Solubilization of binding sites for IAPP and CGRP from rat lung

Functional binding sites for [¹²⁵I]IAPP and [¹²⁵I]CGRP were solubilized from rat lung membranes with CHAPSO (10 mM). Rat IAPP had a higher affinity (K_i = 22.9 nM) for [¹²⁵I]IAPP binding and rat CGRP (K_i = 0.904 nM) had a higher affinity for [¹²⁵I]CGRP binding over related peptides. [¹²⁵I]IAPP binding was unaffected by GTPγS, but [¹²⁵I]CGRP binding was 50% inhibited, indicating solubilization of a G-protein-receptor complex for CGRP but not IAPP binding. Wheat germ agglutinin affinity columns gave a 25-fold purification of IAPP binding sites, but no CGRP binding sites were eluted from the column, indicating different patterns of glycosylation of the two sites.     

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [¹²⁵I]rAM revealed the presence of a single class of high-affinity (K_d1.3 × 10⁻⁸ M) binding sites for rAM in VSMC. The apparent K_i of rat calcitonin gene-related peptide (rCGRP) was 3 × 10⁻⁷ M. Affinity labeling of VSMC membranes with [¹²⁵I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC₅₀ of 10⁻⁸ M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.     

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [¹²⁵I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K⁺-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca²⁺-channel activator BAYK-8644, and blocked K⁺- and BAYK-8644-evoked rise in the intracellular Ca²⁺ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca²⁺ channel-mediated Ca²⁺ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.     

BW1023U90: A new GRP receptor antagonist for small-cell lung cancer cells

Gastrin-releasing peptide (GRP) receptor antagonists were synthesized and their ability to interact with small-cell lung cancer (SCLC) cells determined. [¹²⁵I]BW1023U90, bound with high affinity (K_d = 2 nM) to a single class of sites (Bmax = 55 fmol/mg protein) using SCLC cell line NCI-H345. [¹²⁵I]BW1023U90 binding was time dependent and reversible even at 37°C as the ligand was minimally internalized. Specific [¹²⁵I]BW1023U90 binding was inhibited with high affinity by GRP as well as bombesin (BB) but not neuromedin B (NMB). BW1023U90 inhibited the ability of BB to elevate cytosolic Ca²⁺ and increase the growth of SCLC cells. A BW1023U90 analogue, BW2258U89 (10 μg/day, SC) slowed SCLC xenograft formation in nude mice and [¹²⁵I]BW1023U90 localized to SCLC tumors 1 h after injection into nude mice. BW2258U89 (4% by weight) was placed in microspheres and slowly released over a 3-week period in nude mice bearing SCLC xenografts. The microspheres containing BW2258U89 strongly inhibited SCLC growth in vivo. A radioimmunoassay was developed for the GRP receptor antagonists and the rabbit antiserum cross-reacted totally with BW2258U89 or BW1023U90. BW2258U89 immunoreactivity (5 nM) was detected in the plasma of nude mice containing the microspheres after 1 week. These data suggest that GRP receptor antagonists bind to receptors on SCLC tumors.     

Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38°C. NT contracted the longitudinal muscle (ED₅₀, 0.3 nM), the 2–3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 μM), ≥50% of the response was blocked and the residual effect gave an ED₅₀ of 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 μM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED₅₀, 0.3 nM) and one substance P (ED₅₀, 1.4 nM). Using membrane preparations and ¹²⁵I-labeled NT, specific, high affinty receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (K_ds: 0.1 nM and 2 nM). Sodiu ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg⁺⁺ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.     

Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells

Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor.     

Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca²⁺ and stimulated gastrin release in a dose-dependent manner over the range 10⁻⁵-10⁻³ M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N-Methyl-³H]scopolamine ([³h]nms) binding on cell homogenates was time-dependent, saturable and consistent with a single high-affinity binding class (K_d = 39.5 pM, and B_max = 7.9 fmol/mg DNA). Carbachol competitively inhibited [³H]NMS binding. The potency of inhibition of [³H]NMS binding by subtype selective antagonists was hexahydrodifenidol> pirenzepine> AF-DX 116. These results suggest the M₃, muscarinic receptors may be involved in the carbachol-induced gastrin release from B6 RIN cells.     

Mechanism of action of amylin in bone.

Amylin is a 37 amino acid peptide produced mainly by beta-cells of the endocrine pancreas. Human amylin has 43% homology with human calcitonin gene-related peptide (CGRP) and 13% homology with human calcitonin (CT). Amylin and CGRP have been reported to have CT-like hypocalcemic activity in vivo. To investigate the role of amylin in bone, we examined the mechanisms of action of human amylin, CGRP, and CT in osteoclasts and osteoblasts. Both human amylin and CGRP inhibited 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]- induced bone resorption in an organ culture system, and the potencies of the two peptides were similarly approximately 60-fold lower than that of human CT. Using a recently developed procedure for preparing large numbers of osteoclast-like multinucleated cells (MNCs) formed in co-cultures of mouse osteoblasts and bone marrow cells in the presence of 1 alpha,25(OH)2D3, we found that both human amylin and CGRP stimulated cAMP production in osteoclast-like MNCs, but only at 60-fold higher concentrations than human CT. Specific binding of [125I]-human CT to osteoclast-like MNCs was detected (dissociation constant, 3 x 10(-8) M; binding sites, 3 x 10(7) per cell). To displace the bound [125I]-human CT from osteoclast-like MNCs, about 170-fold higher concentrations of human amylin and CGRP were required. No specific bindings of [125I]-amylin and [125I]-CGRP to osteoclast-like MNCs could be detected. Human CGRP stimulated cAMP production both in established mouse osteoblast-like cells (KS-4) and in mouse primary osteoblast-like cells. Amylin was a weak agonist for cAMP production in KS-4 cells. The increment in cAMP production induced by CGRP and amylin was abolished by the addition of human CGRP(8-37), a selective antagonist for CGRP receptors. CT did not stimulate cAMP production in KS-4 cells. Amylin, but not CT, displaced the bound [125I]-human CGRP from rat brain membranes. These results indicate that amylin binds not only to CT receptors in osteoclast-like MNCs but also to CGRP receptors in osteoblasts. The relative potencies of these compounds to induce cAMP production was CT greater than amylin not equal to CGRP in osteoclast-like MNCs and CGRP greater amylin much greater than CT in osteoblast-like cells.     

Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

Parathyroid hormone binding sites in the brain

Parathyroid hormone (PTH) has been shown to have actions within the brain, suggesting the presence of central PTH receptors. This possibility was examined by determining the binding of ¹²⁵I-labeled [Nle^8,18,Tyr³⁴]bovine PTH to the plasma membranes of rat and rabbit brains. Specific binding of the tracer to membranes of the whole brain was time and tissue dependent, and was greater with membranes from the hypothalamus than with membranes from the cerebellum, cerebrum, or brain stem. The binding of the tracer to rat hypothalamic membranes was saturable and competitively displaced by unlabeled PTH(1–34), PTH(3–34), [Nle^8,18,Tyr³⁴]PTH(1–34), and by PTH-related protein, indicating the presence of a single class of high-affinity (dissociation constant = 2–5 nM), low-capacity (maximum binding capacity, B_max = 110–250 fmol/mg protein) binding site. The binding of radiolabeled PTH to these sites was not displaced by unrelated peptides of comparable molecular size (calcitonin, calcitonin-gene related peptide, adrenocorticotropin). The binding of PTH to these sites did not, however, appear to stimulate adenylate cyclase activity, as in peripheral PTH target sites. Thus, although these results indicate the presence of PTH receptors in the brain, these binding sites have a lower affinity than those in peripheral tissues and may utilize a different signal transduction system.     

Characterization of the novel CCK analogs JMV-180, JMV-320, and JMV-332 in H345 cells

The interaction of the novel CCK analogs JMV-180, JMV-320, and JMV-332 with CCK-B/gastrin receptors on small cell lung cancer (SCLC) cells was investigated. JMV-180, JMV-320, and JMV-332 potently inhibited specific binding of ¹²⁵I-CCK-8 to CCK-B/gastrin receptors expressed on the SCLC cell line NCI-H345 (H345) with IC₅₀ values of 4.9, 1.8, and 7.0 nM, respectively. JMV-320 and JMV-332 stimulated intracellular calcium ([Ca²⁺]_i) release in a dose-dependent manner in cells preloaded with indo-1. JMV-180 did not stimulate [Ca²⁺]_i but inhibited the [Ca²⁺]_i release elicited by 10 nM CCK-8 in a dose-dependent manner. These data indicate that JMV-320 and JMV-332 function as CCK-B/gastrin receptor agonists while JMV-180 functions as a CCK-B/gastrin receptor antagonist in H345 cells.     

Calcitonin gene-related peptide receptor in cultured vascular smooth muscle and endothelial cells

Using 125I-labeled-Tyr0-rat(r)-calcitonin gene-related peptide (CGRP), a potent vasodilatory neuropeptide, we have identified and characterized specific binding sites for CGRP in cultured rat vascular smooth muscle cells (VSMC) and bovine endothelial cells (EC). rCGRP and human (h) CGRP equipotently inhibited 125I-rCGRP binding to both cells, but human calcitonin (hCT) was less potent and other unrelated polypeptides were ineffective. Both rCGRP and hCGRP, but not hCT, equally stimulated intracellular cAMP generation in both cells distinct from beta-adrenergic receptor-mediated mechanism, although they had no effect on cGMP generation in either cell or synthesis of prostacyclin in EC. Autoradiograph of affinity-labeled cell membranes revealed that 125I-rCGRP interacts with a single binding component of almost identical molecular size (approximately 60-kDa) in both cells under reducing and nonreducing conditions. The present study demonstrates for the first time the presence of CGRP receptors in cultured VSMC and EC, functionally coupled to adenylate cyclase system distinct from beta-adrenergic receptors. It is suggested that CGRP-induced vasorelaxation may be mediated partly by cAMP-dependent and/or endothelium-dependent mechanism.     

Differential Calcitonin Gene-Related Peptide (CGRP) and Amylin Binding Sites in Nucleus Accumbens and Lung: Potential Models for Studying CGRP/Amylin Receptor Subtypes

Abstract: Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[¹²⁵I]iodohistidyl¹⁰)humanCGRPα ([¹²⁵I]hCGRPα) binding (IC₅₀ = 0.4–7.7 nM), which was displaced by hCGRP_8–37α with equally high affinity (IC₅₀ = 0.4–7.3 nM). High-affinity binding for [¹²⁵I]Bolton-Hunter human amylin ([¹²⁵I]BH-h-amylin) was also observed in these tissues (IC₅₀ = 0.2–6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC₅₀ = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin_8–37 competed for [¹²⁵I]hCGRPα binding with higher affinity (IC₅₀ = 5.4 nM) compared with [¹²⁵I]BH-h-amylin binding (IC₅₀ = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC₅₀ values for rat amylin_8–37 were 117 and 12 nM against [¹²⁵I]hCGRPα and [¹²⁵I]BH-h-amylin, respectively. Also, functional studies evaluating the activation of adenylate cyclase and generation of cyclic AMP in response to these agonists indicated that hCGRPα (EC₅₀ = 0.3 nM), h-amylin (EC₅₀ = 150 nM), and salmon calcitonin (EC₅₀ = 1,000 nM) activated adenylate cyclase, resulting in increased cyclic AMP production in porcine lung membranes that was antagonized by hCGRP_8–37α. The affinity of hCGRP_8–37α was similar for all three peptides. The cyclic AMP responses to amylin and salmon calcitonin were significantly (p < 0.05) lower than that of hCGRPα and not additive, suggesting that they are acting as partial agonists at the same CGRP1-type receptor in porcine lung membranes. Similar observations were made for guinea pig lung membranes. However, human amylin and salmon calcitonin were weaker than hCGRPα in activating lung adenylate cyclase. None of these peptides activated adenylate cyclase in membranes prepared from the nucleus accumbens of both species. The data from these studies demonstrate both species and tissue differences in the existence of distinct CGRP and amylin binding sites and present a potential opportunity to study further CGRP and amylin receptor subtypes.     

The role of calcitonin gene-related peptide (CGRP) in macrophages: the presence of functional receptors and effects on proliferation and differentiation into osteoclast-like cells

It has been shown that both calcitonin gene-related peptide (CGRP) and amylin bind weakly to calcitonin (CT) receptors in osteoclast-like cells formed in vitro and inhibit bone resorption by a cAMP-dependent mechanism. Osteoclasts are thought to be derived from cells of the monocyte macrophage lineage, in which CGRP, but not CT, induces cAMP production. In this study, we determined the presence of functional receptors for CGRP in mouse alveolar macrophages and the effects of this peptide on proliferation and osteoclastic differentiation in mouse alveolar and bone marrow-derived macrophages. Human CT did not stimulate cAMP production in macrophages. Human CGRP stimulated cAMP production in mouse alveolar macrophages and bone marrow-derived macrophages dose-dependently. Human amylin, which has 43% homology with human CGRP, also stimulated these macrophages to produce cAMP, but only at a 100-fold higher concentration. The increment in cAMP production induced by human CGRP and amylin was abolished by the addition of human CGRP(8–37), a selective antagonist for CGRP receptors. Specific binding of [¹²⁵I]human CGRP to alveolar macrophages was detected (dissociation constant, 2.5 × 10⁻⁸ M; binding sites, 1.4 × 10⁴/cell). Amylin, but not CT, displaced the bound [¹²⁵I]human CGRP from alveolar macrophages, but at a 100-fold higher concentration. No specific binding of [¹²⁵I]human CT and [¹²⁵I]human amylin to alveolar macrophages could be detected. Pretreatment with human CGRP for 24 h dose-dependently suppressed DNA synthesis in alveolar macrophages induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). CGRP also suppressed the number of macrophage colonies formed from bone marrow cells induced by macrophage colony-stimulating factor (M-CSF). Pre-treatment of alveolar macrophages with CGRP inhibited differentiation into osteoclast-like cells in co-cultures with primary osteoblastic cells in the presence of 1α,25-dihydroxy vitamin D₃. These results indicate that specific receptors for CGRP are present in macrophages and that CGRP modulates proliferation and differentiation of macrophages into osteoclast-like cells by a receptor-mediated mechanism involving cAMP.     

Kinetic evidence for isomerization of the dopamine receptor-raclopride complex

The binding kinetics of the specific dopamine D₂ antagonist [³H]raclopride to dopamine D₂ receptors in rat neostriatum were studied. The pseudo-first-order rate constants of [³H]raclopride binding with these membranes revealed a hyperbolic dependence upon the antagonist concentration, indicating that the reaction had at least two consecutive and kinetically distinguished steps. The first step was fast binding equilibrium, characterized by the dissociation constant K_A = 12 ± 2 nM. The following step corresponded to a slow isomerization of the receptor-antagonist complex, characterized by the isomerization equilibrium constant K_i = 0.11. The dissociation constant K_d = 1.3 nM, calculated from these kinetic data, was similar to K_d = 2.4 nM, determined from equilibrium binding isotherm for the radioligand. Implications of the complex reaction mechanism on dopamine D₂ receptor assay by [³H]raclopride were discussed.     

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.     

Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines

To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y₂ receptor than for the Y₁ receptor. The affinity of [Leu³¹,Pro³⁴]NPY is 7–60-fold higher for the Y₁ receptor when compared with the Y₂ subtype. Species selectivity within the Y₂ receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y₂ subtype (K_i of 0.3 nM) compared with the rabbit and rat Y₂ receptor (K_i of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y₂ subtype (K_i of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y₂ receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective.