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Biomedical subjects

M J Welsh

Publications and source records attributed to M J Welsh.

At least 307 records · Page 17Linked to original sources
PubMed1983

Fate of microfilaments in vero cells infected with measles virus and herpes simplex virus type 1.

In herpes simplex virus type 1-infected Vero cells, reorganization of microfilaments was observed approximately 4 h postinfection. Conversion of F (filamentous) actin to G (globular) actin, as assessed by a DNase I inhibition assay, was continuous over the next 12 to 16 h, at which time a level of G actin of about twice that observed in uninfected cells was measured. Fluorescent localization of F actin, using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, demonstrated that microfilament fibers began to diminish at about 16 to 18 h postinfection, roughly corresponding to the time that G actin levels peaked and virus-induced cytopathology was first observable. In measles virus-infected cells, no such disassembly of microfilaments occurred. Rather, there was a modest decrease in G actin levels. Fluorescent localization of F actin showed that measles virus-infected Vero cells maintained a complex microfilament network characterized by fibers which spanned the entire length of the newly formed giant cells. Disruption of microfilaments with cytochalasin B, which inhibits measles virus-specific cytopathology, was not inhibitory to measles virus production at high multiplicities of infection (MOI) but was progressively inhibitory as the MOI was lowered. The carbobenzoxy tripeptide SV-4814, which inhibits the ability of Vero cells to fuse after measles virus infection, like cytochalasin B, inhibited measles virus production at low MOI but not at high MOI. Thus, it appears that agents which affect the ability of Vero cells to fuse after measles virus infection may be inhibitory to virus production and that the actin network is essential to this process.

Actins↗
PubMed1983

Evidence for basolateral membrane potassium conductance in canine tracheal epithelium.

The ionic dependence of the basolateral membrane conductance in canine tracheal epithelium was investigated using intracellular microelectrode techniques. Increasing the K+ concentration in the submucosal bathing solution depolarized the electrical potential difference across the basolateral membrane; neither alteration of the submucosal Na+ concentration nor the mucosal K+ concentration had a significant effect on the cellular electrical potential profile. An increase in the K+ concentration in the submucosal bathing solution also decreased the net rate of Cl-secretion. Addition of ouabain (10(-4) M) to the submucosal bathing solution decreased the short-circuit current and depolarized the intracellular voltage without altering transepithelial resistance or the cell membrane resistance ratio, suggesting that basolateral resistance was unchanged. These findings, together with the previous observation that there is no appreciable basolateral Cl- conductance, indicate that a K+ conductance accounts for the predominance of the electrical conductance at the basolateral membrane. The results also indicate that the basolateral membrane K+ conductance plays a critical role in the generation of the negative intracellular voltage that drives Cl- exit across the apical membrane and thus supports Cl- secretion.

Animals↗
PubMed1983

Barium inhibition of basolateral membrane potassium conductance in tracheal epithelium.

Addition of barium ion, Ba2+, to the submucosal bathing solution of canine tracheal epithelium reversibly decreased the short-circuit current and increased transepithelial resistance. The decrease in short-circuit current represented a decrease in the net rate of Cl secretion with no change in the rate of Na absorption. Intracellular microelectrode techniques and an equivalent electrical circuit analysis were used to localize the effect of Ba2+ to an inhibition of the permeability of the basolateral membrane to K. Ba2+ (2 mM) doubled basolateral membrane resistance, decreased the equivalent electromotive force at the basolateral membrane, and decreased the magnitude of the depolarization of basolateral membrane voltage produced by increasing the submucosal K concentration. The inhibition of the basolateral K permeability depolarized the negative intracellular voltage, resulting in both a decrease in the driving force for Cl exit and an estimated increase in intracellular Cl concentration. These studies indicate that there is a Ba2+-inhibitable K conductance at the basolateral membrane of tracheal epithelial cells and that the K permeability plays an important role in the generation of the negative intracellular electrical potential that provides the driving force for Cl exit from the cell.

Animals↗
PubMed1983

Intracellular chloride activities in the isolated perfused shark rectal gland.

The isolated, perfused shark rectal gland secretes Cl when stimulated with adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of secretion, we used Cl-selective and conventional (KCl-filled) microelectrodes to measure the intracellular Cl activity (aClc). Under nonsecreting conditions, the electrical potential difference across the basolateral membrane (psi b) was -78 m V and aClc was 57 mM, a value seven times greater than predicted for electrochemical equilibrium across the basolateral membrane. When theophylline and 8-bromo-cAMP were added to the perfusate, the transglandular electrical potential difference doubled and the rate of fluid secretion increased 20-fold; however, neither psi b nor aClc changed. During both nonsecreting and secreting conditions the intracellular accumulation of Cl results in an electrochemical potential difference favoring Cl exit across the apical cell membrane. The constancy of aClc despite the variation in secretion rate suggests that stimulation is associated with an equivalent enhancement of net Cl movement across both the apical and basolateral membranes. When stimulated glands were perfused with Na-free (choline) Ringer, secretion was abolished and aClc fell toward the value predicted for electrochemical equilibrium. These findings suggest that the "uphill" step in Cl secretion lies at the basolateral membrane, where cellular Cl accumulation probably involves secondary active transport; i.e., Cl entry is driven by an inwardly directed electrochemical potential difference for Na.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Intracellular chloride activities in canine tracheal epithelium. Direct evidence for sodium-coupled intracellular chloride accumulation in a chloride-secreting epithelium.

Canine tracheal epithelium secretes Cl via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. To examine the mechanisms involved, intracellular chloride activity, acCl, was measured with Cl-selective intracellular microelectrodes. The results indicate that when the rate of secretion was minimal acCl was 37 mM; with stimulation of secretion the intracellular voltage depolarized, but acCl was not significantly altered, at 39 mM. These findings indicate that: (a) Cl is accumulated across the basolateral membrane under nonsecreting and secreting conditions at an activity 3.8 and 2.4 times, respectively, that predicted for an equilibrium distribution; (b) Cl exit across the apical membrane may be passive with an electrochemical driving force of 22 mV; and (c) stimulation of secretion enhanced the rate of Cl entry across the basolateral membrane, since Cl transport increased without a change in acCl. In the absence of Na in the extracellular fluid, acCl approached the value expected for an equilibrium distribution. This finding suggests that "uphill" entry of Cl into the cell against its electrochemical gradient is dependent upon, and energized by, the entry of Na down its gradient. Submucosal bumetanide, a loop diuretic, also decreased the rate of Cl secretion and decreased acCl, indicating an inhibition of Cl entry. These findings indicate that Cl entry into the cell is directed against its electrochemical gradient and is mediated by a Na-coupled, bumetanide-inhibitable, transport process at the basolateral membrane and that Cl may exit passively down a favorable electrochemical gradient across the apical membrane.

Animals↗
PubMed1983

Cigarette smoke inhibition of ion transport in canine tracheal epithelium.

Inhalation of cigarette smoke is known to impair pulmonary mucociliary clearance. Active ion transport by airway epithelium plays an important role in maintaining effective mucociliary clearance by regulating the volume and composition of the airway secretions. To determine the effect of cigarette smoke on airway epithelial ion transport, the electrical properties and transepithelial Na and Cl fluxes were measured in canine tracheal epithelium. In vivo, the inhalation of the smoke from one cigarette acutely and reversibly decreased the electrical potential difference across the tracheal epithelium. In vitro, exposure of the mucosal surface of the epithelium to cigarette smoke decreased the short circuit current and transepithelial resistance. The decrease in short circuit current was due to an inhibition of the rate of Cl secretion with minimal effect on the rate of Na absorption. The effect of cigarette smoke was reversible, was not observed upon exposure of the submucosal surface to smoke, and was most pronounced when secretion was stimulated. The particulate phase of smoke was largely responsible for the inhibitory effect, since filtering the smoke minimized the effect. The effect of cigarette smoke was not prevented by addition of antioxidants to the bathing solutions, suggesting that the inhibition of Cl secretion cannot be entirely attributed to an oxidant mechanism. These results indicate that cigarette smoke acutely inhibits active ion transport by tracheal epithelium, both in vivo and in vitro. This effect may explain, in part, both the abnormal mucociliary clearance and the airway disease observed in cigarette smokers.

Animals↗
PubMed1982

Crypts are the site of intestinal fluid and electrolyte secretion.

The site of adenosine 3',5'-monophosphate-mediated fluid and electrolyte secretion across mammalian large intestine was found to be the crypts of Lieberkühn by means of two techniques. First, the formation of fluid droplets was visualized on the oil-covered mucosal surface directly over crypt duct openings when secretion was stimulated. Second, microelectrode impalement of individual surface and crypt cells revealed that only crypts cells produced a pattern of secretagogue induced alterations in membrane potential and resistance that was characteristic of secretory epithelia.

Amiloride↗
PubMed1982

Calmodulin binds to chick lens gap junction protein in a calcium-independent manner.

A biochemically active conjugate of calmodulin and tetramethylrhodamine isothiocyanate (CaM-RITC) was synthesized. When incubated with sections of chick lens, this conjugate bound to the surface membranes of lens fiber cells in the presence of absence of calcium. Incubation of lens sections with antibodies to gap junction protein of lens completely blocked the binding of the conjugate to cell membranes, whereas serum from nonimmunized animals or antibodies to others lens proteins reduced the binding only slightly. By means of a gel overlay procedure, 125I-labeled calmodulin was found to bind to the gap junction protein of lens, also in a calcium-independent manner. These results support the concept that calmodulin may interact with and regulate gap junctions in living cells.

Animals↗
PubMed1982

Chloride secretion by canine tracheal epithelium: I. Role of intracellular c AMP levels.

We measured the short-circuit current (Isc) across canine tracheal epithelium and the intracellular cAMP levels of the surface epithelial cells in the same tissues to assess the role of cAMP as a mediator of electrogenic Cl secretion. Secretogogues fall into three classes: (i) epinephrine, prostaglandin (PG) E1, and theophylline decrease both Isc and cellular cAMP levels; (ii) PGF2 alpha and calcium ionophore, A23187, increase Isc without affecting cell cAMP levels at the doses employed; and (iii) acetylcholine, histamine, and phenylephrine do not alter either Isc or cAMP levels. These findings indicate that: (i) increases in cAMP or Ca activity stimulate electrogenic Cl secretion by the columnar cells of the surface epithelium; (ii) cAMP mediates the effects of PGE1 and beta-adrenergic agonists; (iii) a strict correlation between cAMP levels and Cl secretion rate is not apparent from spontaneous variations in these parameters or from dose-response relations of Isc and cAMP to epinephrine concentration; and (iv) acetylcholine, histamine, and phenylephrine, agents that stimulate electrically-neutral NaCl secretion by submucosal glands, do not evoke cAMP-mediated responses by the surface epithelium. Addition of 10(-6) M indomethacin (or other prostaglandin synthesis inhibitors) to the mucosal solution decreases Isc and cellular cAMP levels and reduces the release of PGE2 into the bathing media by 80%. Indomethacin does not interfere with the subsequent secretory response to PGE1. This suggests that endogenous prostaglandin production underlies the spontaneous secretion of Cl across canine tracheal epithelium under basal conditions.

Acetylcholine↗
PubMed1982

Chloride secretion by canine tracheal epithelium: II. The cellular electrical potential profile.

We used intracellular microelectrode techniques to study the mechanisms responsible for Cl secretion by canine tracheal epithelium. Tissues were treated with indomethacin (10(-6) M, added to the mucosal solution) to reduce the baseline rate of Cl secretion and then stimulated by addition of epinephrine (10(-6) M) or prostaglandin E1 (10(-6) M) to the submucosal solution. Three conclusions emerged from our findings: First, secretagogues enhance the rate of transepithelial Cl transport primarily by increasing apical membrane Cl permeability, since: (i) stimulation of secretion produced parallel decreases in transepithelial resistance (Rt) and the membrane resistance ratio Ra/Rb, where Ra and Rb refer to the membrane resistance ratio Ra/Rb, where Ra and Rb refer to the resistances of the apical and basolateral membranes; (ii) there was an inverse relation between the short-circuit current and Ra/Rb; (iii) secretagogues depolarized the electrical potential difference across the apical membrane (psi a) and produced an equivalent hyperpolarization of the transepithelial electrical potential difference (psi) t) so that, in the steady-state, the basolateral membrane potential (psi b) was unchanged; and (iv) substitution of sulfate or gluconate for Cl in the bathing solutions prevented secretagogue-induced changes in Rt, Ra/Rb, psi a, and psi t. Second, Cl entry into the cell across the basolateral membrane appears to be electrically-neutral since omission of Cl from the submucosal solution had no effect on psi b and did not decrease Ra/Rb as would be expected if Cl entered the cell by a conductive process. Third, secretagogues decrease Rb. Approximately 20 sec after the onset of the secretory response Ra/Rb underwent a secondary increase while Rt continued to fall. The decrease in Rb may reflect an increase in basolateral membrane K permeability.

Alprostadil↗
PubMed1981

Hormonal control of chloride secretion by canine tracheal epithelium: an electrophysiologic analysis.

A principal event in the stimulation of cyclic AMP-induced active C1 secretion across canine tracheal epithelium is an increase in the C1 permeability of the apical cell membrane. This permits C1 to leave the cell down its electrochemical potential difference across this barrier. C1 transport across the basolateral membrane appears to be nonconductive and may be mediated by NaC1 cotransport. Increased C1 entry across the basolateral membrane would enhance Na entry into the cell and secondarily stimulate Na extrusion from cell to submucosal solution. Increased Na-K-pump activity or an increase in the K permeability of the basolateral membrane may be responsible for the (Formula in text) reduction in basolateral membrane resistance that accompanies stimulation of C1 secretion. Analysis of the electromotive force across the apical cell membrane under conditions where C1 secretion is inhibited or stimulated provides reasonable estimates of intracellular Na and C1 activities for these conditions.

Alprostadil↗
PubMed1981

Regulation of protein kinase inhibitor by follicle-stimulating hormone in Sertoli cells in vitro.

The heat-stable protein inhibitor of cAMP-dependent protein kinase is specifically regulated by hormones in cultures of rat Sertoli cells maintained under completely defined conditions. Hormones that are known to elevate Sertoli cell cAMP concentrations, namely FSH and isoproterenol, produce a 4- to 5-fold increase in the specific activity of protein kinase inhibitor, whereas testosterone and LH have no effect. The stimulatory effects of FSH or isoproterenol on protein kinase inhibitor are completely mimicked by dibutyryl cAMP. The ability of FSH to stimulate protein kinase inhibitor is dependent upon the age of the animals used as a source for the Sertoli cells. FSH stimulates protein kinase inhibitor activity in cells from 9- and 16-day-old animals, but not in cells from 32-day-old animals. On the other hand, isoproterenol or dibutyryl cAMP will stimulate protein kinase inhibitor in cells from both young and old animals. FSH can stimulate protein kinase inhibitor activity in older cells only in the added presence of the phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine. Using specific antibodies to protein kinase inhibitor, we have shown that this protein is regulated by hormones via preferential stimulation of de novo synthesis of the inhibitor. (Endocrinology 108: 427, 1981)

Aging↗
PubMed1981

Electrophysiology of chloride-secreting epithelia.

A primary event in the stimulation of cyclic AMP-mediated active Cl secretion across epithelial tissues is an increase in the Cl permeability of the apical cell membrane. A rapid reduction in apical cell membrane resistance to Cl movement permits Cl to leave the cell down its electrochemical potential difference across this barrier. Since cell Cl activity is not markedly influenced by changes in the rate of Cl secretion, the cyclic nucleotide may also increase the ease with which Cl gains access to the secretory cells across the basolateral membrane. This Cl entry process appears to be mediated by NaCl co-transport since the maintenance of a high cell Cl activity depends upon the presence of sodium in the serosal bathing solution. Increased Cl entry across the basolateral membrane would promote enhanced Na entry into the cell and a secondary stimulation of active Na extrusion from cell to serosal solution. Increased Na-K-pump activity and/or a change in the K permeability of the basolateral membrane may be responsible for the augmented conductive ion flow across this barrier which accompanies the stimulation of active Cl secretion.

Adenosine Triphosphatases↗
PubMed1980

Protein inhibitor of cAMP-dependent protein kinase: production and characterization of antibodies and intracellular localization.

Multiple forms of protein kinase inhibitor exist in mammalian testis. Specific antibodies to testicular protein kinase inhibitor (PKI) have been raised in sheep. The antibody to the smallest of the inhibitors (9300 daltons) has been purified by antigen-affinity chromatography and shown to give a precipitin band with the inhibitor by double immunodiffusion. The antibody does not recognize any of the subunits of cyclic nucleotide-dependent protein kinases, namely cGMP-dependent protein kinase or the catalytic or regulatory subunits from type I or type II cAMP-dependent protein kinases. The biological activity of the 9300 dalton PKI is blocked completely by a 5 fold molar excess of antibody. Furthermore, the antibody can also block the activity of all other forms of testicular PKI. Using the antibody in indirect immunofluorescence microscopy, PKI localization was examined during interphase and mitosis in a variety of cell types. Our observations indicate that PKI is localized on microtubules in the cytoplasmic microtubule complex during interphase and in the spindle apparatus during mitosis. We suggest that PKI may play a role in the cAMP-dependent regulation of microtubule structure and/or function.

Animals↗
PubMed1980

Anion selectivity of the chloride-transport process in dog tracheal epithelium.

We have measured the fluxes of 82Br or 131I across short-circuited dog tracheal epithelium following complete replacement of chloride in the bathing medium by either bromide or iodide. In addition, we have partly replaced the bathing chloride with bromide, iodide, or thiocyanate and studied the effects of these anions on 36Cl fluxes. Our results show that the affinity sequence for the chloride transport process is I > Br congruent to Cl, that Br is transported, but that I is not. Thiocyanate is a potent inhibitor of chloride transport, and its effects are greater than those predicted by a simple competitive interaction with chloride for transport sites.

Animals↗
PubMed1980

Pathways of ion movement in the canine tracheal epithelium.

The pathways of ion movement across canine tracheal epithelium, a Cl-secreting tissue, were examined by three techniques. First, the measurement of simultaneous, unidirectional fluxes of Na or Cl and mannitol, a large hydrophilic molecule that serves as a marker of the paracellular pathway, indicated that a significant fraction of both the Na flux from submucosa to mucosa (J Na sm) and the flux of Cl from mucosa to submucosa (J Cl ms) traverse the cellular pathway. The ratio of the Na-to-Cl diffusion coefficients through the paracellular pathway was 0.23, in contrast to the free solution ratio of 0.63. Second, in voltage-clamp experiments we examined the effect of transepithelial voltage differences on the unidirectional fluxes of Na and Cl. The results agree with the previous findings, suggesting that there are voltage-independent, or transcellular, backfluxes of Na and Cl, and that the relative permeability of Na to Cl through the voltage-dependent (presumably paracellular) pathway was 0.28. Third, measurement of transepithelial diffusion potentials gave a Na-to-Cl permeability ratio of 0.31 +/- 0.02 (mean +/- SE). These results suggest that there are significant transcellular backfluxes of Na and Cl and that the paracellular pathway in the canine trachea is anion selective. An anion-selective pathway would tend to shunt the secreted Cl back through the paracellular pathway, thus minimizing the net ion and fluid movement across the tissue in the open-circuit condition.

Aminophylline↗