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.     

Association of brain ankyrin with brain membranes and isolation of active proteolytic fragments of membrane-associated ankyrin-binding protein(s)

An assay has been developed to measure association of brain ankyrin with protein site(s) in brain membranes that are independent of spectrin and tubulin, behave as integral membrane proteins, and appear to be similar in several respects to the erythrocyte anion channel. Brain membranes were depleted of ankyrin, spectrin, and other peripheral membrane proteins by a brief incubation in 0.1 M sodium hydroxide. Binding of ankyrin to these membranes fulfilled experimentally testable criteria for a specific protein-protein association. Binding was optimal at physiological values for ionic strength and pH, was of high affinity (Kd = 20-60 nM), and the capacity of 25 pmol/mg of brain membrane protein is in the same range as the number of spectrin tetramers (30 pmol/mg). The membrane-binding site(s) for brain ankyrin are likely to be related in some way to the cytoplasmic domain of the erythrocyte anion channel since binding was inhibited by the anion channel domain and by erythrocyte ankyrin. The binding site(s) for brain ankyrin were released from the membrane by limited proteolysis as active water-soluble fragments capable of inhibiting binding of ankyrin to membranes. Ankyrin-binding fragments of Mr = 40,000 and 68,000 were selectively bound to an erythrocyte ankyrin affinity column. The fragment of Mr = 40,000 is close to the size of the cytoplasmic domain of the erythrocyte anion channel. It is likely based on these results that membrane attachment proteins for ankyrin are present in brain and other tissues and that these membrane proteins have domains homologous at least in conformation to the ankyrin-binding site of the erythrocyte anion channel.     

High affinity binding sites for basic fibroblast growth factor in rat hepatic plasma membranes

The binding of [125I]-recombinant basic FGF (rec bFGF) to rat hepatic plasma membranes was investigated. [125I] rec bFGF bound to an apparent single class of high affinity binding sites (KD = 69 pM; Bmax = 9.61 fmoles/mg proteins). The absence of low affinity sites was confirmed by the inability of sulphated polysaccharides and heparinase to interfere with FGF binding. A good correlation existed between the ability of bovine pituitary-derived bFGF, rec bFGF and bovine brain-derived aFGF to displace [125I]rec bFGF from these binding sites and their in vitro potency on bovine aortic endothelial cell proliferation.     

Multiple thyroid hormone binding sites on rat liver nuclear envelopes

Nuclear envelopes relatively free of plasma membrane contamination were isolated from the male rat liver. Equilibrium binding of T3 to nuclear envelopes occurred after incubation for 3 h at 20 degrees C. Scatchard analysis revealed two classes of binding sites; a high affinity site having a KD of 1.8 nM with a maximum binding capacity of 14.5 pmol/mg protein and a low affinity site having a KD of 152.1 nM with a maximum binding capacity of 346.8 pmol/mg protein. No degradation of the radioligand occurred during incubation with the nuclear envelope. T4, rT3 and Triac competed effectively for the binding of T3 to the high affinity site whereas only T4 competed well for binding to the lower affinity site. The binding site was protease sensitive but not salt extractable. Multiple T3 binding sites having similar affinities have been reported on plasma membranes. An intriguing possibility is that membrane binding sites may be involved in translocation of thyroid hormone across membrane barriers.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

Distribution of basic fibroblast growth factor binding sites in various tissue membrane preparations from adult guinea pig

In order to localize a rich source of basic FGF receptor, we examined the distribution of basic FGF binding sites in brain, stomach, lung, spleen, kidney, liver and intestine membrane preparations from adult guinea pig. Comparative binding studies using iodinated basic FGF showed that a specific binding was detected in all the membrane preparations tested. Scatchard plots from iodinated basic FGF competition experiment with native basic FGF in various membrane preparations, suggested the presence of one class of binding sites in some tissues such as liver, kidney, spleen, lung, stomach, and intestine with an apparent dissociation constant (appKD) value ranging from 4 to 7.5 nM and the existence of a second class of higher affinity sites in brain membranes with appKD value of 15 pM. Characterization of these basic FGF high affinity interaction sites was performed using a cross-linking reagent. These results show for the first time that specific interaction sites for basic FGF are widely distributed, suggesting that this growth factor might play a role in the physiological functions of a number of adult organs.     

Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin

Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.     

Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel

Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.     

Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung

Pituitary-adenylate-cyclase-activating polypeptide (PACAP) is a novel 38-amino-acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27-amino-acid N-terminal derivative of PACAP [PACAP(1-27)] labelled with 125I. Particularly high specific binding sites of 125I-PACAP(1-27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I-PACAP(1-27)-binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I-PACAP(1-27) was observed at pH 7.4. Interaction of 125I-PACAP(1-27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1-27) is more potent than PACAP in displacing the binding of 125I-PACAP(1-27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 +/- 1.52 nM and 11.45 +/- 3.65 nM, respectively; mean +/- SEM, n = 4] and lung membranes (IC50 = 4.41 +/- 0.87 nM and 10.68 +/- 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I-PACAP(1-27) in lung membrane (IC50 = 16.88 +/- 5.14 nM) but not in brain membranes. The equilibrium binding of 125I-PACAP(1-27) at 4 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax of 2.84 +/- 0.72 pmol/mg protein and 9.13 +/- 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP-binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.     

Glutamate-binding membrane proteins from human platelets]

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.     

Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes.

An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.     

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.     

Saturable binding to cell membranes of the presynaptic neurotoxin, beta-bungarotoxin.

Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Action of modifiers of a radiation lesion on the binding of prostaglandin E2 with liver membranes in F1 CBA X C57BL strain mice

Prostaglandin E2 (PGE2) was specifically bound by the membrane fraction prepared from the mouse liver. The binding constants indicate the presence of high-affinity PGE2 binding sites with an apparent dissociation constant (Kd) of 0.82 X 10(-9) M and a capacity of 0.36 X 10(9) M/mg protein and a lower affinity PGE2 binding site with Kd = 15.73 X 10(-9) M and a capacity of 5.31 X 10(9) M/mg protein. The radioprotectors, MEA and APAETP inhibit PGE2 binding and alter its kinetics. Apparently the mechanism of PGE2 binding by membranes is related to interaction of prostaglandins with thiols and sufhydryl groups of membrane lipoproteins, while the radioprotectors modify the functional groups participating in receptor PGE2 binding.     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

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.     

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.     

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.     

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.