta at various stages of gestation, in human term placenta, and in choriocarcinoma grown in culture. From Day 15 to Day 20 of gestation the specific activity (units/mg protein) of MAO in rat placenta increased at least 3-fold; from Day 20 to the time of parturition, it decreased about 50%. The specific activity of MAO in human placenta at term was about 50%. The specific activity of MAO in human placenta at term was about 8 times higher than that of rat placenta at term. No MAO activity was found in choriocarcinoma grown in culture.     

Metabolism of biogenic amines in neuroblastoma and glioma cells in culture

The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyltransferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8-14 microM for tryptamine to 510-580 microM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for 1-norepinephrine (200 microM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2-10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2'-dibutyryl adenosine-3',5'-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.     

Prostaglandin F2alpha specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF2alpha specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 X 10(-9)M and the concentration of binding sites of -0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF2alpha specific binding sites indicates specificity for the 9alpha-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF2alpha analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF2alpha binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific 3H-PGE1 binding could be demonstrated, no high affinity sites could be quantitated. 3H-PGE1 binding appeared unaffected by changes in temperature or time of incubation, whereas PGF2alpha specific binding was significantly modified by both these factors.     

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.     

Identification and characterization of prostaglandin-binding components in rat ileum

A specific prostaglandin E2 (PGE2) binding component was identified in the membrane fractions of rat ileum. Two ligands, 3H-PGE2 and 14C-PGE2, competed for the binding sites in crude homogenates of ileum during competition experiments, demonstrating the presence of a limited number of binding sites. There was enhancement in the specific 3H-PGE2 binding to particulate fractions over control when endogenous prostaglandin synthesis was blocked by the administration of indomethacin to rats prior to sacrifice. The specific prostaglandin-binding protein was purified by a combination of [NH4]2SO4 fractionation and Sephacryl S-200 column chromatographic techniques, and shown to be active after these biochemical steps.     

Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the type A and B forms of monoamine oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8-day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [³H]DA (10^?7 M) was examined in 8-day KA-lesioned rat striatal slices, an increase in [³H]dihydroxyphenylacetic acid (DOPAC) and [³H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [³H]DOPAC formation (but not [³H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [³H]DA (10^?5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA-lesioned striata. These results suggest that KA-sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.     

Effect of N-Methyl-4-Phenylpyridinium Ion on Monoamine Oxidase in a Clonal Rat Pheochromocytoma Cell Line, PC 12h

The effects of the neurotoxin N-methyl-4-phenylpyridinium ion (MPP+) on the enzymes involved in synthesis and catabolism of catecholamines were examined using a clonal rat pheochromocytoma cell line, PC12h, as a model of dopaminergic neurons. MPP+ added in the culture medium was found to be accumulated in PC12h cells after 30-min incubation. Monoamine oxidase (MAO) activity in PC12h cells was inhibited by MPP+ in a dose-dependent way from 10 nM to 10 microM, but concentrations of MPP+ higher than 100 microM were found to increase the MAO activity. At the lower concentrations MPP+ inhibited MAO noncompetitively with respect to the substrate, kynuramine, and at the higher concentrations it increased both the Km and the Vmax values of MAO toward the substrate. On the other hand, tyrosine hydroxylase activity and the dopamine concentrations in PC12 cells were not changed by incubation with MPP+ for 30 min, 60 min, or 24 h.     

Regulatory Effect of Magnesium Ions on Interaction of Prolactin and Somatotropin with Receptors on Granulosum Cells of the Cow Bos taurus

A comparative study of effects of Mg²⁺ ions on specific and competitive binding of the bovine somatotropic hormone (STH) and bovine prolactin (PL) with cells of cow granulosum is carried out. It is found that Mg²⁺ increases the level of specific binding of ¹²⁵I-PL with the cells at concentrations from 1 to 70 mM and decreases the level of specific binding of ¹²⁵I-STH at a concentration of 70 mM. Analysis of the data by Scatchard's method has shown that the decrease of the level of specific binding of ¹²⁵I-PL in the absence of MgCl₂ and ¹²⁵I-STH at a MgCl₂ concentration of 70 mM is caused mainly by a decrease of the number of active binding sites on the cells. Oppositely directed effects of the studied divalent cations on the capability of unlabelled STH (25 µg/ml) and PL (25 µg/ml) for cross-suppression of specific binding of ¹²⁵I-PL and ¹²⁵I-STH, respectively, with the granulosum cells have been revealed. At the same time, with increase of Mg²⁺ ion concentration, the degree of replacement of ¹²⁵I-STH and ¹²⁵I-PL with unlabelled STH and PL, respectively, did not change. The obtained results are considered in connection with electrostatic model of participation of the divalent cations in interaction of PL and STH with receptors on cells.