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M J Rutten

Publications and source records attributed to M J Rutten.

34 records · Page 2Linked to original sources
PubMed1991

Physiological and cytotoxic effects of Ca(2+) ionophores on Caco-2 paracellular permeability: relationship of 45Ca(2+) efflux to 51 Cr release.

The human intestinal cell line, Caco-2, and the Ca2+ ionophores, A23187 and ionomycin, were used to determine the interrelationships of 45Ca(2+) efflux, transepithelial electrical resistance (Rt), and [3H]-mannitol flux to changes in 51Cr release and lactate dehydrogenase (LDH) activity. Treatment of Caco-2 monolayers with ionomycin at concentrations of between 0.25 and 2.50 mumol/l showed similar 45Ca(2+) efflux rate constants and coefficients. Analysis of the control and ionomycin-induced 45Ca(2+) efflux values showed the data to best fit a three Ca(2+) compartmental model. All changes in Caco-2 Rt and [3H]-mannitol flux were reversible with no significant increases in 51Cr release with ionomycin concentrations of less than or equal to 2.5 mumols/l. Caco-2 monolayers treated with ionomycin at concentrations of between 5.0 and 50.0 mumols/l showed rapid non-exponential 45Ca(2+) effluxes with irreversible changes in Rt, [3H]-mannitol flux, and significant increases in 51Cr release. There was no changes in media LDH activity using either ionomycin or A23187 at concentrations of up to 50 mumols/l for 60 min. The results of our study show that: (1) disruption of Ca(2+) homeostasis in Caco-2 cells will occur with the addition of Ca(2+) ionophores at concentrations of greater than 2.50 mumols/l; (2) high concentrations (greater than 2.5 mumols/l) of ionomycin will cause non-exponential 45Ca(2+) efflux rates with irreversible changes in intracellular Rt and 14C-mannitol flux, and (3) early signs of Ca(2+) ionophore-induced damage can be detected by 51Cr release from Caco-2 cells into the media and not by changes in LDH media activity.

Anti-Bacterial Agents↗
PubMed1991

Gastric fundic mucous epithelial cells isolated from omeprazole-treated guinea pigs have increased basal and hormone-stimulated growth rates.

Guinea pigs were given a daily oral dose of 400 micrograms/kg of omeprazole or the omeprazole vehicle for 6 weeks. At weekly intervals, the animal and stomach weights were recorded, the nonfasted serum gastrins measured, and the total number of gastric fundic epithelial cells were isolated and counted. Gastric mucous epithelial cells were enriched from the total gastric fundic cell population and cultured for 6 days in the absence or presence of hormones (epidermal growth factor and pentagastrin). We found that omeprazole treatment for 1-6 weeks significantly (p less than 0.05) increased the stomach weight, the nonfasted serum gastrin levels, and the total number of isolated gastric fundic and mucous epithelial cells over control animals. A significant (p less than 0.05) increase was also found in the basal and hormone-stimulated cultured growth rates of gastric mucous epithelial cells isolated from the stomachs of omeprazole-treated animals as compared with gastric mucous cells from control animals. We conclude that oral omeprazole treatment of guinea pigs will cause a specific increase in the gastric mucous cell population as well as increase the in vitro cultured gastric mucous cell basal and hormone-stimulated growth rates.

Animals↗
PubMed1991

Insulin enhances epidermal growth factor- and transforming growth factor-alpha-stimulated growth in primary cultures of guinea pig gastric mucous epithelial cells.

The effect of insulin on epidermal growth factor (EGF)- and transforming growth factor-alpha (TGF alpha)-induced cell proliferation was examined in primary cultures of gastric surface mucous epithelial cells by using changes in cell counts and DNA as indices of cell growth. With 1.0% fetal bovine serum and Dulbecco's modified Eagle medium (low-serum DMEM) there was no effect of bovine insulin (0.1-10.0 micrograms/ml) on cell growth. Higher doses of insulin (20.0 micrograms/ml) in low-serum DMEM were able to stimulate growth of these cells compared with controls. In low-serum DMEM with no insulin the lowest effective dose of EGF or TGF alpha for stimulating cell growth was 10 nM and 5 nM, respectively. In low-serum DMEM containing 2.5 micrograms/ml insulin there was a dose-dependent increase in cell growth with EGF and TFG alpha at concentrations of 0.5-10.0 nM. The addition of insulin alone at 2.5 micrograms/ml, EGF alone at 2 nM, or TGF alpha alone at 2 nM in serum-free DMEM media had no effect on gastric surface mucous cell DNA. In serum-free DMEM media with 2.5 micrograms/ml insulin, the addition of 2 nM EGF or 2 nM TGF alpha caused an increase in both cell counts and DNA of 1.80- and 3.50-fold, respectively. The present study demonstrates that insulin, at concentrations that are not mitogenic, will enhance the proliferative effects of EGF and TGF alpha on the growth of primary cultures of guinea pig gastric surface mucous epithelial cells.

Animals↗
PubMed1990

Feeding induces lipid accumulation and increased Na+ transport in in vitro Necturus antrum.

We investigated the effects of feeding on lipid accumulation and transepithelial transport using in vitro Necturus gastric antral mucosae. Antra from fed Necturi were examined for lipid accumulation using light, fluorescence, histochemical, and electron microscopy. Ussing chambers were used for measurement of potential difference (PD), transepithelial resistance (Rt), short-circuit current (Isc), and unidirectional fluxes of 22Na+ and [3H]mannitol. Light microscopy of antra from 2-day postfed animals showed many intracellular lipid granules in surface mucous epithelial cells. These granules could be distinguished from other intracellular organelles by their high affinity for osmium and the lipid fluorescent probe Nile red. Glycoprotein cytochemical staining showed these granules to be distinct from the epithelial cell mucous granules. Electron microscopy showed the lipid granules to be part of a membranous reticular network. Two-day postfed animals also had a approximately 3.5-fold increase in amiloride-sensitive Isc and PD, a decrease in Rt, and an increased luminal-to-serosal Na+ fluxes. Transepithelial [3H]mannitol fluxes were low and remained unchanged in both fasted and 2-day postfed animals. After 2 days of feeding, the PD and Isc began to decrease followed by a secondary increase in Rt. Feeding Necturi a corn oil diet did not induce the appearance of either cellular lipid or alterations in Isc but produced a transient increase in Rt. Our data show that feeding (goldfish) to Necturi causes an increase in both lipid accumulation and amiloride-sensitive Na+ transport in gastric antral cells.

Amiloride↗
PubMed1989

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum.

Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.

Alkaline Phosphatase↗
PubMed1989

Effects of parathyroid hormone on cyclic-AMP concentrations of in vitro Necturus maculosus gastric antrum.

The effects of bovine parathyroid hormone (bPTH1-84) on the stimulation of intracellular cyclic-AMP [cAMP] were investigated in an in vitro preparation of Necturus maculosus antral mucosa. When the antrum was exposed to 1, 5, 10, or 100 nM bPTH1-84, there was an approximately 2-fold nonlinear increase in tissue [cAMP] over basal values. The pretreatment of the antral mucosa with 1 mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) increased with detectability of mucosal [cAMP]. The addition of 1, 5, 10, or 100 nM bPTH1-84 to tissues pretreated with IBMX resulted in an approximately 3.5-fold linear increase in mucosal [cAMP] over basal values. The time course of the generation of mucosal cAMP to 10 nM bPTH1-84 resulted in a small but significant transient increase at 2.5 min after the addition of bPTH1-84 but no change in the medium [cAMP]. In tissues pretreated with 1 mM IBMX the response to 10 nM bPTH1-84 was a large biphasic increase of [cAMP] at 2.5 min that progressively declined to near basal values by 15 min. There was also a significant sustained increase in the [cAMP] in the bathing medium at 2.5 min of tissues pretreated with IBMX followed by 10 nM bPTH1-84. These results suggest the presence of an adenylate cyclase that can be activated by a mammalian bPTH1-84 in elevating intracellular cAMP levels in the N. maculosus antral mucosa.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Effects of cations and pH on apical membrane potential of in vitro Necturus antrum.

The surface epithelial cells of the gastric mucosa transport Na+ from lumen to serosa. The first step in this process is the entry of Na+ through an amiloride-sensitive Na+ channel at the apical membrane. The exact function(s) of this Na+ transport are unknown, but it has been suggested that it might help the stomach to withstand an acid load. The present study was undertaken to examine the effects of low luminal pH and the alkali cations Li+, K+, Rb+, and Cs+ on the amiloride-sensitive Na+ permeability of the apical membrane of Necturus antral cells by measuring the changes in apical membrane voltage (Vmc) using conventional microelectrode techniques. Isosmolar replacement of luminal NaCl (pH 7.25) with LiCl caused a depolarization of the Vmc, a decrease in transepithelial resistance (Rt), and an increase in the transepithelial potential (Vmc), whereas replacement with KCl, RbCl, or CsCl caused a hyperpolarization of the Vmc, an increase in Rt, and a decrease in the Vmc. Luminal acidification from pH 7.25 to pH 3.00 caused very similar changes with all the cation solutions tested, hyperpolarizing the Vmc, increasing Rt, and reducing the Vmc to near 0 mV. Acidification of the luminal NaCl solution from pH 7.25 to pH 2.00 caused a progressive hyperpolarization of the Vmc similar to the effects seen with luminal amiloride (10(-4) M) in pH 7.25 NaCl solutions or luminal Na+-free (N-methyl-D-glucamine) Ringer at pH 7.25. These results demonstrate that 1) of the cations tested only Li+ can substitute for Na+ in maintaining the Vmc, and 2) external H ions (low luminal pH) block cation permeability of the apical membrane.

Amiloride↗
PubMed1987

Electrical resistance and macromolecular permeability of brain endothelial monolayer cultures.

Electrophysiological measurements were made on endothelial cells initially isolated as individual clones from bovine brain microvessels, and then grown as monolayers on a permeable support of glutaraldehyde-treated collagen gel. When transendothelial cell resistance (R) of the clones was measured, there was a range of values from a low of 157.4 +/- 4.5 omega.cm2 (n = 6) to a high of 783.2 +/- 7.0 omega.cm2 (n = 34). With the high-resistance cells, there was also a small potential difference of -0.46 +/- 0.03 mV luminal-side negative (n = 34). In comparison, endothelial cells from bovine aortas and rat epididymal fat pads cultured on the collagen gels had transendothelial R values of 13.5 +/- 0.2 (n = 62) and 0.45 +/- 0.03 (n = 10) omega.cm2, respectively. Exposure of the high-resistance brain endothelial cell monolayers to a Ca2+-free medium for 10 min decreased the R to 75% of the control values. Addition of Ca2+ back to the medium caused a return of the transendothelial R to control values within 1 h. Endothelial cells were also grown to confluency on microcarrier beads for permeability measurements to Evans blue dye-bovine serum albumin. Microcarriers with no cells (control) and microcarriers with bovine and epididymal endothelial cell monolayers showed no difference in the amount of adsorbed dye. Microcarriers with brain endothelial monolayers excluded up to 80% of the dye. This mammalian brain endothelial culture system will be a useful model for studies of the electrophysiological and permeability properties of the blood-brain barrier.

Animals↗
PubMed1986

Structural and functional changes by ethanol on in vitro guinea pig gastric mucosa.

Relationships between morphological and electrophysiological changes with low concentrations of ethanol on in vitro guinea pig gastric mucosa were investigated. Tissues mounted in Ussing chambers allowed recording of transepithelial potential difference (PD), resistance (R), short-circuit current (Isc), and acid secretion (H+). At selected times the mucosae were processed for morphological analysis. With luminal 10% ethanol there was a decrease in PD, R, Isc, and H+ within 1 min, and they eventually went to low steady-state values between 10 and 40 min. At 1 min many surface epithelial cells lifted off from the basal lamina but were still anchored by thin basal cell processes. After 10 min in ethanol many surface cells had completely detached from the basal lamina but remained connected to adjacent cells by their junctions. Numerous cytoplasmic blebs formed on both apical and basal cell surfaces. Concurrently, there was a significant increase in microvillus length. After 40 min most of the surface cells were detached from the basal lamina as sheets forming epithelial blisters. Upon ethanol washout there was epithelial cell reattachment to the basal lamina and a return of the PD, R, and Isc to control values within 40 min. Incubation of the luminal surface with 10% ethanol for 5 h resulted in a gradual rise of the PD, R, Isc, and H+ to control values by 4 h with a coincident return of the normal mucosal morphology. These studies indicate that ethanol has reversible and possibly adaptable effects on the in vitro guinea pig gastric mucosa and that the morphological changes are closely correlated with the decline and recovery of the electrical and secretory activity of the tissue.

Animals↗
PubMed1985

A rapid method for culturing guinea pig gastric mucous cell monolayers.

A method has been developed for growing confluent primary cultured monolayers of guinea pig gastric mucous cells suitable for in vitro electrophysiological, transport, and pharmacological studies. Isolated mucous cells were enriched on a one-step Percoll density gradient and plated on fibronectin-coated plastic dishes or in small cups with holes containing glutaraldehyde-fixed Vitrogen gels. These cups were designed to fit in Ussing chambers. Mucous cells attached, proliferated, and formed confluent monolayers in 3 d. The low cuboidal cells contained periodic acid Schiff-positive mucous granules that were negative by Bowie and indirect immunofluorescent staining for pepsinogen. Electron microscopy revealed polarized mucous cells with microvilli, mucous granules, microfilaments, small mitochondria, some vacuoles, and junctional complexes that excluded wheat germ agglutinin-peroxidase. No basal lamina was present. Monolayers could be maintained for over 2 wk but subcultures were not made. The cultures were virtually free of fibroblasts. Epithelial sheets produced by this simple and rapid method can be used for electrophysiological, ion transport, and pharmacological studies.

Animals↗
PubMed1984

Rapid repair of injured gastric mucosa.

The morphology of the rat gastric mucosa superficially damaged by a 30-45 sec exposure to 100% ethanol caused over 99% of the luminal surface to be necrotic without extensive hemorrhage or hyperemia. However, this damaged area restituted or rapidly healed and the process was described. These findings were compared with in vitro gastric mucosae of bullfrogs and guinea pigs which were damaged with 1 or 1.25 M NaCl and allowed to restitute. The rat gastric mucosa showed evidence of cell migration within 5 min after ethanol damage and after 15 min as much as 50% of the denuded basement membrane was restituted. After 30 min about 75% of the mucosa was covered with cells and by 1 h there were only minor epithelial discontinuities. The in vitro frog mucosal restitution after hypertonic injury was slower and required 1-2 h to show appreciable cell migration and 4-6 h for completion of the repair process. Using chambered guinea pig gastric mucosa damaged with hypertonic NaCl restituted the necrotic surface almost as rapidly as the intact rat stomach necrotized with absolute ethanol. Since prostaglandin treatment did not prevent ethanol or hypertonic salt injury or affect the restitution process it was proposed that the term cytoprotection as it is generally used is not appropriate.

Animals↗
PubMed1983

Morphology and electrophysiology of guinea pig gastric mucosal repair in vitro.

Guinea pig gastric mucosae stripped of their outer muscle layers were studied in Ussing chambers for up to 14 h. Ten minutes after the mucosae were mounted in the chamber, the electrical parameters were low but continued to rise over 90 min until steady-state potential difference (PD), resistance (R), and short-circuit current (Isc) were recorded. Morphological analysis during the first 10 min of the tissue in the chamber revealed gaps in the epithelium due to damaged cells. However, tissues examined after 20 min in the chamber showed little evidence of epithelial discontinuity. Thereafter, the initial rise in the electrical parameters was noted. After steady-state attainment, the lumen was exposed to 1.25 M NaCl for 5 min and then changed back to 150 mM NaCl. Ten minutes after washout and return to control solutions, the PD, R, and Isc had fallen to low values. At 30 min after washout of the NaCl, the PD, R, and Isc began to increase and after 2 h were back to control values. Morphological analysis of mucosae fixed up to 10 min after exposure to 1.25 M NaCl showed extensive damage and exfoliation of surface cells. However, by 30 min the epithelium was restored and had very few discontinuities, which was then followed by the return of the electrical parameters. The conclusions from these studies are 1) guinea pig gastric mucosae exposed to hypertonic NaCl on the luminal side will primarily result in surface epithelial cell destruction with an immediate drop in the transepithelial electrical values; 2) after return to isotonic saline the damaged mucosa can repair itself within minutes, which then allows the reestablishment of the transepithelial electrical parameters by 2 h; and 3) the good viability and reproducibility of this preparation present a suitable mammalian model system for the study of factors of mucosal repair.

Animals↗
PubMed1982

Fetal rabbit gastric epithelial cells cultured on floating collagen gels.

Cells were isolated from approximately 30 d fetal rabbit stomachs and cultured on floating collagen gels. Electron microscopy showed monolayers in which only one cell type persisted. These columnar cells were joined at apical borders by tight junctions and contained an extensive endoplasmic reticular network with an occasional intracellular canaliculus. They also occasionally contained what appeared to be secretory granules (mucus?), and therefore had some characteristics of all the cell types of the intact fetal stomachs, which showed oxyntic, mucous, and undifferentiated cells. In Ussing chambers with Ringer's solution on both sides, cultures developed transepithelial potential (potential difference [PD], mV, mucosa ground) = 13, resistance (resistance [R], omega - cm2) - 285, and short-circuit current (Isc, microA/cm3) = 45 (n = 7), clearly indicating that cellular polarity and junctional integrity were maintained. These transport parameters were somewhat different for intact fetal stomachs (PD = 20, R = 70, and Isc = 220 [n = 4]), which may be due to extensive folding of intact fetal stomachs or the presence of only one cell type in culture, or both. Although gastric stimulants histamine, dibutyryl cycle AMP (dbcAMP), and isobutylmethylxanthine (IMX) (a phosphodiesterase inhibitor) did not elicit H+ secretion or electrophysiological changes in monolayers or intact stomachs, 10-4 M apical amiloride caused a decrease in Isc in cultured monolayers(27%) and intact stomachs (50%). Thus, Na+ transport seems to be a significant fraction of ion transport in both preparations. This culture system may allow the study of oxyntic cell differentiation and the development of H+, Na+, and Cl- transport in the gastric musoca.

Animals↗
PubMed1982

Histamine, cAMP, and activation of piglet gastric mucosa.

The involvement of cAMP as a second messenger for histamine-induced H+ secretion was studied in a physiologically active, in vitro preparation of piglet gastric mucosa. During the first 5--10 min of stimulation with either histamine or the cAMP phosphodiesterase inhibitor 3-isobutyl-1,4-methylxanthine (IBMX), increases (greater than or equal to 5-fold) in tissue cAMP content [(c-AMP]) were well correlated with the characteristic decrease in transepithelial resistance (R); these changes precede H+ secretion by several minutes. Control experiments indicate that, during these treatments, tissue [cAMP] is dominated by the [cAMP] of oxyntic cells alone; change in R and H+ are also related to activity of these cells alone. At the steady state (45 min), histamine and IBMX caused equivalent increases in H+ and decreases in R, but [cAMP] was markedly different in the two cases. With IBMX [cAMP] was elevated at least fivefold, whereas with histamine [cAMP] was less than or equal to 50% above resting levels. The tissue is also stimulated by exogenous additions of dibutyryl cAMP. A histamine-sensitive adenylate cyclase was present in isolated, purified oxyntic cells. The histamine sensitivity of the cyclase was very similar to that which the intact tissue exhibits for histamine-induced changes in H+ and R. The cyclase activity was blocked by cimetidine but not by promethazine. We conclude that during stimulation histamine activates a histamine (H2)-sensitive adenylate cyclase of oxyntic cells, and there is a rapid increase in cellular [cAMP] that is involved in activation of H+ transport and other associated changes of oxyntic cells. An active phosphodiesterase is responsible for reducing [cAMP] to a level much below the "peak" value. Other cellular factors (e.g. protein kinases and Ca2+-calmodulin) must also be involved in the maintenance of the stimulated state of oxyntic cells.

1-Methyl-3-isobutylxanthine↗