PubMed Health⌕ Search

Biomedical subjects

J D Sheridan

Publications and source records attributed to J D Sheridan.

At least 19 recordsLinked to original sources
PubMed1988

Altered junctional permeability between cells transformed by v-ras, v-mos, or v-src.

Junctional permeability in normal and transformed NRK cells was quantitatively assessed by microinjecting fluorescent dye into one cell of a pair, digitizing the changes of fluorescence intensity using video analysis techniques, and applying the digital values to a solution of Fick's diffusion equation. We show that this approach reliably estimates the junctional permeance of a cell pair. Cells that are temperature sensitive for transformation were shown to also be temperature sensitive vis-ă-vis junctional permeance. Thus permeance values were reduced approximately 80-90% on transformation by either a mutant Rous sarcoma virus or a mutant Moloney murine sarcoma virus. Cells transformed by wild-type Kirsten sarcoma virus were also shown to possess levels of junctional permeance significantly lower than nontransformed controls. The transformed junctional phenotype could be observed as early as 15 min after shifting to transformation-permissive conditions. Our results suggest that the oncogenes src, ras, and mos exert their effects on NRK cell junctions via converging pathways, of which one may be phosphorylation of junctional proteins.

Animals↗
PubMed1988

Increased dye coupling in pancreatic islets from rats in late-term pregnancy.

Our previous studies have suggested that elevated lactogen, increased glucose-stimulated insulin secretion, and increased beta-cell coupling are associated. To determine whether this association occurs under conditions of physiologically increased lactogen, we have studied the extent of dye coupling in rat islets during the later stage of pregnancy. These animals have high plasma lactogen levels in the form of placental lactogen, increased plasma insulin, and decreased plasma glucose. The fluorescent tracer, Lucifer yellow CH, was microinjected into central cells of islets from both pregnant and virgin rats, and the extent of transfer was quantitated by determining the projected area of dye spread. Two area measurements were made for each injection, one around the entire discernible fluorescent region ("outer") and another around the distinct brighter region of cells surrounding the injected cell ("inner"). Pregnancy increased dye transfer, as determined by both measurements. The outer area of dye transfer was 9047 +/- 775 microns2 for the islets from pregnant rats and 4699 +/- 391 microns2 for the islets from virgin rats (P less than .001). Similarly, pregnancy increased the inner area of dye transfer, 1447 +/- 161 microns2 for the islets from pregnant rats and 795 +/- 80 microns2 for the islets from virgin rats (P less than .001). These results support the hypothesis that elevated lactogen, increased glucose-stimulated insulin secretion, and increased beta-cell dye coupling are associated under physiological conditions. The study indicates that enhanced beta-cell coupling is part of the structural and functional adaptation that the islets undergo during a subject's pregnancy and demonstrates that the extent of beta-cell coupling is regulated by a physiological condition.

Animals↗
PubMed1988

The aortic intima in organ culture. Response to culture conditions and partial endothelial denudation.

A culture technique for whole blood vessel wall that preserves an intact and regenerative endothelium has been developed. The retention of an intact endothelium allows careful observation of the tissue as it responds to culture conditions, responses that include a change of replication timing and the induction of particulate endocytosis. Complete and normal regeneration of the endothelium in explants has made possible evaluation of the differences between the regeneration of endothelial cell monolayers and regeneration of intima in vivo. From these comparisons it appears that the shorter induction time of endothelial migration and proliferation and the more rapid migration in endothelial cell monolayers are due to effects of the culture medium or plastic culture surface, while the higher than normal cell density found in the intima after wound recovery in vivo probably is due to the dynamics of the blood vessel itself. The ability to control cell-cell interactions on the cultured tissue has made possible the investigation of gap junction-mediated metabolic interactions in regenerating intima. Results indicate that the disruptive effects of intimal regeneration do not depend on or produce an obvious change in junction-mediated nucleotide transfer between endothelial cells.

Animals↗
PubMed1987

Permeance of Novikoff hepatoma gap junctions: quantitative video analysis of dye transfer.

Fluorescent dyes are commonly used to study permeable (gap) junctions, but only rarely have quantitative values for junctional dye permeability been determined. In the present study, junctional permeance (PA, i.e., the product of the junctional permeability coefficient, P, times the junctional area, A) to Lucifer Yellow CH (LY) has been obtained for pairs of Novikoff hepatoma cells. Dye was microinjected into one cell and the subsequent transfer monitored by a SIT camera and recorded on video tape. The intensities of fluorescence in the injected and "recipient" cell were measured using a Digisector (Microworks) digitizing board and an Apple II Plus computer to analyze the video records. These changes in intensity, along with an estimate of volume of the spherical cells, were used to calculate the junctional permeance (PA) of cell pairs according to Fick's diffusion equation. Junctional permeances show considerable variation ranging from 0.08 X 10(-11) to 27.0 X 10(-11) cm3/sec. Using the mean PA and a previous estimate of the mean number of junctional channels per interface in the Novikoff cultures, a value for diffusion coefficient of LY through gap junctions is calculated to be about 1.4 X 10(-6) cm2/sec. There is a general proportionality between mean PA and cell volume for hepatoma cell pairs of a certain size range. Such a relationship between cell volume and junctional capacity suggests one source of variation of PA. Other possible sources, e.g., related to position in the cell cycle, are discussed.

Animals↗
PubMed1987

Decreased glucose stimulation threshold, enhanced insulin secretion, and increased beta cell coupling in islets of prolactin-treated rats.

In order to determine the effect of lactogen on insulin secretion and junctional coupling among islet beta cells, ovine prolactin (oPRL) was infused by Alzet minipumps into female rats for 4 days. This treatment produced an oPRL level of 994 +/- 122 ng/ml which, combined with residual rat PRL (rPRL) (12 +/- 2 ng/ml), represented nearly a 20-fold increase from control (rPRL: 53 +/- 17 ng/ml). In addition, plasma insulin was increased nearly 50% (control: 21.9 +/- 3 microU/ml; experimental: 30.3 +/- 3 microU/ml; p less than 0.05). When pancreata from lactogen-treated and control animals were perfused with linear 30-200 mg/dl glucose gradients, the apparent glucose threshold for insulin secretion in the experimental group was nearly 33% lower than that of the controls (i.e., 70 +/- 4.6 mg/dl vs. 104 +/- 7.5 mg/dl; p less than 0.01). The oPRL treatment also increased dye coupling among beta cells. Central cells in islets isolated from lactogen-treated and control animals were injected with Lucifer Yellow CH to estimate the extent of gap junctional coupling. There was nearly a twofold increase in the projected area of dye transfer per injection in the experimental vs. the controls: 4,607 +/- 575 micron 2 vs. 2,302 +/- 474 micron 2, respectively; p less than 0.02. The effects of oPRL decreased the apparent glucose threshold for insulin release, increased the above-threshold glucose-induced insulin secretion, and increased the extent of dye coupling among beta cells. These changes in insulin secretion and dye coupling closely resemble those observed in islets from pregnant rats.

Animals↗
PubMed1987

Prolactin (in vitro) decreases the glucose stimulation threshold, enhances insulin secretion, and increases dye coupling among islet B cells.

The purpose of this study was to determine the in vitro effect of ovine PRL (oPRL) on the dynamics of insulin secretion and dye coupling among islet B cells. The effect of oPRL (2 micrograms/ml) on insulin secretion was time dependent and reached a maximum on day 4 when there was a 2.4-fold increase in insulin secretion from cultured neonatal rat islets (n = 6, P less than 0.001). When islets cultured in the presence of oPRL for 4 days were perifused, 300 mg/dl glucose stimulation resulted in insulin release of 131 +/- 20 microU/ml.100 micrograms islet tissue as compared to control islets 94 +/- 20 microU/ml.100 micrograms islet tissue (n = 7, P less than 0.02). Stimulation of the islets with a linear 30-250 mg/dl glucose gradient resulted in a threshold for glucose-stimulated insulin secretion of 73 +/- 6 mg/dl glucose for the oPRL treated islets (n = 7) as compared to a threshold of 123 +/- 6 mg/dl glucose for control islets (n = 7, P less than 0.001). Mean islet volume was unchanged after 4 days of oPRL treatment but was 34% greater after 8 days (n = 6, P less than 0.001). Dye coupling among central islet B cells was also increased after in vitro treatment with oPRL for 4 days. The mean projected area of dye spread was 2-fold greater in the oPRL treated islets (n = 33) in comparison to the control islets (n = 33, P less than 0.05). These results indicate that in vitro lactogen treatment, in the form of oPRL, alters insulin secretory behavior and B cell junctional communication and supports our hypothesis that lactogen, insulin secretion, and junctional communication among B cells are related.

Animals↗
PubMed1987

Prolactin enhances cell-to-cell communication among beta-cells in pancreatic islets.

To determine the role of prolactin in increasing junctional communication among islet beta-cells, we studied dye coupling in pancreatic islets exposed to elevated levels of prolactin in vivo and in vitro. Islets were isolated from rats immediately after lactation or from rats bearing mammosomatotropic tumors (MtTW15), conditions involving high levels of prolactin (either 5-fold or 1000-fold control levels, respectively). When beta-cells were microinjected with the gap junction permeant dye Lucifer yellow CH, the mean number of dye-coupled cells per injection was approximately 10-fold greater than in islets from virgin control rats. As a more direct test of the effects of prolactin on beta-cell coupling, islets isolated from virgin rats were treated for 90 min with 500 ng/ml rat prolactin in the presence of low glucose (2.8 mM) and were microinjected with dye. The mean number of dye-coupled cells per injection increased by 6.7-fold over controls with low glucose, demonstrating a direct effect of prolactin on beta-cell coupling. In vitro treatment with high glucose (16.7 mM) resulted in a 2.7-fold increase in dye-coupled cells per injection. We discuss the possible relationship between the effects of glucose and of prolactin on coupling.

Animals↗
PubMed1986

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.

Animals↗
PubMed1985

Junctional transfer in cultured vascular endothelium: II. Dye and nucleotide transfer.

Vascular endothelial cultures, derived from large vessels, retain many of the characteristics of their in vivo counterparts. However, the observed reduction in size and complexity of intercellular gap and tight junctions in these cultured cells (Larson, D.M., and Sheridan, J.D., 1982, J. Cell Biol. 92:183) suggests that important functions, thought to be mediated by these structures, may be altered in vitro. In our continuing studies on intercellular communication in vessel wall cells, we have quantitated the extent of junctional transfer of small molecular tracers (the fluorescent dye Lucifer Yellow CH and tritiated uridine nucleotides) in confluent cultures of calf aortic (BAEC) and umbilical vein (BVEC) endothelium. Both BAEC and BVEC show extensive (and quantitatively equivalent) dye and nucleotide transfer. As an analogue of intimal endothelium, we have also tested dye transfer in freshly isolated sheets of endothelium. Transfer in BAEC and BVEC sheets was more rapid, extensive and homogeneous than in the cultured cells, implying a reduction in molecular coupling as endothelium adapts to culture conditions. In addition, we have documented heterocellular nucleotide transfer between cultured endothelium and vascular smooth muscle cells, of particular interest considering the prevalence of "myo-endothelial" junctions in vivo. These data yield further information on junctional transfer in cultured vascular endothelium and have broad implications for the functional integration of the vessel wall in the physiology and pathophysiology of the vasculature.

Animals↗
PubMed1983

Junctional transfer in cultured vascular endothelium: I. Electrical coupling.

Vascular endothelial cultures are composed of flat, polygonal monolayer cells which retain many of the growth, metabolic and physiological characteristics of the intimal endothelium. However, intercellular gap and tight junctions, which are thought to perform important roles in normal intimal physiology, are reduced in complexity and extent in culture. We have used electrophysiological techniques to test confluent (3- to 5-day) primary cultures of calf aortic (BAEC) and umbilical cord vein (BVEC) endothelium for junctional transfer of small ions. Both cell types are extensively electrically coupled. The passive electrical properties of the cultured cells were calculated from the decrease in induced membrane potential deflections with distance from an intracellular, hyperpolarizing electrode. Data analyses were based on a thin-sheet model for current flow (Bessel function). The generalized space constants (lambda) were 208.6 microns (BAEC) and 288.9 microns (BVEC). The nonjunctional (6.14 and 8.72 X 10(8) omega) and junctional (3.67 and 3.60 X 10(6) omega) resistances were similar for the BAEC and BVEC, respectively. We detected no statistically significant differences in the resistance estimates for the two cell types. In vivo ultrastructural studies have suggested that aortic endothelium has more extensive gap junctions than venous endothelium. We have found that these ultrastructural differences are reduced in culture. The lack of any significant difference in electrical coupling capability suggests that cultured BAEC and BVEC have functionally similar junctional characteristics.

Animals↗
PubMed1983

In vivo modulation of gap junctions and dye coupling between B-cells of the intact pancreatic islet.

The extent of gap junctions and dye coupling between insulin-producing B-cells was analyzed on islets of Langerhans isolated from adult rats treated for one day with glibenclamide, an insulin secretagogue, or diazoxide, a blocker of insulin release, or a combination of the two drugs. Glibenclamide treatment was associated with a marked depletion of the islet insulin content, an effect which was blocked by pretreatment of the rats with diazoxide. Diazoxide alone caused a marked increase in the plasma glucose level, and a decrease in the level of circulating insulin and in the hormone content of the B-cells. Quantitative analysis showed that (1) under control conditions, B-cells are connected by minute gap junctions (as evaluated on freeze-fracture replicas) and show a nonuniform and apparently restricted dye coupling (as determined by microinjection of the low-molecular-weight fluorescent probe Lucifer Yellow CH); (2) each of the three treatments tested significantly increased the relative and absolute gap junction area of the B-cells and the number of detectable, dye coupled B-cells per microinjection. After treatment with glibenclamide alone or with diazoxide plus glibenclamide, a 1.5-1.8-fold increase in gap junction area and a 2.7-3.7-fold increase in the number of dye-coupled B-cells were observed. In contrast, following treatment with diazoxide alone, gap junctions and dye coupling were found increased 1.8 and 8.7 times, respectively, as compared with control values.

Animals↗
PubMed1982

Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures.

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.

Animals↗
PubMed1981

Islets of Langerhans: dye coupling among immunocytochemically distinct cell types.

Cells in isolated rat islets of Langerhans were microinjected with Lucifer Yellow CH in a medium containing 16.7 millimolar glucose. Dye was rapidly transferred from the injected cell to neighboring islet cells without specificity with regard to the immunocytochemical identity of either the donor or the recipient cells. The transfer of dye between the islet cells (types A, B, and D) demonstrates homologous and heterologous cell coupling in a system where the normal proportions and relationships of the cell types are maintained.

Animals↗
PubMed1981

Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus.

The transformed or normal phenotype of cultured normal rat kidney cells infected with a temperature-sensitive mutant of avian sarcoma virus is conditional on the temperature at which the cells are grown. Using dye injection techniques, we show that junction-mediated dye transfer is also temperature-sensitive. The extent and rate of transfer between infected cells grown at the transformation-permissive temperature (35 degrees C) is significantly reduced when compared to infected cells grown at the nonpermissive temperature (40.5 degrees C) or uninfected cells grown at either temperature. Infected cells subjected to reciprocal temperature shifts express rapid and reversible alterations of dye transfer capacities, with responses evident by 15 min and completed by 60 min for temperature shifts in either direction. These results suggest that altered junctional capacities may be fundamental to the expression of the ASV-induced, transformed phenotype.

Alpharetrovirus↗
PubMed1979

Tight junction development between cultured hepatoma cells: possible stages in assembly and enhancement with dexamethasone.

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: 1) "formation zones" (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; 2) linear strands and aggregates of 9--11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; 3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specializations may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 x 10(-6)M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.

Animals↗