RESUMEN
Parathyroid hypertensive factor (PHF) is a newly described hypertensive factor isolated from the plasma of spontaneously hypertensive rats (SHR). Recent studies have suggested that the primary origin of PHF is the parathyroid gland (PG). In the present investigation, PG from spontaneously hypertensive rats (SHR), as well as from normotensive rats, were isolated and maintained in culture. The PG from SHR, but not normotensive rats, released PHF into the culture medium. Omission of calcium from the culture medium stimulated the release of PHF. For purification of PHF, parathyroid gland culture medium (PGCM) was first dialyzed at 1000 mwco, and then ultrafiltered at 5000 molecular weight cut-off (mwco). PHF activity was retained in the fraction that was greater than 1000 daltons and less than 5000 daltons. Dialyzed and filtered SHR PGCM was fractionated by molecular exclusion HPLC. Biologically active PHF was collected in a discrete region. The biologically active molecular exclusion fraction was subsequently fractionated by reverse-phase HPLC (C-8). PHF was collected in a single discrete peak, which did not occur in culture medium prepared from normotensive PG in a similar manner. This biologically active peak occurred in the same position on molecular exclusion and reverse-phase HPLC as PHF purified from SHR plasma using similar procedures. Incubation of PGCM with trypsin inactivates the biological activity of PHF. The UV spectrum of PGCM PHF is identical to that obtained from purified plasma PHF. These results are consistent with the presence of a peptide moiety in PHF, and support the parathyroid origin of plasma PHF.
Asunto(s)
Factores Biológicos/aislamiento & purificación , Glándulas Paratiroides/química , Ratas Endogámicas SHR/anatomía & histología , Animales , Cromatografía Líquida de Alta Presión , Femenino , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas WKY , Ratas Sprague-DawleyRESUMEN
In fresh-water rainbow trout, Oncorhynchus mykiss (formerly called Salmo gairdneri), experimentally induced mild hypercalcemia results in release of immunoreactive stanniocalcin from the corpuscles of Stannius (CS) and stimulated synthetic and releasing activities of the glands as measured in vitro. Pulse-chase experiments showed that stanniocalcin (STC) is a 56-kDa glycoprotein, processed from a 64-kDa precursor, prostanniocalcin (PSTC). PSTC and STC are homodimeric molecules that are readily split into monomers in the presence of reducing agents such as 2-mercaptoethanol. The monomeric form of PSTC and STC contains an approximately 5- to 6-kDa glycomoiety. Neither this sugar residue nor the NH2-terminal amino acid sequences of PSTC or STC proved to contain antigenic sites for the antiserum used in this study. Two-dimensional gel electrophoresis indicated the presence of several isoforms of PSTC and STC molecules that may reflect different stages of maturation of the (pro)hormone.
Asunto(s)
Glándulas Endocrinas/metabolismo , Glicoproteínas/biosíntesis , Hormonas , Salmonidae/metabolismo , Trucha/metabolismo , Animales , Western Blotting , Calcio/sangre , Concanavalina A , Cisteína/farmacología , Electroforesis/métodos , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucosamina/farmacología , Técnicas In Vitro , Masculino , Pruebas de Precipitina , Trucha/sangreRESUMEN
Calcitonin gene-related peptide (CGRP) was found extensively in the small intestine of both non-mammalian and mammalian vertebrates using radioimmunoassay and immunocytochemistry. By radioimmunoassay, the levels of CGRP in rats, mice, chickens, bullfrogs and rainbow trout were found to range from 91.5 to 419.1 ng/g tissue. To localize CGRP in the small intestine, we used three different tissue preparations for immunocytochemistry: whole-mount preparations, and frozen and Paraplast sections. The combination of three tissue preparations made it easier to visualize the three-dimensional structure and reduced the possibility of missing the immunoreaction. Immunoreactive cell bodies were found in the plexi in the mammalian species. Dense and regular networks of CGRP fibers were observed in the smooth muscle layers, when examined in whole-mount preparations. In non-mammalian species, however, immunoreactive cell bodies could not be detected, although immunoreactive fibers were present, forming less dense and regular networks. Our results indicate that CGRP-immunoreactive fibers are present in the smooth muscle layers of the intestine from fish to mammals, suggesting that CGRP may be involved in regulating gastrointestinal smooth muscles in vertebrates.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/análisis , Intestino Delgado/análisis , Fibras Nerviosas/análisis , Animales , Pollos , Cobayas , Iguanas , Técnicas para Inmunoenzimas , Intestino Delgado/inervación , Ratones , Ratones Endogámicos ICR , Plexo Mientérico/análisis , Radioinmunoensayo , Rana catesbeiana , Ratas , Ratas Endogámicas , Plexo Submucoso/análisis , TruchaRESUMEN
Hypercalcemia (induced by CaCl2-injection or seawater exposure of the fish) reduced the hypocalcin content of corpuscles of Stannius (CS) in trout, goldfish and eel; concomitantly the synthetic activity of CS of hypercalcemic fish, as determinedin vitro, was enhanced. The monomeric forms of prohypocalcin and of hypocalcin of trout and goldfish are 32 and 28 kDA Mr glycoprotein species respectively; those of the eel are 2 kDa bigger,viz. 34 and 30 kDa respectively. Moreover, eel CS producein vitro an enigmatic 70 kDa glycoprotein with affinity for concanavalin-A. It is concluded that plasma calcium levels control storage and synthesis rates of hypocalcin in the CS.
RESUMEN
Highly specific antisera were raised against hypocalcin, a 54-kDa glycoprotein purified from the corpuscles of Stannius (CS) of rainbow trout, Salmo gairdneri. The specificity of the antisera was determined by using Ouchterlony's double immunodiffusion test, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunocytochemistry. In the double immunodiffusion test, a single precipitin line was formed between the antisera and hypocalcin. Both RIA and ELISA studies showed that serial dilutions of hypocalcin, teleocalcin, and CS extracts produced dose-response curves, whereas rat FSH, chicken LH, bovine TSH, salmon calcitonin, bovine parathyroid hormone, and human angiotensins I and II failed to cross-react with the antiserum. Immunoreactive hypocalcin was demonstrated in the plasma of a teleost (flounder), but not of an elasmobranch (dogfish). In the immunocytochemistry, most of the gland cells showed strong immunoreaction with the antisera, whereas some cells displayed no immunoreactivity.
Asunto(s)
Glándulas Endocrinas/análisis , Glicoproteínas/análisis , Hormonas , Sueros Inmunes , Animales , Glándulas Endocrinas/citología , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/inmunología , Carpa Dorada , Inmunodifusión , Técnicas para Inmunoenzimas , Radioinmunoensayo , TruchaRESUMEN
1 A new synthetic bradykinin analogue was found to be an antagonist of bradykinin-induced vascular permeability in rabbit skin. It was effective in equimolar concentrations. 2 These analogues also antagonized the action of bradykinin in contracting the guinea-pig isolated ileum. The mean pA2 values of five different antagonists ranged from 5.3-6.4 respectively, on this preparation. 3 Our observations, together with those of others suggest that these antagonists act on the same receptor types, viz., B2, in rabbit blood vessels and in smooth muscle of guinea-pig ileum. 4 Our results support the view that the way is now promising for the synthesis of potent specific antagonists of bradykinin for experimental and therapeutic use.