Localization Of Specific Binding Sites Research Articles

Localization of specific binding sites for125I-TGF-β1 to fenestrated endothelium in bone and anastomosing capillary networks in enamel organ suggests a role for TGF-β1 in angiogenesis

Previous studies have shown endothelial cells to be a major target for endocrine TGF-beta in several soft tissues in the normal growing rat. The potent effect of TGF-beta1 on bone formation prompted us to analyze in detail the localization of specific binding sites for endocrine TGF-beta in hard tissues. At 2.5 minutes after injection of 125I-TGF-beta1, specific binding, as demonstrated by quantitative radioautography, was localized to fenestrated endothelium participating in angiogenesis in the vascular invasion region of the growth plate in bone as well as to anatomizing capillary networks in the maturation zone of the enamel organ. At 15 minutes after injection, the bound ligand was internalized into endocytic vesicles of endothelial cells. In bone, quantitation revealed significant differences in receptor density between endothelia undergoing proliferation vs those in a state of elongation and anastomosis with neighboring endothelial cells. In the rat incisor, specific binding of 125I-TGF-beta1 to endothelium correlated with increased formation of anastomotic capillary networks. These studies identify differential specific binding sites of 125I-TGF-beta1 in angiogenically active endothelium, providing an important link between TGF-beta1, the endothelium, and hard tissue development.

Localization of Endothelin Binding Sites and Endothelin-Like Immunoreactivity in Human Fetal Heart

The localization of [125I]endothelin-1 ([125I]ET-1) and [125I]endothelin-3 ([125I]ET-3) binding sites, as well as ET-like immunoreactivity, was investigated in sections of human fetal heart, using in vitro autoradiographic and immunohistochemical techniques. High-affinity [125I]ET-1 binding sites showed a tissue-specific distribution pattern, with high-density binding to the atria, ventricles (77-100 amol/mm2), and cardiac valve cusps (120.6 +/- 2.6 amol/mm2). Specific high-density binding of [125I]ET-3 was also exhibited on valve cusps (143.2 +/- 2 amol/mm2), whereas a much lower density of binding was displayed on atria and ventricles (10-15 amol/mm2). Microautoradiographic examination demonstrated binding sites on the wall of the aorta, pulmonary and coronary arteries, myocardium, ventricular conduction system, endocardium, and endothelial lining of valve cusps. Regional differences in the density and affinity of ET binding sites suggest that subpopulations of receptors are present in the human fetal heart. ET-like immunoreactivity was localized to a heterogeneous population of endothelial, endocardial, and epicardial mesothelial cells. The concordant localization of specific binding sites and ET-like immunoreactive cells indicates that locally released peptide might have a paracrine or autocrine role, possibly influencing cardiovascular development and function.

Localization of 3H‐Dihydroergotamine‐binding sites in the cat central nervous system: Relevance to migraine

Dihydroergotamine (DHE) is the treatment of choice in aborting the acute attack of migraine. Although its efficacy has been known for 40 years, its mechanism of action is still disputed. Data regarding the site of action of dihydroergotamine may provide an insight into its mechanism of action and thus identify a locus of potentially abnormal pathophysiology in migraine. By using in vitro and ex vivo autoradiographic techniques, the localization of specific binding sites for 3H-dihydroergotamine in the cat brain has been examined. Binding was seen in the dorsal horn of the cervical spinal cord, in the medulla, associated with the nucleus of the tractus solitarius, area postrema, and descending spinal trigeminal nucleus, and in the mesencephalon and the cerebral cortex. The highest density of binding sites was found in the dorsal and medial raphe nuclei of the midbrain. Furthermore, these same brain regions were also labeled after intravenous administration of 3H-dihydroergotamine. It is important that the brain areas specifically labeled are key nuclei involved in cranial pain transmission, suggesting that dihydroergotamine may act at these central sites in migraine.

Reduction of insulin binding in the arcuate nucleus of the rat hypothalamus after 6-hydroxydopamine treatment

Insulin receptors are present in the hypothalamus, but the cell types bearing them are unknown. In order to test the hypothesis that some insulin receptors in the hypothalamus are associated with catecholamine terminals, rats were injected with 50 μg or 75 μg doses (intracerebroventricular) of 6-hydroxydopamine (6-OHDA). Control rats received vehicle only. The animals were sacrificed 7 days after injection, and catecholamine and indolamine levels in the hypothalamus were measured by high performance liquid chromatography with electrochemical detection. Localization of specific binding sites for [ 125I]-insulin in the arcuate (ARC), dorsomedial (DMN) and ventromedial (VMN) nuclei were determined by quantitative film autoradiography. Treatment with 6-OHDA resulted in a 70% reduction in hypothalamic norepinephrine content as compared to vehicle-treated controls ( P < 0.01). A slight depletion of epinephrine, dopamine and indolamines was also detected. Computerized image analysis of the autoradiograms was used to determine radioactivity bound (DPM/mm 2) in each nucleus. Highest binding was in the ARC and DMN, with much lower binding in the VMN. Insulin binding in the ARC of the 6-OHDA-treated group was decreased by 25% compared to controls ( P < 0.01). No significant change in insulin binding was observed in the DMN or VMN. The 6-OHDA treatment had no significant effect on weight gain or on plasma insulin levels. The reduction of insulin binding in the ARC after 6-OHDA treatment supports the hypothesis that some insulin binding sites are located on catecholamine terminals in the arcuate nucleus.

Atrial natriuretic factor binding sites in the jejunum

We have studied the localization and the characterization of atrial natriuretic factor (ANF) binding sites by radioautographic techniques. Quantitative in vitro radioautography with a computerized microdensitometer demonstrated the presence of high-affinity, low-capacity 125I-ANF-(99-126) binding sites (Kd, 48 pM; Bmax, 63 fmol/mg protein) mainly in the villi of 20-microns slide-mounted transverse sections of the rat jejunum. Competition curves showed 50% inhibitory concentrations of 55 and 1,560 pM for ANF-(99-126) and ANF-(103-123), respectively. In vivo electron microscope radioautography showed that 80% of the silver grains were localized on the lamina propria fibroblast-like cells, 18% on mature enterocytes, and 2% on capillaries. Bradykinin and adrenocorticotropin did not compete with ANF binding. These results demonstrate that ANF binding sites in the rat jejunum possess the pharmacological characteristics of functional ANF receptors encountered in other rat tissues, and ultrastructural radioautographs show their cellular distribution. Taken together, these results demonstrate the presence and the localization of specific binding sites for ANF in the jejunal villi of the rat small intestine.

Autoradiographical Localization of Binding Sites for Porcine [125I]Endothelin-1 in Humans, Pigs, and Rats

Endothelin-1 (ET-1) is a recently discovered 21 amino acid peptide with potent vasoconstrictor properties. So far, its expression has been found only in porcine aorta, whereas its putative role as the endothelium-derived constricting factor (EDCF) would require it to be expressed and active in most vascular beds. We have used quantitative receptor autoradiography on pig, rat, and some human tissues to determine the distribution and localization of specific binding sites for ET-1. In some cases where binding sites were found, studies were performed to determine whether these are likely to be functional receptors. Binding sites for ET-1 have been found in heart (nerves greater than atria greater than ventricle greater than coronary arteries), kidney (glomeruli greater than papilla), adrenal (zona glomerulosa greater than medulla), cerebellum, spinal cord, gut, spleen, and lung. The binding of [125I]ET-1 was displaced at all these sites by unlabeled ET-1 but not by nitrendipine, apamin, and other vasoconstrictor peptides. ET-1 contracted strips of human coronary artery at an EC50 of 15 nM, with a maximal contraction 130% that of K+. A positive inotropic effect was found in strips of human atria (EC50 = 1 nM), which was not blocked by alpha- or beta-blockade. The widespread distribution of its binding sites suggests a more extensive role than control of vascular tone.

Localization of specific binding sites for bombesin in the canine gastrointestinal tract

The goal of these studies was to determine the tissue and cell types possessing specific binding sites for bombesin/gastrin-releasing peptide in the canine gastrointestinal tract. Monoiodinated, biologically active (Tyr-4)-bombesin 14 (100 pM) was applied to sections of canine gut and localized using quantitative autoradiography. The highest density of bombesin/gastrin-releasing peptide specific binding sites occurred over endocrine cells in the antral mucosa. Specific binding sites were also found on the circular muscle layer of the gastric fundus, gastric antrum, and ileum, on longitudinal muscle of the gastric fundus and antrum, and on neuronal elements in the myenteric plexus in the gastric fundus, antrum, and small intestine. No evidence for specific binding of 125I-(Tyr-4)-bombesin 14 was found in sections of canine esophagus, gastric cardia, gallbladder, pancreas, or colon. These results suggest sites of direct action of bombesin and endogenous gastrin-releasing peptide for gastrin release and gastrointestinal motility.

Localization of specific binding sites for bombesin and related peptides in the gastrointestinal tract : Patrick W. Mantyh, Christopher R. Mantyh, Andrew S. Giraud, Andrew H. Soll, John H. Walsh, and Steven R. Vigna. CURE/UCLA, VA Wadsworth Medical Center, Los Angeles, CA 90073, USA

Localization of specific binding sites for bombesin and related peptides in the gastrointestinal tract : Patrick W. Mantyh, Christopher R. Mantyh, Andrew S. Giraud, Andrew H. Soll, John H. Walsh, and Steven R. Vigna. CURE/UCLA, VA Wadsworth Medical Center, Los Angeles, CA 90073, USA

Localization of specific binding sites for atrial natriuretic factor in the central nervous system of rat, guinea pig, cat and human

Specific, high-affinity binding sites for atrial natriuretic factor (ANF) were identified and localized in the rat and guinea pig central nervous system (CNS), the cat brainstem, and the rat, guinea pig, cat and human spinal cord using quantitative autoradiographic techniques. The radioligands tested were rat 125I-ANF(1–28) in guinea pig, rat, cat and human tissues, human 125I-ANF in rat and human, and rat [ 3H]atriopeptin III in rat. All 3 radioligands labeled essentially the same structures in the brain and spinal cord of all species in which they were tested. In guinea pig very high concentrations of ANF binding sites were observed in the olfactory bulb, lateral olfactory tract and the granule cell layer of the cerebellum, high concentrations were observed in the fasciculus retroflexus, interpeduncular nucleus and subfornical organ. Moderate concentrations were observed in the nucleus accumbens, dorsomedial and suprachiasmatic hypothalamic nuclei, paraventricular thalamic nuclei, primary olfactory cortex and the subcommissural organ. High concentrations of ANF binding sites were also observed in the choroid plexus and the leptomeninges. Low concentrations were observed in the pineal gland. In the rat the same structures were labeled as in the guinea pig except that suprachiasmatic and dorsomedial hypothalamic nuclei, paraventricular thalamus and cerebellum were unlabeled. In the lower brainstem of the cat and all levels of the rat, guinea pig, cat and human spinal cord, the only site where specific binding was observed was in the pia/arachnoid. These findings suggest that ANF binding sites constitute several functional classes in the CNS as well as in a variety of other tissues. Outside the blood-brain barrier binding sites are prominent in (1) glandular tissues implicated in the production of hormones involved in fluid and electrolyte balance, e.g. adrenal glomerulosa, neurohypophysis and subfornical organ, (2) unstratified epithelia involved in ion gradient exchange, e.g. renal glomerulus, ciliary body, choroid plexus and pia mater; crossing the blood-brain barrier are sites in the (3) anterior hypothalamus, e.g. organum vasculosum, (4) regions of the brain parenchyma associated with angiotensin II binding sites, e.g. dorsomedial nucleus of hypothalamus, some of which may be occupied by brain rather than cardiac synthesized ANF, (5) regions of brain lacking an obvious role in fluid and ion exchange or regulation, e.g. cerebellum, although association with K +, Na +-ATPase in guinea pig cerebellum may be a relevant clue and (6) brain regions possibly implicated in an integrative and/or indirect regulatory role in fluid and electrolyte balance, but in a manner that is not understood, e.g. habenulo-interpeduncular system and nucleus accumbens.

Binding and action of aldosterone, dexamethasone, 1–25(OH) 2D 3, and estradiol along the nephron

The localization of specific binding sites of four steroids—aldosterone, dexamethasone, 1–25-dihydroxycholecalciferol and estradiol—is described along the nephron. This localization has been determined by using an autoradiographic method on dry films, applied to intact microdissected tubular segments. Aldosterone binds specifically to nuclei of the distal and cortical collecting tubule. No specific labeling was observed in the cytoplasm. This localization corresponds to the known sites of action of aldosterone on sodium reabsorption. Specific nuclear binding of dexamethasone is present all along the tubule except the proximal tubule. In this latter part of the nephron, a specific cytoplasmic labeling is observed, in the absence of nuclear labeling. This cytoplasmic binding could correspond to physiological effects in this structure, via non-genomic mechanisms. 1–25(OH) 2D 3 nuclear binding is located mainly in the loop of Henle and medullary collecting tubule, which are the sites of synthesis of calcium-binding proteins. No specific binding of estradiol is present in any part of the nephron.

Subcellular and extracellular localization of specific binding sites for triphenylethylene antiestrogens in human breast cancer

MCF-7 human breast cancer cell homogenates and subcellular organelles were submitted to isopycnic centrifugation on Percoll gradients to investigate the subcellular localization of triphenylethylene antiestrogen specific binding sites (AEBS). Electron microscopy revealed that gradient fractions coincident with the migration of [3H]tamoxifen-AEBS complexes were homogeneously represented by rough and smooth endoplasmic reticulum. Eighty percent of AEBS were localized in the endoplasmic reticulum [45,000 ± 4,000 sites/cell, mean ± S.D.), while 20% of these sites were also found in the nuclear fraction (12,000 ± 1,000 sites/cell, mean ± S.D.). A similar subcellular distribution of AEBS was observed in human breast cancer bioptic specimens. No differences in [3H]tamoxifen binding affinity between microsomal and nuclear AEBS were observed in MCF-7 and bioptic breast cancer. No major differences in microsomal AEBS levels were observed in the limited number of estrogen receptor-positive or -negative breast cancer specimens we have studied, whereas estrogen receptor-negative samples had higher levels of nuclear AEBS with respect to estrogen receptor-positive tumors. The presence of AEBS was also detected in the human serum of healthy and tumor-bearing subjects. The affinity and the binding specificity of serum AEBS were similar to those of intracellular AEBS. No differences in the levels of serum AEBS were observed between healthy and tumor-bearing subjects [19 ± 4and22 ± 4 pmoles/ml (mean ± S.D.) respectively. Human serum AEBS did not appear to be associated to lipoproteins, whereas it migrated as a 5.5 S sedimenting molecule.

Localization of specific binding sites for atrial natriuretic factor in peripheral tissues of the guinea pig, rat, and human.

Specific, high affinity atrial natriuretic factor (ANF) binding sites were identified and localized by autoradiographic techniques in peripheral tissues of the guinea pig, rat, and human. In the guinea pig kidney, high concentrations of ANF binding sites were located in the glomerular apparatus, outer medulla, and small renal arteries. Other peripheral tissues containing ANF binding sites included the zona glomerulosa of the adrenal cortex, the smooth muscle layer of the aorta and gallbladder, the lung parenchyma, the posterior lobe of the pituitary, the ciliary body of the eye, and the leptomeninges and choroid plexus of the brain. The distribution of ANF binding sites in the rat and human kidney was nearly identical to those seen in the guinea pig kidney; high concentrations were present in the glomerular apparatus, outer medulla, and small renal arteries. These results are consistent with earlier physiological and pharmacological studies that suggested that ANF plays a functional role in the regulation of extracellular fluid volume and blood pressure. There appears to be little species variation in the location and concentration of renal ANF binding sites, suggesting that, at least in the kidney, the results in experimental animals are relevant to the actions of ANF in humans. The finding that ANF binding sites were stable and present in high concentrations in human postmortem kidneys further suggests that these tissues may be amenable to testing for the involvement of ANF receptor dysfunction in diseases such as hypertension and congestive heart failure.

Immunocytochemical staining of isolated rat Sertoli cells for anti-FSH beta.

Sertoli cells are a primary target for the action of follicle-stimulating hormone (FSH) in the testis. The purpose of this investigation was to verify ultrastructurally that FSH binds to receptors on the plasma membrane of isolated rat Sertoli cells. A relatively pure aliquot of Sertoli cells was obtained by first dissociating testicular tissue from immature rats with collagenase and then centrifuging the suspension in Percoll density gradients. Pre-embedding staining with the peroxidase anti-peroxidase (PAP) complex technique using anti-FSH beta as the primary antiserum localized endogenous receptor-bound FSH on the plasma membrane of isolated Sertoli cells. Staining was considered to be specific since membranes of Sertoli cells derived from hypophysectomized rats were not stained when subjected to the same procedure. Cytoplasmic vesicles in Sertoli cells from experimental, control, and hypophysectomized groups also stained with PAP. Staining of these structures appeared to be specific since it was obliterated by preabsorption of anti-FSH beta with FSH. Preabsorption with luteinizing hormone (LH) did not affect the staining of cytoplasmic vesicles. The results of this investigation provide the first evidence for ultrastructural localization of specific binding sites for anti-FSH beta on the cell membrane of isolated Sertoli cells using an unlabeled antibody technique, and they further support the contention that Sertoli cells are a primary target for the action of FSH.

Autoradiographic localization of specific binding sites for [ 3H][Arg 8]vasopressin in the septum of the rat brain with tritium-sensitive film

Autoradiographic localization of specific binding sites for [ 3H][Arg 8]vasopressin in the septum of the rat brain with tritium-sensitive film

Radioautographic localization of specific binding sites for blood-borne angiotensin II in the rat brain

Radioautographic localization of specific binding sites for blood-borne angiotensin II in the rat brain

Stimulation of ADH release by the renin-angiotensin system.

Stimulation of ADH release by the renin-angiotensin system.