Properties of vasoactive intestinal peptide-receptor interaction in rat liver membranes

The properties of the specific receptors for vasoactive intestinal peptide (VIP) in rat liver plasma membranes have been studied by using 125I-VIP as a tracer. The binding of the peptide was a reversible, saturable and specific process, as well as time and temperature dependent. Peptide inactivation was also dependent on time and temperature and remained relatively low in the standard conditions used, as it happened in the inactivation of the binding sites. The binding data were compatible with the existence of two classes of VIP receptors: a high affinity (Kd = 4.2 x 10(-10) M) and low binding capacity (1.5 pmol VIP/mg protein) class and another one of low affinity (Kd = 1.7 x 10(-7) M) and high binding capacity (38.6 pmol VIP/mg protein). The specificity of the binding sites of VIP was established from the fact that binding of 125I-VIP was inhibited by native VIP and by 60-fold higher concentrations of secretin but not by the parent hormone glucagon, by insulin or somatostatin at concentrations as high as 10(-6) M.     

Evidence of high and low affinity binding sites for basic fibroblast growth factor in mouse placenta

The placenta has been shown to contain bFGF, but the presence of specific binding sites for this growth factor in this tissue remained to be established. In order to study the role of bFGF in the placenta growth, we looked for specific binding sites on mouse placental cell membranes at days 12, 14, 16, and 18 of pregnancy. At day 12, Scatchard analyses indicated that two classes of specific interaction sites for bFGF were detected. One class of high affinity binding sites was characterized by an apparent Kd of 10 pM and a binding capacity of 10 fmoles per mg of membrane protein. A second class of low affinity binding sites was detected with an apparent Kd of 60 nM and a binding capacity of 26 pmoles per mg of membrane protein. At days 14, 16 or 18, Scatchard analyses only showed low affinity binding sites with an apparent Kd of 24 nM and a binding capacity of 230 pmoles per mg of membrane protein. The characterization of these binding sites was performed by cross linking experiments that revealed two forms of specific complexes. This result suggested that the high affinity binding sites correspond to putative receptors with relative molecular masses equal to 65,000 and 85,000. The dramatic decrease of the high affinity receptor number after the 12th day of pregnancy, which is synchronous with the 9-fold increase of the low affinity binding site number, suggests that the biological activity of bFGF could be regulated by a balance between both the numbers of high and low affinity binding sites on placenta cell membranes. Thus, as it was shown for other growth factors, bFGF could only be involved at specific pregnancy stages.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

Evidence for somatostatin binding sites in rabbit kidney

Specific binding sites for somatostatin have been identified in cytosolic fraction of rabbit kidney (cortex and outer medulla) using 125I-Tyr11-somatostatin. The binding was saturable and reversible, as well as time and temperature dependent. Optimal pH for binding was observed at about 7.4. Scatchard plots were compatible with the existence of two classes of binding sites: a first class with a high affinity (Kd = 40 nM) and a low binding capacity (2.0 pmol somatostatin/mg protein) and a second class with a low affinity (Kd = 222 nM) and a high binding capacity (114.3 pmol somatostatin/mg protein). Vasoactive intestinal peptide, neurotensin, substance P, Leu-enkephalin and vasopressin had practically no effect on somatostatin binding. The properties of these binding sites strongly support the concept that somatostatin could behave as a regulatory peptide on the rabbit kidney.     

The interaction of vasoactive intestinal peptide (VIP) with isolated bovine thyroid plasma membranes

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase were studied in bovine thyroid plasma membranes. The binding depended on time, temperature and was saturable and specific. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 13 nM) and low capacity (6411 sites/pg), and a class with low affinity (Kd = 480 nm) and high capacity (105,300 sites/pg) at 15 degrees C. Secretin, glucagon, insulin and somatostatin did not displace the tracer from the membranes. VIP stimulated cyclic AMP production. Maximal cyclic AMP production (2-fold above basal values) was observed with 100 nM VIP and half-maximal response was obtained at 5 nM VIP at 15 degrees C.     

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.     

Regulation of insulin-like growth factor I binding in the rat uterus by growth hormone and thyroxine.

The insulin-like growth factor-I (IGF-I)-binding sites in the rat uterus were characterized further and the effects of growth hormone and thyroxine examined. The 125I-labelled IGF-I binding sites on uterine membranes demonstrated relative binding affinity of less than 20% for IGF-II, less than 1% for insulin and no affinity for an unrelated peptide, epidermal growth factor, compared with 100% for IGF-I confirming the specificity of these binding sites. Scatchard analysis of the specific binding data revealed the presence of a single class of high-affinity binding sites (Kd = 2.50 +/- 0.68 nmol l-1, with a binding capacity of 1.02 +/- 0.13 pmol mg-1 membrane protein in the uterus of the pituitary-intact ovariectomized rat. After hypophysectomy, the uteri from these rats had significantly (P less than 0.05) increased IGF-I binding sites, without significant changes in their affinity. Administration of growth hormone with or without L-thyroxine reversed this increase in IGF-I binding. Injection of thyroxine alone to the hypophysectomized ovariectomized rats had no significant effects on the uterine IGF-I binding sites. These data show that growth hormone, but not thyroxine, can regulate IGF-I binding sites in the rat uterus, possibly through regulating IGF-I production.     

Interaction of human prothrombin with tissue thromboplastin

The binding of 125I-labeled human prothrombin to native and papain-treated tissue thromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard plots for the protein binding suggest the presence at thromboplastin surface of two types of binding sites, high affinity [Kd(app) = 7.4.10(-8) M] and moderate affinity [Kd(app) = 7.9.10(-5) M]. The removal of Ca2+ did not influence the Kd (values for these) sites but markedly reduced their number. Proteolysis by papain caused a decrease in the affinity of high affinity sites without affecting the Kd values of the moderate affinity sites yet caused a proportional increase in the number of both high and moderate affinity sites in the presence of Ca2+. At low prothrombin concentrations a positive cooperativity of protein binding at high affinity sites in the presence of Ca2+ was observed.     

Epidermal growth factor binding and steroid receptor content in human benign prostatic hyperplasia

The receptor for epidermal growth factor (EGF-R) was characterized on membrane fractions from human benign prostatic hyperplasia (BPH). Specific binding of [125I]EGF reached equilibrium after 40 min at 25 degrees C and was stable for up to 120 min. Saturation analysis of EGF-R, performed by incubating the membranes with 0.0156-15 nM [125I]EGF in the presence and in the absence of 100-fold excess of cold EGF for 60 min, revealed the presence of two classes of binding sites with high and low affinities (Kd = 0.35 +/- 0.23 and 9.60 +/- 2.87 nM respectively). Competition experiments revealed that FSH, insulin and calcitonin did not compete with [125I]EGF. The simultaneous determination of EGF-R and that of estradiol (ER), progesterone (PR) and androgen receptors (AR) was performed using the same buffer to homogenate the tissues and to obtain cellular membranes. The steroid receptors (SR) were determined by means of the dextran-coated charcoal method. There was a significant negative correlation between nuclear SR and binding capacity of EGF-R. The presence of specific and high affinity binding sites for EGF and the modulation of the level of these sites by steroid receptors suggest a possible role of EGF in prostatic hyperplasia.     

The binding of bombesin and somatostatin and their analogs to human colon cancers.

Specific receptors for bombesin/gastrin-releasing peptide, somatostatin, and EGF were investigated in 15 human colon cancer specimens. Eight of 15 clinical specimens (15%) of colon cancer showed the presence of somatostatin receptors. Octapeptide somatostatin analogs, RC-160 and RC-121, showed 10 times higher binding affinity for somatostatin receptors on colon cancer membranes than somatostatin. Analysis of 125I-Tyr4-bombesin binding data revealed the presence of specific binding sites in six (40%) specimens of human colon cancer. Scatchard analysis of 125I-labeled bombesin indicated a single class of receptors in three specimens with an apparent Kd value of 2.5 nM and two classes of receptors with high (Kd = 0.4 +/- 0.2 nM) and low affinity (Kd = 1.6 +/- 0.4 microM) in three other specimens. The 125I-Tyr4-bombesin binding capacities in the colon cancers for high affinity binding sites were from 6 to 228 fmol/mg protein and for low affinity binding sites 76 +/- 15 pmol/mg protein. None of the membrane preparations made from normal colonic mucosa specimens showed specific binding for 125I-Tyr4-bombesin. Five pseudononapeptide (psi 13-14) bombesin (6-14) antagonists, with different modifications at Positions 6 and 14, synthesized in our laboratory, inhibited the binding of 125I-Tyr4-bombesin in nanomolar concentrations. No correlation was found between the degree of differentiation and the presence of binding sites for somatostatin or bombesin. Specific binding of EGF was detected in 80% of colon cancer specimens. EGF binding capacity in colon cancer membranes was on average twice as high as in normal colon mucosa (50 +/- 21 vs 28 +/- 14 fmol/mg protein, respectively). Specific binding sites for somatostatin and EGF, but not bombesin, were also demonstrated in human colon cancer cell line HT-29. In HCT-116 colon cancer line only EGF receptors were found. These receptor findings and our in vivo studies on inhibition of colon cancer growth support the merit of continued evaluation of somatostatin analogs and bombesin/gastrin-releasing peptide antagonists in the management of colonic carcinoma.     

Characterization of somatostatin binding sites in cytosolic fraction of rat intestinal mucosa

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rat intestinal mucosa by using 125I-labelled Tyr11-somatostatin and a variety of physicochemical conditions. The binding depended on time, temperature and pH, and was reversible, saturable and specific. At apparent equilibrium, the specific binding of 125I-Tyr11-somatostatin was competitively inhibited by native somatostatin in the 1 nM-4 microM concentration range. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 0.07 microM) and low capacity (4.6 pmol/mg protein) and a class with low affinity (Kd = 1.05 microM) and high capacity (277 pmol/mg protein) at 25 degrees C. Somatostatin exhibited competitive inhibition of tracer binding, while neuropeptides such as neurotensin, substance P, Leu-enkephalin, and vasoactive intestinal peptide were ineffective. The presence of somatostatin binding sites in cytosolic fraction of intestinal mucosa, together with the known occurrence of somatostatin in D-cells and nerve endings in the small intestine, strongly suggest that this peptide may be involved in the physiology and physiopathology of intestinal epithelium.     

Characterization of the beta-adrenergic receptors of hamster white fat cells. Evidence against an important role for the alpha 2-receptor subtype in the adrenergic control of lipolysis

The binding characteristics of the beta-adrenergic antagonist, [3H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity (Kd = 1.6 +/- 1.3 nM) but low capacity (32 +/- 17 fmol/mg membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both beta-antagonists and beta-agonists, competition at the low-affinity sites occurred only with beta-antagonists and was non-stereoselective. Thus, the beta-agonist (-)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity (Kd = 1.6 +/- 0.5 nM) binding sites with a binding capacity of 53 +/- 13 fmol/mg membrane protein (corresponding to 4000 +/- 980 sites per cell), and independent kinetic analysis provided a Kd value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a beta 1-receptor subtype, yielding Kd values in good agreement with the Kact and the Ki values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of beta-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [3H]dihydroalprenolol binding sites defined as the binding displaceable by (-)-isoproterenol represent the physiologically relevant beta-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (-)-norepinephrine showed that the inhibitory effect of the alpha 2-component of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the alpha 2-receptors in the adrenergic control of lipolysis in hamster white adipocytes.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

Selective modulation by guanine nucleotides of the high affinity subset of plasma membrane receptors for leukotriene B4 on human polymorphonuclear leukocytes

Isolated human polymorphonuclear (PMN) leukocyte plasma membranes express high affinity (mean Kd = 0.12 nM) and low affinity (mean Kd = 50 nM) receptors for the chemotactic factor leukotriene B4 (5(S),12(R)-dihydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid; LTB4) that are similar to those on intact PMN leukocytes. A portion of high affinity LTB4-R on PMN leukocyte membranes were converted to the low affinity state by GTP (mean +/- SE = 28.6 +/- 14.0%) and nonhydrolyzable GTP analogues, such as 5'-guanylylimidodiphosphate (GMP-PNP), in a concentration-dependent, nucleotide-specific, and reversible manner, without altering the intrinsic binding affinities of either class. [3H]GMP-PNP bound specifically to one class of receptors (mean Kd = 13 nM) on PMN leukocyte membranes. The interdependence of the LTB4-binding membrane protein and guanine nucleotide-binding protein was suggested by the capacity of LTB4 to enhance by a maximum of 150% the binding of [3H]GMP-PNP to PMN leukocyte membranes by increasing the number, but not altering the affinity, of receptors for GMP-PNP. Pertussis toxin, but not cholera toxin, reversed the enhancement of binding of [3H]GMP-PNP produced by LTB4. Guanine nucleotide-binding proteins and high affinity LTB4-R thus exhibit a mutual regulation that differs mechanistically from that of peptide chemotactic factor receptors on PMN leukocytes.     

Down-regulation and recycling of high affinity cholecystokinin receptors on pancreatic acinar cells

First incubating dispersed acini from rat pancreas with monensin, a cation ionophore that can inhibit recycling of receptors, inhibited binding of 125I-cholecystokinin 8 (125I-CCK-8) measured during a second incubation by as much as 50%. A maximal effect of monensin required 90 min of first incubation. Detectable inhibition of binding of 125I-CCK-8 occurred with 300 nM monensin, and inhibition increased progressively with concentrations of monensin up to 25 microM. Pancreatic acini possess two classes of receptors that bind 125I-CCK-8. One class has a high affinity (Kd = 461 pM) and a low capacity for CCK (512 fmol/mg DNA); the other class has a low affinity (Kd = 47 nM) and a high capacity for CCK (18 pmol/mg DNA). First incubating acini with monensin caused an 84% decrease in the number of high affinity CCK receptors with no change in the number of low affinity CCK receptors or the values of Kd for either class of receptors indicating that there is recycling of high affinity CCK receptors but not low affinity CCK receptors. First incubating acini with monensin did not alter CCK-stimulated amylase secretion indicating that in contrast to previous conclusions, occupation of low affinity CCK receptors mediates CCK-stimulated enzyme secretion. Moreover, the biphasic dose-response curve for CCK-stimulated enzyme secretion from monensin-treated acini suggests that pancreatic acini also possess a third, previously unrecognized class of very low affinity CCK receptors.     

Interaction of Leu-enkephalin with isolated enterocytes from guinea pig: binding to specific receptors and stimulation of cAMP accumulation

The specific binding of Leu-enkephalin and the stimulatory effect of the peptide on cAMP accumulation have been assessed in isolated enterocytes of guinea pig. The binding was reversible as well as time and temperature dependent. Two classes of binding sites could be defined: a class with a relatively high affinity (Kd = 0.7 microM) that represented 1% of total binding capacity, and another class with low affinity (Kd = 55.5 microM). The stimulation of cAMP accumulation was also shown to depend on time and temperature and was potentiated by a phosphodiesterase inhibitor. Half-maximal stimulation of cAMP accumulation was observed at 119 microM and maximal stimulation (27-fold basal level) at 300 microM Leu-enkephalin. Both steps of the interaction were not modified by Na+ but exhibited a high specificity since modification in the structure of Leu-enkephalin resulted in an important loss of binding affinity and stimulatory activity.     

Studies on adrenocorticotropic hormone receptor using isolated rat adrenocortical cells.

To clarify the function of ACTH receptors, the actions of ACTH on cyclic AMP formation, Ca2+-influx across cell membrane, and corticoidogenesis were examined using dispersed adrenocortical cells prepared from the rat adrenal gland. 1) There are two types of ACTH receptors from Scatchard analysis of 125I-ACTH1-24 binding to the cell, the one receptor is of high affinity and low capacity (dissociation constant (Kd1) = 2.6 x 10(-19) M and 7,350 sites per cell), and the other one is of low affinity and high capacity (dissociation constant (Kd2) = 7.1 x 10(-9)M and 57,400 sites per cell). 2) Both apparent dissociation constants derived from the effects of ACTH on corticoidogenesis and Ca2+ influx well correspond with Kd1 of the high affinity receptor, 3) Apparent dissociation constant obtained from the effect of ACTH on cyclic AMP formation is in good agreement with Kd2 of the low affinity receptor. Thus it could be deduced from these data that the high affinity receptor is concerned with an increased Ca2+-influx to regulate corticoidogenesis at physiological levels of ACTH, whereas the low affinity receptor is coupled to adenylate cyclase at supraphysiological concentrations of ACTH.     

Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages.

Functional vasoactive intestinal peptide (VIP) receptors have been characterized in rat peritoneal macrophages. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Scatchard analysis of binding data suggested the presence of two classes of binding sites: a class with high affinity (kd = 1.1 +/- 0.1 nM) and low capacity (11.1 +/- 1.5 fmol/10(6) cells), and a class with low affinity (kd = 71.6 +/- 10.2 nM) and high capacity (419.0 +/- 80.0 fmol/10(6) cells). Structural requirements of these receptors were studied with peptides structurally or not structurally related to VIP. Several peptides inhibited 125I-VIP binding to rat peritoneal macrophages with the following order of potency: VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, somatostatin, pancreastatin and octapeptide of cholecystokinin (CCK 26-33) were ineffective. VIP induced an increase of cyclic AMP production. Half-maximal stimulation (ED50) was observed at 1.2 +/- 0.5 nM VIP, and maximal stimulation (3-fold above basal levels) was obtained between 0.1-1 microM. Properties of these binding sites strongly support the concept that VIP could behave as regulatory peptide on the macrophage function.     

Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin

Freshly prepared plasma membranes from rat corpora lutea were examined for the presence of low density lipoprotein (LDL) and high density lipoprotein (HDL) receptors by determining the specific binding of 125I-LDL and 125I-HDL. These membranes have two types of binding site for 125I-LDL, one with high affinity (Kd = 7.7 micrograms of LDL protein/ml), the other with low affinity (Kd = 213 micrograms of LDL protein/ml) and one type of binding site for 125I-HDL with Kd = 17.8 micrograms of HDL protein/ml. LDL receptor is sensitive to pronase and trypsin; HDL receptor, however, is resistant. The binding reaction was further characterized with respect to effect of time and temperature of incubation, requirement of divalent metal ion, influence of ionic strength, and binding specificity. In vivo pretreatment of rats with human choriogonadotropin (hCG) resulted in induction of both LDL and HDL receptors in a dose- and time-dependent manner when compared with saline-injected controls. The induction of lipoprotein receptors by hCG treatment is target organ-specific since the increase was seen only in the ovarian tissue. Membranes prepared from liver, kidney, and heart did not show an increase in lipoprotein receptors after hCG injection. An examination of the equilibrium dissociation constants for 125I-LDL and 125I-HDL binding after hCG administration revealed that the increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, rat corpus luteum possesses specific receptors for both LDL and HDL and these receptors are regulated by gonadotropins.     

Interaction of vasoactive intestinal peptide (VIP) with rat lymphoid cells

Receptors for vasoactive intestinal peptide (VIP) have been characterized in rat lymphoid cells. The interaction of [125I] VIP with blood mononuclear cells was rapid, reversible, specific and saturable. At apparent equilibrium, the binding of [125I] VIP was competitively inhibited by native VIP in the 0.01-100 nM range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.050 +/- 0.009 nM and a low binding capacity (2.60 +/- 0.28 fmol/10(6) cells), and a low-affinity class with a Kd = 142 +/- 80 nM and a high binding capacity (1966 +/- 330 fmol/10(6) cells). Secretin, glucagon, insulin and somatostatin did not show any effect at a concentration as high as 100 nM. With spleen lymphoid cells, stoichiometric studies were performed. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.100 +/- 0.033 nM and a low binding capacity (4.60 +/- 1.07 fmol/10(6) cells), and low-affinity class with a Kd = 255 +/- 110 nM and high binding capacity (2915 +/- 1160 fmol/10(6) cells). With thymocytes, no binding was obtained under different conditions.