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C Yagil

Publications and source records attributed to C Yagil.

34 records · Page 2Linked to original sources
PubMed1990

Expression of glutamine/glutamic acid-rich proteins in rat submandibular glands.

The expression of GRP transcripts was found to be highly specific to the rat submandibular gland. GRP cross-reactive species were detected in the saliva of both inbred and outbred rat strains. There was no evidence of GRP transcripts in RNA prepared from bovine, ovine, porcine or murine submandibular glands. Thus the GRPs differ from the family of PRPs that are expressed in several species. The restriction of GRP expression to the rat suggests a relatively recent origin for a functional GRP gene, presumably after rat-mouse divergence. The ontogeny of the relative steady-state levels of GRP transcripts was assessed by dot blot analysis. Maximal levels of RNA-encoding GRP were detected at 6 months; there was a significant age-related decline at both 12 and 18 months. There was, however, no significant age-related alteration in the size of transcripts which encode this protein family.

Animals↗
PubMed1990

Osmotic regulation of vasopressin and oxytocin release is rate sensitive in hypothalamoneurohypophysial explants.

Explants of the hypothalamoneurohypophysial system (HNS) from rats were maintained in perifusion culture and exposed to 15-18 mosmol/kgH2O changes in the osmolality of the culture medium achieved by increasing or decreasing the NaCl concentration. The rate of change in osmolality was either 0.14 +/- 0.01 mosmol/min (2%/h), 0.27 +/- 0.02 mosmol/min (5%/h), or 1.7 +/- 0.2 mosmol/min (30%/h). Vasopressin (VP) and oxytocin (OT) release into the culture medium was determined. Increased VP release was detected after all three rates of increase, but the peak response occurred sooner and in response to a smaller increment in osmolality during the 5% rate of increase compared with the 2% rate of increase. Peak VP release occurred during the first 10 min of the 30% pulse and was significantly greater than the response to the 2 and 5% rates (P less than 0.05). The increase in VP release was sustained throughout the 3 h of hypertonicity during the 2% pulse, but not during the 5 and 30% osmotic pulses. A significant decrease in VP release was observed on returning the osmolality to basal at both the 2 and 5% rate, but this inhibition was followed by a rebound in VP release that started during the decrease in osmolality and significantly exceeded basal VP release. A significant inhibition of VP release also was observed when explants were exposed to hypotonic pulses at the 2 and 5% rate. At both rates, the inhibition of VP release corresponded to a 5- to 7-mosmol/kgH2O decrease in osmolality.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effect of extended exposure to hypertonicity on vasopressin messenger ribonucleic acid content in hypothalamo-neurohypophyseal explants.

The feasibility of using organ-cultured explants of the rat hypothalamo-neurohypophyseal system (HNS) to study the mechanisms regulating the vasopressin (VP) mRNA content of the HNS was examined by evaluating the effect of exposure to hypertonicity on the VP mRNA content of these explants. Different effects were observed after a step increase in osmolality and a gradual increase in the same amount over 24 h. The VP mRNA content of control HNS explants determined from a RNA protection assay was 22 +/- 6 pg. It gradually decreased to 23% and 9% of the control value during 24 and 48 h in culture, respectively. Northern blot analysis revealed a single band of VP mRNA approximately 700 bases long in explants cultured for 36 h. Explants exposed to the step increase in osmolality were maintained in static culture. The control explants were placed directly into isotonic medium (299 mosmol/kg H2O). The explants exposed to the step increase were placed directly into hypertonic medium (greater than 304 mosmol/kg H2O). After 24 h in culture, basal VP release was measured, and all explants were then exposed to a further acute 15 mosm/kg H2O increase in osmolality. The highest basal release of VP was observed in the explants maintained under isotonic conditions (299 mosm/kg H2O). These explants significantly increased VP release in response to the acute increase in osmolality. Basal VP release was lower in explants maintained in hypertonic medium (greater than 304 mosmol/kg H2O), and these explants did not respond to the acute hypertonic pulse. VP mRNA content was significantly decreased in explants maintained for 24 or 48 h in hypertonic medium compared to that in explants maintained in isotonic medium (47 +/- 10% and 57 +/- 6%, respectively; P less than 0.01). No significant difference existed in the VP content of the posterior pituitary between the groups. To achieve a slow increase in osmolality, explants were perifused in individual chambers with medium at 2.1 ml/h. A gradual increase in osmolality (16 mosmol/kg H2O medium) was achieved by increasing the NaCl concentration in the perifusion medium. In response to this stimulus there was a significant increase in VP release, which was sustained for 9 h. VP mRNA content in the hypertonic group was 165 +/- 19% of that in control explants (P less than 0.001), but no difference existed in VP content in the posterior pituitary compared to that in time control explants.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells.

In vivo, filtered insulin is absorbed and degraded in proximal tubules after binding to the apical membrane. Peritubular removal also occurs and involves basolateral receptor binding and degradation. Whether basolateral degradation proceeds within the cell or on the cell surface is unknown. Because of the difficulties in addressing this question in vivo, this study was carried out with a cultured opossum kidney epithelium cell line with proximal-like features and insulin receptors. Cells were grown in partitioned wells on polycarbonate filters and, when confluent, the monolayer effectively separated the culture well into apical and basolateral compartments. Apical and basolateral binding, internalization, and degradation were studied separately by incubating monolayers with 125I-insulin added to either the apical or basal compartment. At 37 degrees C insulin associated with either pole in a time-dependent manner. This interaction was specific, for it was competitively inhibited by cold insulin but not by unrelated peptides. Separation of surface-bound from internalized insulin was achieved by lowering extracellular pH. At 4 degrees C, 92% of the radioactivity added to either side of the monolayer was surface-bound, whereas at 37 degrees C and after 1 h, 57% was surface-bound and 43% internalized. Affinity of apical and basolateral receptors were similar (1-2 nM), but basolateral receptor number was greater, for at high insulin concentrations (5 x 10(-8) M) basolateral membrane binding exceeded apical by fivefold (250 +/- 81 vs. 56 +/- 11 fm/10(6) cells). Degradation followed exposure to either pole of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1988

Internalization and catabolism of insulin by an established renal cell line.

Proximal renal tubules are a key site of insulin metabolism. To explore the kinetics and metabolic requirements of insulin internalization and catabolism, we used the opossum kidney cell line, which has proximal tubular-like features and possesses insulin-specific receptors. Internalization was determined by separating membrane-bound insulin from intracellular insulin by exposure to an acidified medium. Internalization of membrane-bound insulin was rapid, and half-maximal internalization occurred within 2.5 min. Degradation products did not accumulate in the cell but appeared in the medium after a delay of 5 min from the onset of internalization. In other experiments, addition of KCN (2 mM) or omission of glucose did not alter degradation, but KCN, combined with the omission of glucose, inhibited degradation by 64%. This was associated with a 240% increase in membrane-bound insulin and an 81% decrease in intracellular insulin. Accordingly, it appears that under these circumstances impaired degradation was a consequence of impaired internalization. In contrast, although 0.1 mM chloroquine, an endosomal-lysosomal inhibitor, also depressed degradation (by 57%), intracellular insulin increased fourfold, indicating failure of intracellular processing. We conclude that these cultured kidney cells rapidly internalize and degrade insulin and that internalization, a prerequisite for degradation, is dependent on energy that can be derived from anaerobic glycolysis or oxidative metabolism. Furthermore, the intracellular degradative processing of insulin by these cells involves a chloroquine-sensitive pathway.

Animals↗
PubMed1988

Insulin binding, internalization, and degradation by a cultured kidney cell line.

Proximal tubules are a key site of insulin metabolism, which is in part a receptor-mediated process. To explore the interaction between insulin and the kidney and to evaluate the role of receptors in insulin uptake and processing, a study was carried out with a cultured proximal-like opossum kidney (OK) cell line. 125I-insulin associated with confluent monolayers in a specific manner, and this interaction was competitively inhibited by insulin; unrelated peptides were relatively ineffective. Insulin degradation exhibited time and temperature dependency and up to a concentration of 5 X 10(-8) M was not saturable. Degradation exhibited partial hormone specificity. Separation of plasma membrane bound from internalized insulin was achieved by lowering extracellular pH. At 4 degrees C, 94% of cell-associated radioactivity was membrane bound, whereas at 37 degrees C, in the steady state, 33% was membrane bound and 67% was internalized. There was a significant correlation between membrane-bound insulin and the rate of degradation. These findings reveal that the binding and processing of insulin by the kidney cell line are compatible with the description of the uptake of filtered insulin by the proximal tubule in the intact kidney. Accordingly we conclude that this cell line provides a good model for studying renal epithelial uptake and metabolism of insulin.

Adrenocorticotropic Hormone↗
PubMed1988

High performance liquid chromatographic analysis of insulin degradation products from a cultured kidney cell line.

The kidney is a major site for insulin removal and degradation, but the subcellular processes and enzymes involved have not been established. We have examined this process by analyzing insulin degradation products by HPLC. Monoiodoinsulin specifically labeled on either the A14 or B26 tyrosine residue was incubated with a cultured kidney epithelial cell line, and both intracellular and extracellular products were examined on HPLC. The products were then compared with products of known structure generated by hepatocytes and the enzyme insulin protease. Intracellular and extracellular products were different, suggesting two different degradative pathways, as previously shown in liver. The extracellular degradation products eluted from HPLC both before and after sulfitolysis similarly with hepatocyte products and products generated by insulin protease. The intracellular products also eluted identically with hepatocyte products. Based on comparisons with identified products, the kidney cell generates two fragments from the A chain of intact insulin, one with a cleavage at A13-A14 and the other at A14-A15. The B chain of intact insulin is cleaved in a number of different sites, resulting in peptides that elute identically with B chain peptides cleaved at B9-B10, B13-B14, B16-B17, B24-B25, and B25-B26. These similarities with hepatocytes and insulin protease suggest that liver and kidney have similar mechanisms for insulin degradation and that insulin protease or a very similar enzyme is involved in both tissues.

Animals↗
PubMed1988

Effect of bacitracin on binding and processing of insulin by established renal cell line.

The effect of bacitracin on the binding and processing of 125I-labeled insulin was studied in a proximal tubular epithelium-like opossum kidney cell line. This cultured cell line handles insulin in a manner comparable to the in vivo situation, which requires membrane binding, internalization, and intracellular degradation. The addition of bacitracin inhibited insulin degradation significantly and delayed the time of appearance of products in the medium (22 min) compared with control cells (14 min). Maximum total cell-associated radioactivity increased from 1.5 +/- 0.19% in the control cells to 2.5 +/- 0.17% in the treated cells. Separation of cell membrane from internalized radioactivity was achieved by acid washing and showed no change in membrane-bound radioactivity or rate of internalization, but a significant increase in intracellular radioactivity was noted. Gel-filtration chromatography revealed that this was due to an accumulation of chromatographically intact insulin. Accordingly, we conclude that bacitracin inhibits insulin degradation in cultured kidney cells by perturbing the intracellular processing of insulin, not by altering the binding or internalization of the hormone or by inhibiting the release of small degradation products. Because of the multiple actions of this agent, the exact site in these kidney cells at which intracellular degradation is inhibited remains to be established. However, in contrast to studies with lysosomes isolated from cells of other tissues, this study showed that when lysosomes isolated from rat renal cortex were exposed to bacitracin, insulin degradation was inhibited markedly (81%).

Animals↗
PubMed1986

Immunocytochemical localization of a developmentally regulated, isoproterenol-inducible protein (LM protein) in rat submandibular gland.

Administration of the beta-adrenergic drug isoproterenol (IPR) produces hyperplastic and hypertrophic enlargements of the submandibular gland of the rat and induces the synthesis of specific proteins in this organ. One of these proteins, the LM (large mobile) protein, was demonstrated immunocytochemically in the submandibular glands of developing untreated and IPR-treated rats. Immunoreactive LM protein was absent in the glands of 20-day-old fetuses and 1- and 2-day-old rats. It was localized in the proacinar and immature acinar cells in the glands of 6- to 21-day-old animals, but it was undetectable at 28 days of age. In the glands of adult rats, secretory granules of the granular convoluted tubule cells showed immunostaining for the LM protein which was also present in trace amounts in the acinar cells. Daily administration of IPR for 5 days to newborn or 8- or 15-day-old rats caused an apparent acceleration of proacinar/acinar cell differentiation, and consequently it increased the frequency of cells immunostained for the LM protein as well as the amount of immunoreactive material in these cells. Thus, the expression of LM protein in the submandibular gland is developmentally regulated, and it is restricted to the stage of differentiation of proacinar cells from terminal tubule cells. IPR is capable of inducing this protein in fully differentiated acinar cells in 3-week-old or older animals.

Animals↗
PubMed1986

Induction of a specific (LM) protein in the submandibular gland of the rat by repeated amputation of the lower incisor teeth.

Chronic administration of the beta-adrenergic agonist isoproterenol (IPR) leads to marked hyperplastic/hypertrophic enlargements of the parotid and submandibular glands in rats and mice with concomitant changes in the composition of both the glands and the saliva. Conspicuous among the alterations of the submandibular saliva is the appearance of a 13,000 Mr protein, termed LM (large mobile) protein. Repeated amputation of the lower incisor teeth also causes enlargements of the major salivary glands in rats. In this study, we have compared the enlargements of submandibular glands of rats produced by IPR administration or teeth amputation with respect to the relative levels of the LM protein in gland extracts and saliva. Administration of IPR-HCl (40 mg/kg) twice daily for 5 days or amputation of the lower incisor teeth 3 times a week for 3 weeks resulted in a 2.2-fold increase in the weight of the submandibular gland. Amputation for one week led to a 1.4-fold increase in gland weight. Double immunodiffusion in agar antibodies against the purified LM protein gave a single precipitin line with gland extracts and saliva of IPR-treated and teeth-amputated rats, indicating immunological identity of the reacting antigens. No precipitin lines were seen with gland extracts or saliva of untreated rats. Immunoblots of pooled saliva obtained from IPR-treated or teeth-amputated rats revealed a single protein band of the same electrophoretic mobility in SDS-polyacrylamide gels when stained using anti-LM antibodies. The relative concentrations of LM protein in gland extracts and saliva were measured by a solid-phase enzyme-linked immunoabsorption assay using antibodies against the purified LM protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Induction of the synthesis of a specific protein in rat submandibular gland by isoproterenol.

Prolonged treatment of rats with the beta-adrenergic agonist, isoproterenol (IPR), produces hypertrophy and hyperplasia of the parotid and submandibular glands. The drug induces the synthesis of several secretory proteins that are absent or occur at very low concentrations in the gland or saliva of the untreated rat. We have measured the relative concentrations of one of these proteins (termed "large mobile" (LM) protein, Menaker et al. (1974) Lab. Invest. 30, 341-349) by using a solid phase enzyme-linked immunoabsorption assay. LM protein was not measurable in gland extracts of 20-day-old fetuses or 2-day-old rats. Its concentration was very low in the glands of 6- and 13-day-old and adult rats. Administration of IPR for 4 to 7 days to adult or 6-day old rats increased the levels of the LM protein by 20 to 25-fold. The LM protein was localized immunocytochemically primarily in the acinar cells in the glands of control and IPR-treated adult rats. In vitro translation studies using a mRNA-dependent reticulocyte lysate system and labeling with [35S]methionine showed: little synthesis of pro-LM from poly(A+) RNA from glands of adult rats and none from 13-day-old animals, and that, in comparison, poly(A+) RNA from glands of adult or 13-day-old IPR-treated rats directed the synthesis of a much greater concentration of pro-LM. The in vitro precursor of the LM protein migrated electrophoretically as a single band in anti-LM immunoprecipitates, and had a molecular weight of 14,000. The LM protein, which appears to be a single, unique polypeptide induced by IPR in the submandibular glands of developing and adult rats, will be useful in studies examining the effects of catecholamine beta-agonists on gene expression in an exocrine cell.

Animals↗
PubMed1985

The streaming submandibular gland.

Twenty female young adult rats were injected with tritiated thymidine (3HTdR). The animals were then killed in groups of five, at the following times: 1 hour, and 4, 16, and 23 days. Autoradiograms of sections through the submandibular gland were prepared, and the location of labelled cells in relationship to tubuli and acini was recorded. The different tubular and acinar cross sections could be distinguished by their cell number. Narrow tubuli had fewer nuclei than the wider ones. The nuclear number of a cross section was defined as its class and the location of a labelled epithelial cell was expressed in relationship to the class where it was found. The location of a labelled stromal cell was determined by the class of its neighboring tubular or acinar cross sections. The mean cell numbers of intercalated, granular, and striated duct cross sections were, respectively, 4.7, 10.5, and 10.2, while the average cell content of acini was 4.7 cells. One hour after labelling most labelled tubular epithelial and stromal cells were found in tubular cross sections (or low tubular classes), while in the acini, labelled epithelial and stromal cells were found mainly in wider cross sections (or higher acinar classes). Within the next 23 days labelled tubular cells and stroma proceeded into higher classes, while labelled acinar epithelium and stroma cells were displaced into narrower cross sections (or lower classes). The displaced tubular epithelium and stroma covered daily 0.26 classes. At this velocity the cell will reach the highest tubular class in 62 days and the estimated maximal tubular cell life span is 62 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Compensatory proliferative response of the rat submandibular salivary gland to unilateral extirpation.

The present experiment was conducted in order to identify the progenitor compartment of the submandibular salivary gland (SSG) and to explore the proliferative activity of this gland in response to unilateral extirpation. Left submandibular and retrolingual glands were extirpated in 30 rats (B.W. 200 +/- 12 g). The rats were killed 0, 1, 2, 3, 5 and 7 days after surgery. Five intact rats served as controls. The animals were given intraperitoneal injections of 3HTdR (0.5 microCi/grB. W) 1 h before they were killed. The contralateral SSG's were subjected to routine histological procedures and embedded in glycol methacrylate. Selected sections (2 micron thickness) were processed for autoradiography. In each gland, labelled and unlabelled nuclei were counted in 50 random microscopic fields and sorted according to their parenchymal histomorphological features and "nuclear class" (number of nuclei/cross section/feature). In the control glands the total labelling index (LI) was 0.18%; during acute compensatory stimulation, however, the total LI reached a maximum of 0.86% on day 3 after surgery. suggesting that the SSG, which normally undergoes a slow turnover, is capable of elevated proliferation in response to a stimulus. In both normal and stimulated glands, the LI was higher in the intercalated ducts (1.1%-5.85%) than in the granular ducts (0.17%-0.93%) and acini (0.05%-0.36%). This consistency of LI ratio between the various histomorphological features in the normal and experimental glands indicates that the glandular progenitor compartment is located in the intercalated ducts, which supply cells to both the ductal system and acini.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Building on the past--challenging the future: will genomics provide the solution to hypertension?

Hypertension is one of the major common polygenic and multifactorial diseases, and continues to constitute a major cause of morbidity and mortality worldwide. The pathophysiological mechanisms underlying hypertension have not been elucidated, which significantly limits our ability to treat and prevent hypertension. A frequently asked question is whether genomics will provide the solution to the many remaining unanswered questions as to the causes of hypertension. Genomics, an art by its own virtue, holds its promises, yet the questions we are asking of genomics must be well defined for genomics to meet at least some of our expectations. The hopes and promises of genomics are high and one of the major hopes is that genomics will improve our understanding of the pathophysiology of hypertension and enable us to classify hypertensive syndromes more succinctly and enhance our ability to provide patients with specific anti-hypertensive therapy as well as predict outcome. There is still much to be done but perseverance in the task and a firm belief in genomics and in the immense potential of the human genome is essential.

Genetic Predisposition to Disease↗