Identification and characterization of a homogeneous population of beta 2-adrenergic receptors on human alveolar macrophages

Human alveolar macrophages obtained by bronchoalveolar lavage were studied with the high specific activity beta-adrenergic ligand [125I]pindolol and found to possess a moderate density of beta-adrenergic receptors. Using macrophage membranes, the receptor density (Bmax) was 42 +/- 9 fmol/mg protein with an apparent equilibrium dissociation constant (Kd) of 44 +/- 9 pM (mean +/- SEM). With intact macrophages, the Bmax = 5,643 +/- 942 sites/cell with Kd = 29 +/- 9 pM. Competition binding studies with subtype-specific antagonists revealed an exclusive population of beta 2-adrenergic receptors. Incubation of intact macrophages with the beta-adrenergic agonist isoproterenol caused a 6-fold increase in intracellular cyclic AMP (cAMP). Prostaglandin E1 and forskolin, which activate adenylate cyclase via different mechanisms, afforded typical marked increases in macrophage cAMP. Saturation binding, competition binding, and cAMP accumulation studies may all be performed from a single sample of about 2 x 10(7) cells, which can be obtained by bronchoalveolar lavage. This should facilitate studies of in vivo regulation of human alveolar macrophage beta-adrenergic receptors with regard to immune function and mediator release, and as a possible reflection of lung parenchymal receptors.     

Aldosterone and dexamethasone binding in human arterial smooth muscle cells

Regulation of blood pressure by direct action of corticosteroids on blood vessel walls was first hypothesized in 1952 [1]. The presence of receptors specific for these hormones within the peripheral vessel walls is a pre-requisite of this hypothesis. This report documents specific binding of both aldosterone (AL) and dexamethasone (DM) in cultured human arterial smooth muscle cells. After the arterial smooth muscle cultures were incubated with tritiated AL or DM at 37 degrees C for 30-60 min, the cells were sonicated and the protein bound steroid fraction isolated on a Sephadex G25 column. Using ion exchange chromatography of this fraction, each corticosteroid receptor complex displayed a distinct, reproducible elution pattern. Aldosterone showed a single peak at 0.096 +/- 0.005 mol/l sodium phosphate and DM had two peaks at 0.029 +/- 0.003 and 0.050 +/- 0.004 mol/l sodium phosphate. The Scatchard plots of specific binding from AL saturation curves are linear and revealed mean +/- s.d. steady state binding parameters of dissociation constant (Kd) = 0.35 +/- 0.15 nmol/l and maximum binding capacity (Bmax) = 98 +/- 53 X 10(-18) mol/micrograms DNA. Similarly, the mean +/- s.d. steady state binding parameters for DM are Kd = 4.4 +/- 2.0 nmol and Bmax = 3031 +/- 1385 X 10(-18) mol/micrograms DNA. Therefore, there are approximately 1150 AL binding sites and 30000 DM binding sites per cell. The Kd and Bmax values are similar to those previously described for corticoid receptors in rat aortic smooth muscle cells and are consistent with physiological steroid concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of negative cooperativity to the thyrotropin-receptor interaction in normal human thyroid: kinetic evaluation.

The kinetics of 125I-labeled thyrotropin (125I-TSH) binding to human thyroid receptors are presented. At pH 6.0, binding was maximal (30--35%) and there was one class of binding sites [Kd = 6.8 X 10(-9) M; binding capacity (Ro) = 57 pmol/mg of protein]. At pH 7.4, Scatchard plots of binding were nonlinear, indicating either a single class of negatively cooperative sites (Kd = 3.7 X 10(-9) M; Ro = 26 pmol/mg of protein) or, alternatively, independent high- (Kd = 5.0 X 10(-10) M; Ro = 3 pmol/mg of protein) and low-affinity (Kd = 1.7 X 10(-8) M; Ro = 26 pmol/mg of protein) binding sites. The role of negative cooperativity was evaluated from the rates of association and dissociation at pH 7.4. The kinetically determined binding constants (Kd = 1.7 X 10(-11) M; Ro = 2 pmol/mg of protein) were more similar to those determined for the high-affinity component than to those predicted from the negative cooperativity model. Dissociation of bound TSH was independent of initial site occupancy over a 40-fold range, corresponding to a 100-fold range of free TSH concentration. The dissociation rate of 125I-TSH was enhanced by unlabeled TSH to a similar degree, irrespective of initial binding site occupancy. Because the negative cooperativity model does not accommodate these data, it is concluded that TSH receptors in human thyroid behave kinetically and at equilibrium as a single class of high-affinity sites up to TSH concentrations well above the physiological range.     

Interleukin 2 binding molecule distinct from the Tac protein: analysis of its role in formation of high-affinity receptors.

Interleukin 2 (IL-2) receptors on activated T cells exist in high- and low-affinity configurations, both of which share a ligand-binding component known as the Tac protein. Although almost all binding of IL-2 to such cells was inhibited by an antibody to Tac, the predominant component of binding on the natural killer (NK)-like cell line YT was resistant to this reagent. The ligand-binding component on YT cells also differed from Tac in its affinity constant (Kd approximately 8.2 X 10(-10) M vs. Kd approximately equal to 1.1 X 10(-8) M for low-affinity Tac sites) and in its susceptibility to inhibition by certain antibodies to IL-2. When the YT cells were stimulated in a manner that induced expression of the Tac protein, the IL-2 binding sites were converted to a high-affinity configuration (Kd approximately 1.8 X 10(-11) M). Thus, the original binding component on unstimulated YT cells appeared to combine with Tac and IL-2 to produce a high-affinity receptor complex. Use of bifunctional crosslinking agents following ligand binding to unstimulated YT cells yielded covalent IL-2-receptor complexes of 83 and 90 kDa. These complexes were similar in size to those derived from high-affinity receptors on activated T cells and shared a similar fragmentation pattern upon proteolysis. These results demonstrate the existence of a second IL-2 binding component in addition to the Tac protein and suggest that this component combines with Tac and IL-2 to form high-affinity receptor sites.     

Expression of melatonin receptors in arteries involved in thermoregulation

Melatonin binding sites were localized and characterized in the vasculature of the rat by using the melatonin analogue 2-[125I]iodomelatonin (125I-melatonin) and quantitative in vitro autoradiography. The expression of these sites was restricted to the caudal artery and to the arteries that form the circle of Willis at the base of the brain. The arterial 125I-melatonin binding was stable, saturable, and reversible. Saturation studies revealed that the binding represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 3.4 x 10(-11) M in the anterior cerebral artery and 1.05 x 10(-10) M in the caudal artery. The binding capacities (Bmax) in these arteries were 19 and 15 fmol/mg of protein, respectively. The relative order of potency of indoles for inhibition of 125I-melatonin binding at these sites was typical of a melatonin receptor: 2-iodomelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine. Norepinephrine-induced contraction of the caudal artery in vitro was significantly prolonged and potentiated by melatonin in a concentration-dependent manner, suggesting that these arterial binding sites are functional melatonin receptors. Neither primary steps in smooth muscle contraction (inositol phospholipid hydrolysis) nor relaxation (adenylate cyclase activation) were affected by melatonin. Melatonin, through its action on the tone of these arteries, may cause circulatory adjustments in these arteries, which are believed to be involved in thermoregulation.     

Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand [125I]pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed.     

Localization and quantification of high-affinity melatonin binding sites in Rana pipiens retina

Melatonin binding was localized to the inner plexiform layer (IPL) of the frog retina by in vitro autoradiography, using 2-125I-melatonin as the radioligand. Radioreceptor binding assays of frog retinal homogenate demonstrated saturable melatonin binding. Scatchard analysis revealed a single population of binding sites with an apparent dissociation constant (Kd) of 125 pM, with a Bmax of 0.138 fmoles/mg of protein. These results suggest that high-affinity melatonin binding sites are present in the IPL of the frog retina, which may reflect the presence of melatonin receptors in this synaptic layer.     

Beta-adrenergic receptors in catfish liver membranes: characterization and coupling to adenylate cyclase.

beta-Adrenergic binding sites in catfish liver membranes have been characterized by centrifugal assay, using a beta-adrenergic receptor antagonist, (-)-[3H]dihydroalprenolol ([3H]DHA). Binding of the radioligand was saturable and reversible. At 22 degrees equilibrium conditions were established in 15 min and the half-time for dissociation of bound [3H]DHA was approximately 4 min. Analysis of binding data was compatible with the existence of two classes of binding sites: a low-affinity site had a Kd of 62.3 nM and a Bmax of 452.0 fmol/mg protein, while the high-affinity site had a Kd of 2.04 nM and a Bmax of 46.7 fmol/mg protein. The dissociation constant of (-)-alprenolol for the beta-adrenergic receptors was about 2 nM as determined independently by direct kinetic studies and by inhibition of isoproterenol-stimulated adenylate cyclase activity. Phenylephrine was as potent as other catecholamines in inhibiting [3H]DHA binding, indicating that fish adrenoceptor subtyping is different from that of mammals.     

Age-related changes in glucocorticoid and androgen receptors of cultured human pubic skin fibroblasts

In order to evaluate age-related changes in glucocorticoid receptor and androgen receptor of cultured human pubic skin fibroblasts in young and aged men, we determined both [3H]dexamethasone binding and [3H]methyltrienolone (R1881, potent androgenic steroid) binding, using dispersed whole cell assay. Scatchard analyses of specific [3H]dexamethasone binding to the fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean (+/- SD) binding site concentration (Bmax) of 12.69 +/- 2.36 X 10(4) and 12.87 +/- 12.21 X 10(4) sites/cell, respectively, and mean (+/- SD) dissociation constant (Kd) of 5.60 +/- 0.41 and 7.36 +/- 2.17 nM, respectively. Scatchard analyses of specific [3H]R1881 binding to the same cultured skin fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean Bmax of 5.77 +/- 1.02 X 10(3) and 2.82 +/- 0.97 X 10(3) sites/cell, respectively, and a mean Kd of 0.56 +/- 0.23 and 0.50 +/- 0.28 nM, respectively. These findings indicate that there were no significant age-related changes in binding site and affinity of glucocorticoid receptor in cultured human pubic skin fibroblasts, whereas binding sites of androgen receptor significantly decreased in those of aged men as compared to young men, without significant change in affinity.     

Increase in the Bmax of gamma-aminobutyric acid-A recognition sites in brain regions of mice receiving diazepam.

gamma-Aminobutyric acid (GABA) receptors were characterized in vivo by studying ex vivo the binding of [3H]muscimol to cerebellum, cortex, hippocampus, and corpus striatum of mice receiving intravenous injections of tracer doses of high-specific-activity (approximately equal to 30 Ci/mmol) [3H]muscimol. This ligand binds with high affinity (apparent Kd, 2-3 X 10(-9) M) to a single population of binding sites (apparent Bmax, 250-180 fmol per 10 mg of protein). Pharmacological studies using drugs that selectively bind to GABAA or GABAB receptors suggest that [3H]muscimol specifically labels a GABAA recognition site. Moreover, diazepam (1.5 mumol/kg, i.p.) increases the Bmax but fails to change the affinity of [3H]muscimol binding to different brain areas. This diazepam-elicited increase in Bmax is blocked in mice receiving the diazepam antagonist Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4] benzodiazepine-3-carboxylate). Since the diazepam-induced increase of [3H]muscimol binding is paralleled by a significant potentiation of the inhibitory effect of muscimol on locomotor activity, it is proposed that the facilitatory action on GABAergic transmission elicited in vivo by diazepam is mediated by an increase in the Bmax of the binding sites of GABAA receptors.     

Bovine pituitary folliculo-stellate cells have beta-adrenergic receptors positively coupled to adenosine 3',5'-cyclic monophosphate production

The specific beta-adrenergic radioligand [125I]iodocyanopindolol (ICYP) was used to identify and characterize beta-adrenergic receptors in bovine pituitary folliculo-stellate cells (bFSC) grown in culture. Saturation analysis demonstrated the binding of ICYP to bFSC particulate fractions to be of high affinity (apparent Kd = 80 pM) and low capacity (Bmax = 37 fmol/mg protein). The specific beta-adrenergic radioligand [3H] dihydroalprenolol also bound to bFSC particulate preparations with parameters compatible with binding to the beta-adrenergic receptor (Kd = 3.0 nM; Bmax = 52 fmol/mg protein). No specific binding was observed with either the dopamine receptor radioligand [3H]spiperone or the alpha-adrenergic radioligand [3H]dihydro-alpha-ergocryptine. The bFSC beta-adrenergic receptors were further characterized by computer modeling of competition studies with a variety of agonists and antagonists selective for beta-adrenergic subtypes. The pharmacological profiles of ICYP binding obtained from these studies indicated that approximately equal proportions of both beta 1- and beta 2-adrenergic subtypes are expressed in cultured bFSC. Bovine FSC beta-adrenergic receptors are functionally coupled to activation of cAMP. The beta-adrenergic agonists isoproterenol, epinephrine, and norepinephrine provoked a rapid and marked stimulation of intracellular cAMP accumulation. The approximately equipotent effect of epinephrine and norepinephrine indicated that the beta-adrenergic effect on cAMP production is principally mediated via the beta 1-adrenergic receptor. The identification of beta-adrenergic receptors on bFSC positively coupled to adenylate cyclase provides a possible regulatory control pathway for the proposed role of pituitary FSC in the modulation of anterior pituitary hormone secretion.     

Presence of a specific binding site of endothelin on cultured smooth muscle cells

The 21 amino-acids endothelium-derived peptide, endothelin, recently isolated by Yanagisawa et al. (Nature 1988; 33, 411-5) possesses potent vasoconstrictive properties in vivo and in vitro. In the present study, we investigated the binding of endothelin on cultured rat aortic smooth muscle cells using 125I-iodotyrosyl-endothelin labelled by the chloramine T method. 125I-endothelin bound to a single class of hight affinity binding sites in vascular smooth muscle cells. After 2 hours incubation at 37 degrees C, dissociation constant (Kd) was 1.2 +/- 0.3 nM and binding capacity (Bmax) was 59 +/- 11 fmol/10(6) cells (n = 5). 125I-endothelin was displaced by unlabelled endothelin with a inhibition constant (Ki) of 0.2 nM, whereas an absence of competition was observed with 1 microM of vasoactive substances such as angiotensin II, arg-vasopressin, atrial natriuretic factor, histamine, epinephrine and norepinephrine, and with the calcium entry blocks nifedipine, diltiazem and D 600. 125I-endothelin binding was not reversible by addition of unlabelled endothelin (1 microM) and not dissociable by acetic acid (10 mM) or trypsin (0.1 p. 100) treatment of the cells. Furthermore, preincubation of vascular smooth muscle cells with endothelin (1 nM) at 37 degrees C induced a rapid down-regulation of endothelin binding capacity by about 50 p. 100. These data indicate that specific endothelin bindind sites are present in smooth muscle cells, and suggest a tight binding or a rapid captation of endothelin into the cell membrane leading to contractile events.     

Epidermal growth factor binding sites on human erythrocytes in donors with different ABO blood groups.

Analysis of epidermal growth factor (125J-EGF) binding to human red cells revealed the presence of two classes of binding sites with apparent equilibrium dissociation constants (app.Kd) in the 10(-10)-10(-9) M and in the 10(-8) M range, respectively. The number of binding sites/cell ranged between 600 and 2,400 for the high-affinity binding site and between 7,200 and 23,000 for the low-affinity site. No differences were seen in the apparent Kd values for both types of binding sites between red cells obtained from donors with different ABO-blood groups. An increase in the number of high affinity EGF binding sites was observed in donors with blood group A1-erythrocytes as compared to red cells taken from donors with blood groups O and B.     

Distribution of angiotensin II receptors and renin in the mouse fetus

To investigate the ontogeny of the renin-angiotensin system we studied the characteristics and location of angiotensin II (AII) receptors in mouse fetuses and examined sites of renin mRNA expression by in situ hybridization and Northern blot analysis. Autoradiographic analysis of the binding of 125I-[Sar1,Ala8]AII to slide-mounted frozen sections of 17-day-old DBA/2N mice revealed abundant AII receptors widely distributed throughout the body. High receptor density was found in primitive mesenchymal tissue under the epidermis and surrounding muscle and cartilage, in skeletal and smooth muscle, and in all layers of the adrenal cortex. Lower receptor density was seen in the kidney, liver, and lungs. The autoradiographic staining was abolished by incubation of the sections with excess unlabeled AII. Scatchard analysis of the binding of 125I-[Sar1,Ala8,]AII to membrane-rich fractions of eviscerated fetuses showed a single type of high affinity receptors with a Kd of 2.9 x 10(-9) M and a receptor concentration of 3300 fmol/mg protein. Localization of renin mRNA was analyzed by in situ hybridization using an antisense 35S-labeled riboprobe transcribed from a mouse renin2 cDNA clone. Hybridization to fetal tissue sections showed high intensity staining in the kidney and adrenal cortex. Northern blot analysis confirmed the high expression of renin mRNA in the fetal kidney. The presence of an active renin-angiotensin system in the fetus was confirmed by the demonstration of renin-like activity and bioactive AII in fetal extracts. The widespread distribution of AII receptors in the fetus, compared to the discrete localization to specialized tissues in the adult, may indicate a unique role for the peptide during development.     

Glucocorticoid modulation of beta-adrenergic receptors of cultured rat arterial smooth muscle cells

Since both glucocorticoids and catecholamines are involved in the regulation of normal blood pressure, we investigated the modulation of beta-adrenergic receptors of cultured rat arterial smooth muscle cells by glucocorticoids. The synthetic glucocorticoids dexamethasone and RU 28362, at 10(-8) M concentration, increased maximum beta-adrenergic binding but had no effect on the dissociation constant (Kd). Each steroid caused an increase in maximum [3H]dihydroalprenolol binding over the concentration range of 10(-8) to 10(-6) M, but not at 10(-9) M. The glucocorticoid effect on beta-adrenergic receptors of arterial smooth muscle cells required a minimum of 20 hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml), indicating that the glucocorticoid effect required protein synthesis. The effect of dexamethasone on [3H]dihydroalprenolol binding was significantly inhibited by the glucocorticoid antagonist RU 38486. Basal and agonist-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels in arterial smooth muscle cells, before and after glucocorticoid treatment, were measured as an indicator of the physiological significance of the observed glucocorticoid-induced increase in beta-adrenergic receptor binding. While causing no change in the basal cAMP level, treatment of arterial smooth muscle cells with 10(-6) M dexamethasone for 24 hours increased the 10(-6) M isoproterenol-stimulated cAMP levels.     

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125I-labeled melatonin (125I-Mel), a potent melatonin agonist. 125I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent Kd of 2.3 +/- 1.0 x 10(-11) M and 2.06 +/- 0.43 x 10(-10) M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125I-Mel from its receptor. Furthermore, GTP[gamma S] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radio-activity corresponding to Mr greater than 400,000 and Mr ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[gamma S] before solubilization; only the Mr 110,000 peak was present in GTP[gamma S]-pretreated membranes. The results strongly suggest that 125I-Mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.     

Isolation and characterization of an olfactory receptor protein for odorant pyrazines.

The highly potent bell pepper odorant 2-isobutyl-3-[3H]methoxypyrazine [( 3H]IBMP) binds specifically and saturably to bovine and rat nasal epithelium. Specific binding is not detected in 11 other tissues assayed, and in the rat binding is 9 times higher in olfactory than in respiratory epithelium. We have purified to apparent homogeneity a soluble pyrazine odorant binding protein that constitutes approximately equal to 1% of the total soluble protein in bovine nasal epithelium. Polyacrylamide gel electrophoresis shows a single band of 19,000 Da and gel filtration data suggest that the native protein is a dimer of 38,000 Da. Binding of [3H]IBMP to the purified protein reveals two binding sites (Kd = 10 X 10(-9) M, Bmax = 135 pmol per mg of protein; Kd = 3 X 10(-6) M, Bmax = 25 nmol per mg of protein). The binding affinities of a homologous series of pyrazine odorants correlate with the human odor detection thresholds of these compounds. This correlation, together with the regional distribution of the protein, suggests that the protein is a physiologically relevant olfactory receptor.     

Regulation by progesterone of the high-affinity state of myometrial beta-adrenergic receptor and of adenylate cyclase activity in the pregnant rat.

The effects of pregnancy or progesterone dominance on the beta-adrenergic responsiveness of the uterus were studied in myometrial membranes from mid- and late-pregnant rats (day 15 and on the 16th h of day 22 of pregnancy respectively) or 24 h after administration of progesterone. Levels of the high (RH)- and low (RL)-affinity states of the beta-adrenergic receptor were determined by competition experiments between 125I-labelled cyanopindolol binding and the selective beta-agonist isoproterenol. The ratio KL/KH (respective dissociation constants) was determined since it also reflects the degree of formation of the high-affinity state of the beta-adrenergic receptor. From day 15 to the 10th h of day 22 of pregnancy, two distinct affinity states were apparent: 80-55% RH (KH = 0.31-0.21 microM) and 45-20% RL (KL = 14-5 microM) with a ratio of KL/KH of 55-34. In the last 6 h before birth, beta-adrenergic receptors underwent uncoupling which was paralleled by decreased responsiveness of myometrial adenylate cyclase to isoproterenol (maximum velocity (Vmax) = 17 +/- 3 vs 44 +/- 3 fmol cyclic AMP/10 min per mg protein on day 15). At this stage of pregnancy, previous exposure to progesterone resulted in a 1.8-fold increase in 125I-labelled cyanopindolol-binding sites (Bmax) and the reappearance of the high-affinity state (67% RH, KH = 0.19 +/- 0.04 (S.E.M.) microM, ratio KL/KH = 81.1 +/- 16.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of angiotensin II with dispersed cells from the anterior pituitary of the male rat

Membranes from 6-week-old male rat anterior pituitaries possess saturable binding sites for angiotensin II (AII; Kd = approximately 2 X 10(-9) M). The binding is specific since it can be competed for with [Sar1,Leu8]AII and is unaffected by the presence of insulin or cholecystokinin octapeptide at nanomolar concentrations. To find out which cell types specifically interact with AII, rat anterior pituitaries were enzymatically dispersed and exposed to [125]iodo-AII (2 nM) in the absence or presence of [Sar1,Leu8]AII (400 nM). The cells were then washed free of unbound ligand and processed for light and electron microscopic autoradiography. Distribution of autoradiographic grains revealed that three cell types were specifically labeled with [125I]iodo-AII, namely mammotrophs, corticotrophs, and presumptive thyrotrophs. These cells were all labeled in the presence of [125I]iodo-AII alone (experimentals), whereas only 10-30% of them were labeled when 400 nM [Sar1,Leu8]AII was included in the binding reaction (controls). The number of grains over the labeled cells in the controls was 20% of that found in the experimental cells. These results may imply that AII can regulate anterior pituitary functions under physiological conditions by interacting directly with its secretory cells.     

Receptor regulation of atrial natriuretic factor

Two atrial natriuretic factor (ANF) receptor subtypes are present in vascular smooth muscle cells: the B receptors (or biologically active) coupled to a guanylate cyclase and the C receptors (clearance) representing 95% of the total number of ANF binding sites but noncoupled to a guanylate cyclase. Using binding experiments with 125I-ANF and measurement of cGMP production stimulated by ANF, we were able to demonstrate that ANF receptors are sensitive to homologous (induced by ANF) and heterologous regulation (induced by angiotensin II, AII) in rat cultured vascular smooth muscle cells. The effect of the two hormones showed marked differences, in their time course, their reversibility and their consequence on guanylate cyclase activity. Although both ANF and AII reduced the total number of ANF binding sites after 18 h, ANF induced a desensitization of the guanylate cyclase whereas AII elicited a potentialization of this system. From these results, we have concluded that in vascular cells B receptors are sensitive to homologous regulation and C receptors are sensitive to heterologous regulation by AII. This also highlights a specific interaction between ANF and AII at the receptor level.