Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney.

In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney

Summary In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

Muscarinic binding to mouse brain receptor sites

In mouse brain the binding of [³H]-Atropine to the muscarinic receptor seems to be a simple mass-action determined process as gauged both by approach to equilibrium kinetics and binding at equilibrium. In contrast, using isotopic dilution technique, dissociation measurements indicate the existence of two receptor-ligand complexes. It would appear that association and dissociation rates of binding of the muscarinic antagonists atropine, scopolamine, N-methyl-4-piperidyl benzilate (4NMPB) and 3-quinuclidinyl benzilate (QNB) decrease with increasing affinity based on comparisons of kinetic binding data. The differences between the association rate constants are small whereas those between the dissociation rate constants differ markedly. This kinetic behavior is similar to the well-known time profile of antimuscarinic activity in isolated tissues. These phenomena are discussed in terms of possible isomerization of the receptor-ligand complex, as has been proposed recently for [³H]-scopolamine and [³H]-4NMPB binding.     

Atrial natriuretic peptide binding sites in the brain and pituitary gland of the toad, Bufo marinus: localisation and receptor characterisation

The distribution and nature of 125I-atrial natriuretic peptide binding sites have been examined in the brain and pituitary gland of the toad, Bufo marinus, using tissue section autoradiography, affinity cross-linking and electrophoresis, guanylyl cyclase assays and molecular analysis of natriuretic peptide receptor C (NPR-C) and NPR-GC mRNA expression. The highest density of 125I-atrial natriuretic peptide binding sites occurred in the dorsal pallium, the habenular region, the torus semicircularis, the choroid plexus, and the pituitary gland. Less dense binding was observed in the medial pallium, the thalamic region, the hypothalamus, the optic tectum, and the interpeduncular nucleus. The natriuretic peptide receptor-C specific ligand, C-ANF, displaced the binding in all brain regions; however, some residual binding was observed in the habenular region, the hypothalamus, the choroid plexus, and the pituitary gland. In isolated brain membranes, 1 microM rat atrial natriuretic peptide increased cyclic guanosine monophosphate levels to 90% above basal. Affinity cross-linking followed by reducing electrophoresis showed that 125I-atrial natriuretic peptide bound to proteins of 65 kDa and 135 kDa respectively. Furthermore, molecular analysis demonstrated that natriuretic peptide receptor-C and guanylyl cyclase messenger ribonucleic acid are expressed in the brain. In combination with the autoradiography, the data indicated that atrial natriuretic peptide acting via specific receptors could be important in natriuretic peptide regulation of the brain.     

Age dependence of somatostatin levels and somatostatin binding in the rat brain

1. Somatostatin-like immunoreactivity (SLI) and 125I-Tyrl-somatostatin binding were measured from the brains of rats aged 1, 8 and 18 months. 2. Somatostatin binding was reduced in the striatum, frontal cortex, hypothalamus and hippocampus of the 8-month-old rats compared to the 1-month-old group. 3. Somatostatin binding was reduced in the striatum, frontal cortex and hippocampus of the 18-month-old rats compared to the 1-month-old group. 4. The reduction (40%) was most striking in the frontal cortex. 5. In no area of the brain did changes in SLI differ significantly between the different age groups.     

Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart

Since there exist some obscurities in the expression of mRNAs and their receptors in the heart, we have investigated the gene expression (mRNA levels) of adrenoceptors (alpha1A-, alpha1B-, beta1-, beta2-, beta3-) and muscarinic receptors (M2) and the density of receptor binding sites (alpha1A-, alpha1B-, beta1-, beta2-adrenoceptors, muscarinic receptors). Moreover, the heart regions consist of tissue rich in ganglion cells (that are of importance in heart neural circuits) and those virtually free of them (myocytes). Therefore, we have examined the differences in the distribution of mRNAs/receptor binding sites in the atrial samples of the heart rich in ganglion cells vs. those are virtually free of them. Binding sites and mRNAs of muscarinic receptors and alpha1B-adrenoceptors differ in their distribution in different heart regions. The mRNAs for beta1- and beta2-adrenoceptors were almost equally distributed herein, while the amount of beta-adrenoceptors significantly differs in the heart regions. The alpha1A- and beta3-adrenoceptors mRNAs were also found in all investigated heart regions, but at significantly lower level and have not shown region differences. This is a new finding, especially to beta3-adrenoceptors, as they were not regularly found in each heart regions. alpha1B-adrenoceptors have similar distribution of their mRNAs and binding sites in some heart parts. Thus, we can conclude that there are noticeable differences in the presence of receptors in heart regions that contain ganglion cells in comparison to those are virtually free of them.     

Distribution and characterisation of immunoreactive somatostatin in human gastrointestinal tract

Immunoreactive somatostatin (IRS) was measured in acid extracts of human gastrointestinal tissue. The highest levels were found in the duodenum, pancreas, jejunum and stomach with lower levels in the ileum and colon. In the antrum, pylorus, duodenum and pancreas the main peak of IRS (1.6K IRS) coeluted with synthetic somatostatin-14 on both gel filtration chromatography and HPLC. In the body of stomach, jejunum, ileum and colon, a large peak coeluting with synthetic somatostatin-28 (3.5K IRS) on both chromatographic systems was also identified, while minor peaks of IRS assigned molecular weights of 6000 (6K) and greater than 15 000 (15K) were seen in some extracts. The total IRS content and pattern of molecular forms were similar in tissues obtained from adults at surgery or rapid post mortem, and in tissue taken from human fetuses after prostaglandin termination of pregnancy. When tissues were divided into mucosal and muscle layers, greater than 90% of the IRS was in the mucosa with less than 10% in the muscle layer. In the muscle layer the IRS was almost entirely the 1.6K form in all tissues. Immunohistochemical studies showed the IRS in the mucosa to be localised in endocrine-type cells, while in the muscle layer the IRS is present in nerve fibres and neurones of the myenteric plexus. It is suggested that (1) different mechanisms may control the biosynthesis of somatostatin-14 and somatostatin-28 in mucosal cells in different parts of the gut, (2) different biosynthetic controls may operate in endocrine-like and neuronal cells in the same region of the gut.     

Characterization and visualization of neurotensin binding to receptor sites in human brain

The binding of monoiodo [125I-Tyr3]-neurotensin to human brain was characterized and visualized using radioreceptorassay and autoradiographic techniques. Specific binding to homogenates of human substantia nigra at 25 degrees C was maximal at 20 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two populations of binding sites with Kd values of 0.26 nM and 4.3 nM. Corresponding binding capacities were 26 and 89 fmol/mg of protein. Neurotensin analogs inhibited the binding of iodinated neurotensin with relative potencies that demonstrated the crucial role of the C-terminal hexapeptide portion of neurotensin for binding to its receptors. Autoradiography of human substantia nigra sections incubated with iodinated neurotensin revealed high levels of specific binding in the nucleus paranigralis and substantia nigra, pars compacta, and low levels in the substantia nigra, pars reticulata.     

Distribution of neuromedin U receptor subtype 2 mRNA in the rat brain

Neuromedin U (NMU) is a family of peptides found in the gut and the central nervous system [Neuroscience 25 (1988) 797; Biochem. Biophys. Res. Commun. 130 (1985) 1078]. While several peripheral activities such as uterus stimulating and hypertensive effects have been described for NMU [Biochem. Biophys. Res. Commun. 130 (1985) 1078], its role in the CNS remains poorly understood. Recently, we reported the identification of two receptors for NMU (NMU1R and NMU2R), and demonstrated that NMU may play a role in regulating feeding behavior. The central effect of NMU is likely mediated primarily via NMU2R, since NMU1R is detectable only in the periphery, but not in the brain [Nature 406 (2000) 70]. In this report, we describe detailed mapping of NMU2R mRNA expression in the rat brain by in situ hybridization. The most intense signals were observed in the ependymal cell layer along the wall of the third ventricle in the hypothalamus, CA1 region of the hippocampus, indusium griseum and septohippocampal nucleus. Moderate expression was detected in the hypothalamic paraventricular nucleus, dorsal raphe nucleus as well as a number of other brain structures. The presence of NMU2R in the hypothalamus is consistent with its role in energy balance. Significant levels of expression of NMU2R elsewhere in the brain may suggest additional physiological functions for this neuropeptide.     

Evidence for somatostatin binding sites in rabbit kidney

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

Characterization of somatostatin binding sites in isolated rat adipocytes

Somatostatin binding sites were characterized in isolated rat adipocytes. The binding was found to be saturable, reversible, and time- and temperature-dependent. The somatostatin binding sites are principally located on the cell surface. 125I-[Tyr11]somatostatin binding was not inhibited by glucagon and angiotensin II. By contrast, native somatostatin and somatostatin-28 displaced labeled peptide with a similar ED50: 50 nM. Scatchard analysis pointed to the existence of two classes of binding sites, with a Kd of 7.64 nM for the high-affinity sites and a Kd of 295 nM for the low-affinity ones. Comparison of somatostatin receptor binding and its lipolytic action in isolated rat adipocytes suggested that the spare receptor phenomenon cannot be applied to the lipolytic action of somatostatin in rat adipose tissue.     

Atrial natriuretic factor mRNA and binding sites in the adrenal gland.

The factor inhibiting aldosterone secretion produced by the adrenal medulla may be atrial natriuretic factor (ANF), since the latter abolishes aldosterone release in response to a number of secretagogues, including angiotensin II and K+. In this study we have shown that cells in the adrenal medulla contain ANF mRNA and therefore have the potential to synthesize this peptide. The presence of binding sites for ANF predominantly in the adrenal zona glomerulosa suggests that, if ANF is synthesized in the medulla and transferred to the cortex, it may affect mineralocorticoid status.     

Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor.

The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.     

Species variations of somatostatin concentrations and binding sites in jejunal mucosa

1. Somatostatin-like immunoreactivity (SLI) and specific binding of 125I-Tyr11-somatostatin were measured in jejunal mucosa of the mouse, rat, hamster, rabbit and guinea-pig. 2. The SLI concentrations in guinea-pig and rabbit were much greater than those in other rodents considered. 3. Somatostatin binding varied greatly with the species examined, the highest values being observed in cytosolic fraction of guinea-pig and rabbit jejunal mucosa, but the lowest ones in mouse. 4. The observed differences in somatostatin binding were not related to varying extents of degradation by the diverse cytosolic preparations studied. 5. The binding sites were highly specific for somatostatin in all rodent species studied. 6. There appears to be a direct relationship between somatostatin levels and somatostatin binding sites in jejunal mucosa when considering a variety of rodent species usually employed as laboratory animals.     

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.     

Affinity labeling of melatonin binding sites in the hamster brain.

N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin, was used to identify melatonin binding proteins in synaptosomes from Syrian hamster brain. Incubation of the synaptosomes with BIM resulted in a concentration dependent, irreversible inhibition of 2-125I-iodomelatonin binding. The radioactive form of BIM, N-Bromoacetyl-2-125I-iodo-5-methoxytryptamine (125I-BIM), became covalently attached to three proteins in the synaptosomes, in a concentration dependent manner. These proteins had apparent molecular weight values of 92, 55 and 45 kilodaltons. The incorporation of 125I-BIM into all three proteins was inhibited by BIM greater than 2-iodomelatonin greater than melatonin whereas the melatonin antagonist N-(1,4 dinitrophenyl)- 5-methoxytryptamine (ML-23) selectively inhibited the labeling of the 45 kDa protein. These results indicate that the 92, 55 and 45 KDa polypeptides are melatonin binding proteins.     

beta-Endorphin: characteristics of binding sites in the rat brain.

Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using $\text{[}{}^3\text{H-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the ligand. The binding is time dependent and saturable with respect to β_h-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of β_h-endorphin, is presented.     

Characterization of specific somatostatin binding sites in guinea pig lung

Somatostatin binding sites have been demonstrated in the cytosolic fraction of guinea-pig lung. Binding of 125I-Tyr11-somatostatin was dependent on time and temperature, saturable, reversible and highly specific. Under equilibrium condition, i.e. 60 min at 25 degrees C, native somatostatin inhibited tracer binding in a dose-dependent manner. Two types of somatostatin binding sites were defined by Scatchard analysis: a small population with a high affinity (Kd = 23.4 nM) and a large population with a low affinity (Kd = 253.5 nM) for somatostatin. The biphasic nature of the dissociation process confirmed the heterogeneity of somatostatin binding sites. Apart from somatostatin, no peptide (1 microM) tested influenced the binding of 125I-Tyr11-somatostatin. The present data represent the first analysis of somatostatin binding sites in lung.