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M J Welsh

Publications and source records attributed to M J Welsh.

At least 289 records · Page 16Linked to original sources
PubMed1986

Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium.

Adenosine is a local regulator of a variety of physiological functions in many tissues and has been observed to stimulate secretion in several Cl-secreting epithelia. In canine tracheal epithelium we found that adenosine stimulates Cl secretion from both the mucosal and submucosal surfaces. Addition of adenosine, or its analogue 2-chloroadenosine, to the mucosal surface potently stimulated Cl secretion with no effect on the rate of Na absorption. Stimulation resulted from an interaction of adenosine with adenosine receptors, because it was blocked by the adenosine receptor blocker, 8-phenyltheophylline. The adenosine receptor was a stimulatory receptor as judged by the rank-order potency of adenosine and its analogues and by the increase in cellular adenosine 3',5'-cyclic monophosphate levels produced by 2-chloroadenosine. Adenosine also stimulated Cl secretion when it was added to the submucosal surface, although the maximal increase in secretion was less and it was much less potent. Part, but not all, of the lower potency of submucosal adenosine resulted from submucosal uptake and metabolism of the drug. The observation that mucosal 8-phenyltheophylline blocked the effect of submucosal 2-chloroadenosine, whereas submucosal 8-phenyltheophylline did not prevent a response to mucosal or submucosal 2-chloroadenosine, suggests that adenosine receptors are located on the mucosal surface. Thus submucosal adenosine may stimulate secretion by crossing the epithelium and interacting with receptors located on the mucosal surface. Because adenosine can be released from mast cells located in the airway lumen in response to inhaled material, and because adenosine stimulated secretion from the mucosal surface, it may be in a unique position to control the epithelium on a regional level.

2-Chloroadenosine↗
PubMed1986

Two-dimensional gel analysis of Sertoli cell protein phosphorylation: effect of short term exposure to follicle-stimulating hormone.

The response of cultured Sertoli cells to short term FSH stimulation was studied to elucidate early events involved in the hormone response of this cell type. The phosphorylation of proteins by [32P]orthophosphate-labeled cells was examined using two-dimensional polyacrylamide gel electrophoresis and autoradiography. FSH stimulation resulted in a variety of changes in the phosphoprotein labeling pattern. Within 5 min, unique phosphoproteins appeared in autoradiograms of treated cells. Increased labeling of vimentin was also noted. After 25 min of FSH stimulation, increases and decreases in apparent labeling intensity were evident in additional phosphoproteins that were constitutively labeled in control cultures. Changes in protein patterns in stained gels were also noted after acute hormone treatment. These observations demonstrate that Sertoli cells respond to hormonal stimulation in a detectable manner within 5 min. Cytoskeletal involvement in initial phases of hormone response is indicated. Mechanisms such as increased protein kinase activity, changes in protein kinase substrate affinity, increased protein turnover, or phosphatase activation may all contribute to the early events involved in Sertoli cell hormone response.

Animals↗
PubMed1986

Mechanisms of airway epithelial ion transport.

Airway epithelia contain the transport mechanisms required to effect either chloride secretion or sodium absorption. Thus, depending on the local neurohumoral environment, they have the ability to either secrete or absorb fluid. Therefore, they are in a unique position to precisely regulate the quantity and composition of the respiratory tract fluid. The importance of the respiratory tract fluid as a pulmonary defense mechanism is clear. This point is emphasized by the substantial morbidity and mortality that result from abnormalities of airway epithelial function in controlling the quantity and composition of fluid in diseases such as cystic fibrosis and asthma, and in conditions such as cigarette smoking. Therefore, it is essential to understand the function of the epithelium, its regulation, and the mechanism of ion transport at the level of the individual transport processes on the two cell membranes. As our understanding of the mechanism of ion transport increases, we may be able to define a more rational therapy of disease.

Animals↗
PubMed1986

Cell interactions in the rat seminiferous epithelium with special reference to the cellular distribution of calmodulin.

Spermatogenesis is dependent on stimulation by pituitary gonadotropins, FSH and LH. Targets for these hormones are Sertoli and Leydig cells, respectively. The effect of LH on spermatogenesis is mediated by testosterone. In addition to hormones, interactions between neighbouring cells seem to regulate spermatogenesis. This is reflected by cyclic secretion of several proteins by the seminiferous epithelium, of which plasminogen activator is a good example. While it is controlled by FSH a factor in preleptotene spermatocytes may also influence its cyclic secretion pattern. Both testosterone and FSH have a cyclic action in the seminiferous epithelium. The androgens seem to predominate in stages where spermiation, onset of meiosis and the highest rate of RNA transcription occur (VII-XI). FSH is most active in stages that contain meiotic divisions and early spermiogenesis (XIII-V), greatly stimulating the production of cyclic AMP. To investigate further the "second messengers" of FSH action in the seminiferous epithelium, the cellular distribution of calmodulin was analyzed using an indirect immunocytochemical method. In addition to their clear cyclic distribution in primary spermatocytes and in spermatids, Sertoli cells also showed a bright calmodulin immunofluorescence that was apparently cyclic. These observations suggest a local calmodulin and calcium regulation of spermatogenesis.

Androgens↗
PubMed1985

Ion transport by primary cultures of canine tracheal epithelium: methodology, morphology, and electrophysiology.

Canine tracheal epithelial cells were isolated by enzymatic and mechanical dispersion and cultured on permeable supports. The cells formed confluent monolayers and retained most of the morphologic characteristics of the intact epithelium, including apical microvilli, apical tight junctions, and a moderately interdigitated lateral intercellular space. The cells also retained the functional properties of the epithelium. The monolayer responded to addition of isoproterenol with the characteristic changes in cellular electrical properties expected for stimulation of C1 secretion: isoproterenol increased transepithelial voltage, depolarized apical membrane voltage, and decreased both transepithelial resistance and the ratio of apical-to-basolateral membrane resistance. Examination of the cellular response to ion substitutions and inhibitors of C1 secretion indicate that the cultured monolayers retain the same cellular mechanisms of ion transport as the intact epithelium. Thus, primary cultures of tracheal epithelium may provide a useful preparation for future studies of the mechanism and regulation of C1 secretion by airway epithelia.

Animals↗
PubMed1985

Basolateral membrane potassium conductance is independent of sodium pump activity and membrane voltage in canine tracheal epithelium.

When secretagogues stimulate Cl secretion in canine tracheal epithelium, apical membrane Cl conductance (GCla) increases, and then basolateral membrane K conductance (GKb) increases. Conversely, inhibition of GCla results in a secondary decrease in GKb. The coordination of the two membrane conductances and regulation of GKb is critical for maintaining constant intracellular ion concentrations and transepithelial Cl secretion. The purpose of this study was to test two hypotheses about the regulation of GKb. First, we asked whether GKb is directly linked to the activity of the Na,K-ATPase. We found that pump activity could be dissociated from K conductance. Inhibition of the Na pump with ouabain, in nonsecreting tissues led to an increase in Gb. Elevation of the bathing solution K concentration produced a similar effect. Addition of ouabain to secreting tissues did not appear to alter Gb. These results indicate that GKb does not directly parallel Na pump activity. Second, we asked whether changes in GKb are voltage dependent. We prevented secretagogue-induced depolarization of the electrical potential difference across the basolateral membrane psi b by clamping psi b at its resting value during stimulation of Cl secretion with epinephrine. Despite maintaining psi b constant, the typical changes in transepithelial resistance and the ratio of membrane resistances persisted. This observation indicates that depolarization is not required for the secretagogue-induced increase in GKb. In addition we examined the effect of depolarizing and hyperpolarizing psi b by passing transepithelial current in secreting and nonsecreting epithelia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium.

The tracheal mucosa from a 12-year-old girl was digested with collagenase 4 hr after her death from cystic fibrosis. Forty million viable cells were obtained. The cells, plated at 10(6) per cm2 onto four Nuclepore filters coated with human placental collagen, formed confluent monolayers after 1 day. Their ultrastructure was similar to that of normal human cells. They were studied in conventional Ussing chambers or with intracellular microelectrodes on days 5-7 after plating. The monolayers displayed resistance of 380 +/- 50 omega X cm2 and short-circuit current (Isc) of 1.8 +/- 0.4 microA X cm-2. This resistance is similar to that obtained for dog or normal human monolayers. The Isc is less than normal human (approximately 3 microA X cm-2) or dog (approximately 10 microA X cm-2) cells. The cystic fibrosis cells resembled normal monolayers in that serosal ouabain and mucosal amiloride inhibited Isc, while mucosal ouabain or serosal amiloride had no effect. They differed from normal human or dog cells in that Isc was not inhibited by bumetanide and the stimulation of Isc by isoproterenol or prostaglandin E2 was greatly reduced or abolished. Addition of isoproterenol depolarized apical membrane potential (psi a) and decreased fractional resistance (fR) in normal human and dog but had no effect on psi a or fR in cystic fibrosis cells. Reduction of mucosal chloride from 120 to 5 mM by replacement with gluconate increased fR of dog and normal human monolayers and depolarized psi a by 22 (dog) or 30 (human) mV. In cystic fibrosis monolayers, chloride replacement hyperpolarized psi a by 2 mV and had little effect on fR. These results suggest that the primary defect in cystic fibrosis is reduced apical membrane chloride conductance.

Animals↗
PubMed1985

Intracellular calcium regulates basolateral potassium channels in a chloride-secreting epithelium.

The two individual cell membranes of epithelia are functionally coupled, so that changes in apical membrane conductance are paralleled by changes in basolateral K+ conductance. However, the signal that regulates basolateral K+ conductance, thereby coupling the two membranes, is unknown. We tested the hypothesis that the cellular calcium concentration, [Ca2+]c, may regulate basolateral K+ conductance in canine tracheal epithelium, a Cl- -secreting epithelium that shows marked membrane coupling. Three findings support the hypothesis. First, the intracellular Ca2+ antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) attenuated the secretory response. Second, the secretagogue epinephrine increased [Ca2+]c, as measured with quin-2. Third, we found a K+ channel that was activated by Ca2+ on the cytosolic side of the membrane. Thus, cytosolic Ca2+ regulates the basolateral K+ conductance and may be the signal responsible for functional coupling of the two cell membranes.

Animals↗
PubMed1985

Stimulation of rat Sertoli cell adenylate cyclase by germ cells in vitro.

The effect of germ cells or germ cell fractions on adenylate cyclase (AC) activity in membrane preparations from cultured rat Sertoli cells has been examined. Whole germ cells or 30,000 X g pellet or supernatant fractions of germ cells have the ability to stimulate Sertoli cell AC to levels comparable to those measured in follicle-stimulating hormone-stimulated Sertoli cell membranes. Treatment at 100 degrees C but not 60 degrees C for 1 min abolished the ability of germ cell preparations to stimulate Sertoli AC. Germ cell stimulation of Sertoli cell AC was not calcium dependent, was not blocked by propranolol, and was observed to be dose dependent.

Adenylyl Cyclases↗
PubMed1985

Oxidants increase paracellular permeability in a cultured epithelial cell line.

Inflammation of epithelia is an important step in the pathophysiology of a wide variety of diseases. Because reactive oxygen metabolites are important effector molecules of acute inflammation, we examined the effect of oxidants on the barrier function of a cultured epithelium, Madin Darby Canine Kidney cells, by measuring the transepithelial electrical conductance, Gt, of monolayers grown on permeable supports. We found that H2O2, added directly or generated with glucose oxidase, increased Gt. Similar effects were observed with addition of xanthine and xanthine oxidase, a system which enzymatically generates superoxide radical O2-. The oxidant-induced increase in Gt was reversible if the exposure to oxidants was not prolonged (less than 20 min), and if the concentration of H2O2 was less than 5 X 10(-3) M. The increase in Gt suggested that oxidants increase the permeability of the paracellular pathway, a suggestion supported by an oxidant-induced increase in the permeability to 14C-mannitol, which primarily crosses epithelia via the extracellular route. In addition to functional changes in the epithelial monolayer, oxidants changed the cell morphology; after H2O2 exposure, the cells tended to pull apart, most prominently at their basolateral surfaces. These changes were heterogeneous with most areas showing no changes. Some of the morphologic changes could be reversed if the exposure to H2O2 was limited. We also observed a disruption of the normal pattern of the actin-cytoskeleton, particularly in the area of cell to cell junctions, as demonstrated by fluorescent staining of f-actin with rhodamine phallicidin. These functional and structural findings indicate that oxidants increase the permeability of the paracellular pathway in a cultured epithelium. The changes can be reversible, and are accompanied by alterations in organization of the cell cytoskeleton. These studies demonstrate the dynamic nature of the interaction between epithelial cells and oxygen metabolites.

Animals↗
PubMed1984

Identification of spectrin and protein 4.1-like proteins in mammalian lens.

Human, bovine, canine, and rabbit lenses were found to contain proteins which cross-react with anti-4.1 serum and which have molecular weights similar to erythrocyte proteins 4.1a and 4.1b (approximately 80 kd). Additionally, bovine, canine, and rabbit lenses contain a 4.1-like protein of approximately 125 kd which is absent from human lens. Proteins which cross-react with antibody to human erythrocyte spectrin were also detected. The human lens showed weak cross-reaction of bands of 240 kd and 225 kd, and a more intense cross-reaction of a band of 235 kd. Canine and bovine lenses showed weak cross-reaction with only the bands at 240 kd and 235 kd. The lens 240 kd band of all species also demonstrated calcium-dependent binding of calmodulin. Our results indicate that proteins related to, but distinct from, erythrocyte protein 4.1 and spectrin are found in mammalian lens.

Animals↗
PubMed1984

Anthracene-9-carboxylic acid inhibits an apical membrane chloride conductance in canine tracheal epithelium.

Canine tracheal epithelium secretes Cl from the submucosal to the mucosal surface via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. Cl exit across the apical membrane is thought to be a passive, electrically conductive process. To examine the cellular mechanism of Cl secretion we studied the effect of anthracene-9-carboxylic acid (9-AC), an agent known to inhibit the Cl conductance of muscle membrane. When added to the mucosal solution, 9-AC rapidly and reversibly decreases short-circuit current and transepithelial conductance, reflecting a reduction in electrogenic Cl secretion. The inhibition is concentration-dependent and 9-AC does not appear to compete with Cl for the transport process. The decrease in current and conductance results from a decrease in the net and both unidirectional transepithelial Cl fluxes without substantial alterations of Na fluxes. Furthermore, 9-AC specifically inhibits a Cl conductance: tissues bathed in Cl-free solutions showed no response to 9-AC. Likewise, when the rate of secretion and Cl conductance were minimized with indomethacin, addition of 9-AC did not alter transepithelial conductance. In contrast, neither removal of Na from the media nor blockade of the apical Na conductance with amiloride prevented a 9-AC-induced decrease in transepithelial conductance. We also found that the effect of 9-AC is independent of transepithelial transport: 9-AC decreases transepithelial conductance despite inhibition of Cl secretion with ouabain or furosemide. Intracellular electrophysiologic techniques were used to localize the effect of 9-AC to a reduction of the electrical conductance of the apical cell membrane: 9-AC hyperpolarizes the electrical potential difference across the apical membrane and decreases its relative conductance. 9-AC also prevents the characteristic changes in the cellular electrical potential profile, transepithelial conductance, and the ratio of membrane conductances produced by a reduction in mucosal bathing solution Cl concentration. These results indicate that 9-AC inhibits Cl secretion in tracheal epithelium by blocking an electrically conductive Cl exit step in the apical cell membrane. Thus, they support a cellular model of Cl secretion in which Cl leaves the cell across a Cl permeable apical membrane driven by its electrochemical gradient.

Animals↗
PubMed1984

The effect of 2-methoxyethanol and methoxyacetic acid on Sertoli cell lactate production and protein synthesis in vitro.

Exposure to 2-methoxyethanol (ME) or its major metabolite, methoxyacetic acid (MA), results in spermatocyte depletion and testicular atrophy in experimental animals. The site of spermatogenesis is within the seminiferous tubule. Sertoli cells support spermatogenesis, synthesizing and secreting proteins, and metabolic substrates for utilization by differentiating germ cells in the seminiferous tubule lumen. One of these substrates, lactate, is preferentially metabolized by spermatocytes. Therefore, because germ cells are dependent upon the metabolic products of Sertoli cells, the effect of ME and MA on production of lactate and protein synthesis was measured in cultured rat Sertoli cells. Cell cultures were incubated with ME or MA at 0, 3, or 10 mM for up to 12 hr. No significant difference was seen in total protein synthesis as measured by [3H]leucine incorporation. ME and MA had no apparent effect on cell viability. However, lactate concentrations and rates of lactate accumulation were significantly decreased by MA, but not ME, at both 3 and 10 mM following incubation for 6, 9, and 12 hr. The results suggest that inhibition of Sertoli cell lactate production resulting from ME or MA exposure could account for the inhibitory action of these compounds on spermatogenesis.

Acetates↗
PubMed1984

Energetics of chloride secretion in canine tracheal epithelium. Comparison of the metabolic cost of chloride transport with the metabolic cost of sodium transport.

Canine tracheal epithelium secretes Cl and absorbs Na; the energy for both is derived from the activity of the basolateral membrane Na-K-ATPase. These properties allowed us to examine the energetics of Cl secretion by directly comparing the metabolic cost of Cl transport with the metabolic cost of Na transport. We measured the change in short-circuit current and O2 consumption rate that was produced by a variety of maneuvers that alter Na and/or Cl transport rate. The experimental interventions included the use of the secretagogue epinephrine, the Cl transport inhibitor bumetanide, the Na transport inhibitor amiloride, the Na-K-ATPase inhibitor ouabain, and ion substitutions. The O2 consumption rates required for Na and Cl transport were compared in each individual tissue. The results indicate that the oxygen cost of Cl transport is significantly less than the oxygen cost of Na transport: two to four Cl ions are transported for the same metabolic cost that is required to transport one Na ion. These findings suggest that the basolateral membrane Na-dependent Cl entry step couples the entry of more than one Cl ion to each Na ion.

Amiloride↗
PubMed1983

The distribution of calmodulin in living mitotic cells.

Calmodulin has been labeled with rhodamine isothiocyanate (CaM-RITC) and used as a probe for the location of calmodulin in vivo. CaM-RITC retains its capacity to regulate the activity of brain phosphodiesterase in a Ca2+-dependent manner in vitro, indicating that the labeled protein is still active. After injection into living mammalian cells CaM-RITC incorporates rapidly into the mitotic spindle; the details of its localization there mimic closely the distribution of calmodulin seen by immunofluorescence. In interphase cells the CaM-RITC is excluded from the nucleus, but shows no region of specific concentration within the cytoplasm. Neither a 2-fold increase in cellular CaM nor the injection of anti CaM has any observable effect on the progress of mitosis.

Animals↗