Specific Binding Of 125I Research Articles

Relationship between specific binding of 125I- ω-conotoxin GVIA and GTP binding protein: effects of the GTP analogues, mastoparan and AlF 4−

We investigated whether the specific binding or labeling of 125I- ω-CgTX on crude membranes from chick whole brain was affected when endogenous GTP binding protein (G protein) was activated by GTP analogues, mastoparan (MP) and aluminum fluoride (AlF 4 −; AlCl 3+NaF). Both GTP γS and Gpp(NH)p attenuated the inhibitory effect of selective N-type Ca channel inhibitors such as aminoglycoside antibiotics (AGs) or dynorphine (1-13)(Dyn) on specific 125I- ω-CgTX binding in a dose-dependent manner. On the other hand, the inhibitory effects of the divalent metal cations Cd 2+, Co 2+, Mg 2+ and Mn 2+ on such binding were not attenuated by GTP γS. MP and AlF 4 − also attenuated the inhibitory effect of Neo on this binding similar to GTP γS. The attenuating effect of MP was enhanced by the presence of Mg 2+ in a dose-dependent manner. However, GTP analogues, MP and AlF 4 −, did not affect binding or labeling without AGs or Dyn. GTP γS, MP and AlF 4 − also attenuated the specific labeling of a 215-kDa band in crude membranes with 125I- ω-CgTX using the cross-linker DSS (non-reduced condition) in the presence of Neo. These results indicate that there are direct or indirect relationships between N-type Ca channels and G proteins via binding sites for AGs or MP.

A Human Orphan Calcitonin Receptor-like Structure

A novel calcitonin receptor-like protein of 461 amino acids with seven putative transmembrane domains has been identified through molecular cloning in a cDNA library of the human cerebellum. 91% and 56% of the amino acids are identical in a rat orphan calcitonin receptor-like sequence and the human calcitonin receptor, respectively. 5.2 kb mRNA is predominantly expressed in the lung, heart and kidney. Specific binding of 125I -labeled salmon calcitonin and human calcitonin gene-related peptide-I to COS-7 cells transiently transfected with the receptor cDNA was less then 0.5%. Cellular cAMP accumulation was indistinguishable in cDNA transfected and non-transfected control COS-7 and renal tubular cells from the American opossum stimulated with human and salmon calcitonin, human calcitonin gene-related peptide-I and -II, human amylin, human adrenomedullin, lizard helodermin, salmon stanniocalcin and chicken parathyroid hormone-related protein. The receptor-like protein whose ligand remains to be discovered belongs to the family of receptors of calcitonin, parathyroid hormone, secretin, vasointestinal peptide and pituitary adenylate cyclase-activating polypeptide.

Characterization of specific calcitonin gene-related peptide receptors present in hamster pancreatic beta cells.

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of 125I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound 125I-[His]hCGRP I could be induced in the presence of 1 microM chicken CGRP (cCGRP). The specific 125I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC50) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0-2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of 125I-[His]hCGRP I and sCT was only effective at a high concentration (1 microM). Binding of 125I-[His]hCGRP I was dose dependently inhibited by guanosine-5'-O-(3-thiotriphosphate) or (GTP gamma S) and a 70% reduction of binding was obtained with 0.1 mM GTP gamma S. The IC50 value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTP gamma S. Human CGRP I and cCGRP at 2.5 microM did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 microM) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster beta cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.

C-type natriuretic peptide and atrial natriuretic peptide receptors of rat brain

Natriuretic peptide receptors in rat brain were mapped by in vitro autoradiography using 125I-labeled [Tyr0]CNP-(1-22) to bind atrial natriuretic peptide receptor (ANPR)-B and ANPR-C receptors selectively, and 125I-labeled alpha-ANP to select ANPR-A and ANPR-C receptors. Des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4- 23)-amide (C-ANP) was used for its selectivity for ANPR-C over ANPR-A. Specific binding of 125I-[Tyr0]CNP-(1-22) with a dissociation constant (Kd) approximately 1 nM occurred in olfactory bulb, cerebral cortex, lateral septal nucleus, choroid plexus, and arachnoid mater. This binding was abolished by C-type natriuretic peptide [CNP-(1-22)], alpha-ANP and C-ANP, and conformed to ANPR-C. 125I-alpha-ANP bound to all structures that bound 125I-[Tyr0]CNP-(1-22). This binding was also inhibited by both CNP-(1-22) and C-ANP, confirming the presence of ANPR-C-like binding sites. However, ANPR-C-like binding sites were heterogenous because only some had high affinities for 125I-[Tyr0]CNP-(1-22) and CNP-(1-22). 125I-alpha-ANP also bound sites without affinities for C-ANP or CNP-(1-22). These sites were consistent with ANPR-A. They occurred mainly on the olfactory bulb, the choroid plexus, and the subfornical organ. Guanosine 3',5'-cyclic monophosphate production was strongly stimulated by alpha-ANP but not by CNP-(1-22) in olfactory bulb. Neither ligand stimulated it in cortical tissue. Thus the natriuretic peptide binding sites of rat brain conformed to ANPR-A and to heterogenous ANPR-C-like sites. No ANPR-B were detected.

Effects of angiotensin II and nonpeptide receptor antagonists on transduction pathways in rat proximal tubule.

Because the presence of the angiotensin II (ANG II)-dependent phosphoinositide hydrolysis has been questioned from studies in proximal cells in culture, we looked for this transduction pathway in suspension of freshly isolated rat proximal tubule fragments. ANG II-receptor activation induced a prompt (within 15 s) and sustained increase in [3H]inositol phosphates (IPs; inositol trisphosphate, inositol bisphosphate, and inositol monophosphate). In fura-2-loaded tubules, it elicited a rapid and biphasic rise in cytosolic free calcium ([Ca2+]i) with an early peak (within 15 s) followed by a plateau. The peak was maintained in the absence of extracellular calcium. ANG II-induced inositol trisphosphate and [Ca2+]i rises showed a similar dose dependency, with a 50% effective concentration (EC50) of 2.9 and 5.5 nM, respectively. We checked that ANG II inhibited basal (EC50 4.4 nM) and parathyroid hormone- and forskolin-stimulated cAMP production, the latter effect being inhibited by pertussis toxin pretreatment. The effects of ANG II on IPs and [Ca2+]i were inhibited by the ANG II receptor subtype 1 (AT1) antagonist losartan and not by the ANG II receptor subtype 2 (AT2) antagonists PD 123177 and PD 123319. The effect of ANG II on forskolin-stimulated cAMP was inhibited by losartan and not by PD 123319. In agreement with these results, specific binding of 125I-[Sar1,Ile8]ANG II was markedly inhibited by losartan, whereas PD 123319 had no effect. These results demonstrate that AT1 receptor subtypes are present in intact rat proximal tubule cells and are coupled to both IPs-Ca2+ and cAMP signaling pathways. No evidence for AT2 receptor subtype is found.

Specific binding of 125I-LHRH agonist to hippocampal membranes: Fluctuations during the estrous cycle

Specific binding of 125I-[D-Ala 6-CH 3-Leu 7-Pro 9,NHET]LHRH, a LHRH agonist, to hippocampal membranes prepared from ovariectomized female rates was examined. One high affinity binding site was observed with a K d of 0.12 ± 0.01 nM and an apparent B max of 13.0 ± 3.8 fmol/mg . Luteinizing hormone-releasing hormone and the behaviorally active Ac-LHRH(5–10) were able to compete for the agonist binding site. Native LHRH had an apparent K i of 1.73 nM , while AC-LHRH(5–10) was 30 times less potent. Competition studies examined over the rat estrous cycle revealed an eighteenfold decrease in apparent affinity during diestrus I and estrus compared with ovariectomized animals. Tissue from animals in proestrus had a K i of 5.0 nM . Specific binding studies indicate that receptor concentration is highest in proestrus ( 6.11 ± 0.90 fmol/mg ) and significantly lower during estrus ( 2.4 ± 0.29 fmol/mg ). These data suggest that at least one fragment of native LHRH can interact with neuronal LHRH receptors and that these receptors, like those in the pituitary, can be modulated by circulating steroid hormones.

Biochemical and autoradiographic analysis of β-adrenoceptors in rat nasal mucosa

The specific binding of 125I-(−)-cyanopindolol ( 125I-(−)-CYP) to homogenates and cryostat sections of rat nasal mucosa was saturable, stereoselective and of high affinity (K d = 5.0 ± 0.4 pM, B max = 204 ± 12 fmol/mg protein and K d = 7.2 ± 0.7 pM; B max = 15 ± 1 fmol/mg protein respectively). The subtype-selective antagonists, LK203-030 and ICI118,551, inhibited specific 125I-(−)-CYP binding according to a two-binding site model, indicating the presence of 57 and 45% β 1-adrenoceptors in homogenates and cryostat sections, respectively. Competition of isoprenaline for antagonist binding to homogenates demonstrated 30 ± 3% high-affinity agonist binding sites. A steepening of the curve was observed in presence of guanine nucleotides. In vitro labelling of cryostat sections of rat nasal mucosa was combined with autoradiography. The autoradiographs generated after incubation with 20 pM 125I-(−)-CYP showed specific labelling of the epithelium and glandular excretory ducts. It appeared from autoradiographs generated with subtype-selective antagonists in addition to the radioligand that β 1-and β 2-adrenoceptors were present in both structures.

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.

Different ionic requirements for somatostatin receptor subpopulations in the brain

Two populations of brain somatostatin (SS) receptors, one with high affinity for the somatostatin octapeptide analogue SMS 201-995 (SS 1 type) and one poorly sensitive to this analogue (SS 2 type) have been characterised in regard to their ionic requirements using two radioligands, the iodinated Tyr 3 derivative of the octapeptide SS analog SMS 201-995 and the iodinated [Tyr 11]-SS. Specific binding of 125I-[Tyr 11]-SS to rat cortex membrane homogenates can be increased by approximately 180% in presence of 5 mM Mg 2+. The increase in number of binding sites seen by Mg 2+ is not accompanied by a marked increase in affinity for SS but for SMS 201-995: the low affinity binding for SMS 201-995 seen in absence of Mg 2+ is replaced in part by higher affinity binding in presence of these ions. SMS 201-995 sensitive SS 1 receptor subpopulation measured with 125I-204-090, a specific ligand for SS 1 subpopulation, is massively increased in presence of Mg 2+. However, SMS 201-995 insensitive SS 2 receptor population measured with 125I-[Tyr 11]-SS in presence of excess SMS 201-995 is unchanged in presence of Mg 2+. The Mg 2+-dependency can also be observed with autoradiography for extra cortical, i.e. hippocampal, brain SS receptors. 120 mM Na + does not affect the total brain SS receptor population, but reduces the specific binding of SS 1 receptors and increases that of SS 2 receptors. Therefore, the rat brain, in particular the cortex, possesses a SMS 201-995-sensitive, Mg 2+-dependent SS receptor subpopulation (SS 1) as well as a SMS 201-995-insensitive, Mg 2+-independent SS population (SS 2).

β-Adrenergic control of motility in the rat colon. II. Proportions of β1- and β2-adrenoceptors identified with 125I-(−)pindolol binding

The specific binding of 125I-(−)pindolol to membranes prepared from rat colon displayed high affinity (Kd = 75 ± 3 pM) and relatively low capacity (Bmax = 48 ± 3.8 fmol/mg protein). Furthermore, the binding was reversible and possessed properties expected of a β-adrenoceptor. Thus, for example, the potency of agonists in displacing 125I-(−)pindolol was, in decreasing order, isoproterenol > epinephrine > norepinephrine, and the negative isomer of propranolol was two orders of magnitude more potent than the positive isomer. Displacement of specific binding by the β1-adrenoceptor antagonist pafenolol and the β2-adrenoceptor antagonist ICI 118.551 revealed that the colon preparation possessed a heterogenous β-adrenoceptor population. Analysis of the inhibition curves using computerassisted curve fitting suggested that the sites consisted of a small (14%–21%) β1-adrenoceptor population and a large (79%–86%) β2-adrenoceptor population. The coexistence of β1- and β2-adrenoceptors is discussed in relation to evidence of a functional linkage of both subtypes with colon motility.

Pulsatile insulin has greater hypoglycemic effect than continuous delivery.

The relative hypoglycemic effects of pulsatile versus steadily infused insulin have been examined in six normal subjects in whom pancreatic insulin output was suppressed by somatostatin-14. Soluble insulin was infused continuously overnight on one occasion and on another occasion the same quantity was given in pulses of 2-min duration with a gap of 11 min. The mean plasma glucose concentrations were lower when pulsed insulin was given [mean for the last hour: 4.66 +/- 0.08 mmol/L (+/- SEM) versus 5.53 +/- 0.06 mmol/L (+/- SEM) for steady infusion], diverging significantly (P less than 0.05 paired t test) 7 h after the start of the study. The specific binding of 125I(A14)mono-iodo-insulin to monocytes was greater after pulsed insulin (2.9% with pulsed versus 2.4% with steadily infused insulin at tracer-only point; P less than 0.02 paired t test). Thus, intravenous insulin has greater hypoglycemic effect when pulsed, possibly mediated by greater insulin receptor binding.