Search PubMed⌕ Search

Biomedical subjects

M J Welsh

Publications and source records attributed to M J Welsh.

At least 271 records · Page 15Linked to original sources
PubMed1988

Calmodulin stabilization of kinetochore microtubule structure to the effect of nocodazole.

To investigate the function of calmodulin (CaM) in the mitotic apparatus, the effect of microinjected CaM and chemically modified CaMs on nocodazole-induced depolymerization of spindle microtubules was examined. When metaphase PtK1 cells were microinjected with CaM or a CaM-TRITC conjugate, kinetochore microtubules (kMTs) were protected from the effect of nocodazole. The ability of microinjected CaM to subsequently protect kMTs from the depolymerizing effect of nocodazole was dose dependent, and was effective for approximately 45 min, with protection decreasing if nocodazole treatment was delayed for more than 60 min after injection of CaM. The CaM-TRITC conjugate, similar to native CaM, displayed the ability to activate bovine brain CaM-dependent adenylate cyclase in a Ca++-dependent manner and showed a Ca++-dependent mobility shift when subjected to PAGE. A heat-altered CaM-TRITC conjugate also protected kMTs from the effect of nocodazole. However, this modified CaM was not able to activate adenylate cyclase nor did it display a Ca++-dependent mobility shift when electrophoresed. In a permeabilized cell model system, both CaM analogs were observed to bind to the spindle in a Ca++-independent manner. In contrast, a performic acid-oxidized CaM did not have a protective effect on spindle structure when microinjected into metaphase cells before nocodazole treatment. The oxidized CaM did not activate adenylate cyclase and did not exhibit Ca++-dependent mobility on polyacrylamide gels. These results are interpreted as supporting the hypothesis that CaM binds to the mitotic spindle in a Ca++-independent manner and that CaM may serve in the spindle, at least in part, to stabilize kMTs.

Animals↗
PubMed1988

Evidence that the 90-kilodalton heat shock protein is associated with tubulin-containing complexes in L cell cytosol and in intact PtK cells.

It has been established that the 90-kilodalton murine heat shock protein, hsp90, is associated with the untransformed, non-DNA-binding form of the glucocorticoid receptor in L cell cytosol. In this work, we show that incubation of L cell cytosol with Affi-Gel-coupled monoclonal antibodies directed against either alpha-tubulin alone or both alpha- and beta-tubulin results in the immune-specific adsorption of hsp90 identified by Western blotting with the AC88 monoclonal antibody. Similarly, the AC88 antibody, which is specific for hsp90, causes the immune-specific isolation of both alpha- and beta-tubulin from hypotonic cytosol. The distribution of hsp90 in cultured Potorous tridactylis kidney cells was examined by indirect immunofluorescence using the AC88 monoclonal as primary antibody. In interphase cells, AC88-dependent fluorescence was distributed like antitubulin antibody-dependent fluorescence in a fibrillar array located in the cytoplasm and around the periphery of the nucleus. In cells undergoing mitosis, AC88 fluorescence was located in the mitotic spindle. These observations suggest that a significant portion of hsp90 is associated with a tubulin-containing complex both in a hypotonic cytosol preparation from mouse fibroblasts and in intact marsupial kidney epithelial cells. The distribution of AC88 fluorescence in interphase Potorous tridactylis kidney cells is similar to the distribution of glucocorticoid receptor demonstrated by Wikstrom, A. C., Bakke, O., Okret, S., Bronnegard, M., and Gustafsson, J. A in rat hepatoma and human uterine cells.

Animals↗
PubMed1988

Calmodulin colocalization with cold-stable and nocodazole-stable microtubules in living PtK1 cells.

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of both CaM and tubulin were examined in mitotic PtK1 cells in which MT subclasses had been selectively removed or altered by treatment with cold or with the MT inhibitor, nocodazole. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) was microinjected into living cells, and the CaM distribution in the living cell was compared to the distribution of MTs indicated by tubulin immunofluorescence. In cells which had been treated for 2 h at 0 to 4 degrees C or with a low (0.03 micrograms/ml) dose of nocodazole, the only MTs remaining appeared to be kinetochore MTs (kMTs). The distribution of microinjected CaM-TRITC in these cells was indistinguishable from that found in untreated cells and appeared to be colocalized with the kMTs. In cells which were treated with a high (3.0 micrograms/ml) dose of nocodazole, only short MTs remained. When CaM-TRITC was injected into these cells, it formed a somewhat punctate distribution near the chromosomes and, after tubulin immunofluorescence processing, colocalized with what appeared to be remnants of kMTs. We believe that these observations support the hypothesis that CaM exists in the MA in a structural association with kMTs.

Animals↗
PubMed1987

Neuroleptics antagonize a calcium-activated potassium channel in airway smooth muscle.

We examined the effect of neuroleptics on Ca-activated K channels from dog airway smooth muscle cells. Because these agents inhibit a variety of other Ca-mediated processes, it seemed possible that they might also inhibit Ca-activated K channels. In excised, inside-out patches, several neuroleptics potently and reversibly inhibited the K channel from the internal but not the external surface of the patch. Measurements of the effect on open probability and open- and closed-state durations support a simple kinetic model in which neuroleptics bind to and block the open channel. Inhibition by neuroleptics was moderately voltage dependent, with blockers less potent at hyperpolarizing voltages. The relationship between voltage and the dissociation constant for the blocker suggests that the binding site is one-third of the way across the channel's electrical field. Equilibrium dissociation constants for the drug-channel complex were: haloperidol, 1.0 +/- 0.1 microM; trifluoperazine, 1.4 +/- 0.1 microM; thioridazine, 2.4 +/- 0.1 microM; and chlorpromazine, 2.0 microM. This rank-order potency is different from their potency as calmodulin inhibitors, which suggests that neuroleptics bind to the channel rather than a calmodulin-channel complex.

Animals↗
PubMed1987

Effect of phorbol ester and calcium ionophore on chloride secretion in canine tracheal epithelium.

The control of Cl- secretion was examined by two of the terminal events in the phosphoinositide regulatory pathway: activation of protein kinase C and an increase in cell Ca2+. The phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, stimulated Cl- secretion in both native and cultured monolayers of tracheal epithelium. Approximately 1.5 nM of mucosal PMA was required for half-maximal stimulation. Stimulation was not dependent on prostaglandin production nor was it accompanied by an increase in cellular levels of cAMP. Although the maximal rate of PMA-induced Cl- secretion was less than that induced by cAMP, there was a synergistic effect between PMA and forskolin, an agent that activates adenylate cyclase. The response to PMA was at least partly transient and PMA may also attenuate Cl- secretion under some circumstances. Thus the response to PMA, and presumably protein kinase C activation, may be complex. An increase in cell Ca2+ produced by addition of the Ca2+ ionophore A23187 also stimulated Cl- secretion. However, the effect was at least partly indirect. A23187 enhanced prostaglandin E2 production and the prostaglandin synthesis inhibitor, indomethacin, blocked A23187-induced secretion in native epithelia and attenuated the effect of A23187 in cultured monolayers. These results indicate the presence of a non-cAMP-dependent regulatory pathway capable of controlling Cl- secretion in tracheal epithelium. Activation of protein kinase C may stimulate secretion directly or modulate the response to cAMP. An increase in cell Ca2+ may stimulate secretion at least partly by stimulating endogenous prostaglandin production.

Calcimycin↗
PubMed1987

Regulation of sodium absorption by canine tracheal epithelium.

To regulate the quantity of respiratory tract fluid, the airway epithelium either secretes chloride, Cl-, or reabsorbs sodium, Na+. Many secretagogues inhibit Na+ absorption, but the decrease may result from a fall in the electrochemical gradient for Na+ absorption. We examined regulation of Na+ absorption independent of Cl- secretion, by bathing canine tracheal epithelium in Cl--free, gluconate Ringers solution. Prostaglandin E2, 2-chloroadenosine, and isoproterenol increased short-circuit current (Isc) and the rate of Na+ absorption. In contrast, indomethacin, which inhibits endogenous prostaglandin production, decreased Isc. These agents regulate cellular levels of cAMP; direct addition of 8-Br-cAMP also acutely increased Isc. We examined chronic regulation of Na+ absorption in cell monolayers grown on permeable supports in serum-free media. Exposure to aldosterone for two days increased baseline Isc by 50% and the amiloride-inhibitable current by 55%. These data indicate that Na+ absorption is both acutely and chronically regulated in the airway epithelium.

2-Chloroadenosine↗
PubMed1987

Cystic fibrosis.

Explore the source record for details and available documents.

Cystic Fibrosis↗
PubMed1987

Germ cell stimulation of Sertoli cell protein phosphorylation.

Cultures of Sertoli cells were treated with freshly isolated, intact germ cells to determine if germ cells were capable of influencing Sertoli cell function. By using two-dimensional polyacrylamide gel electrophoresis and autoradiography, germ cells were found to increase several-fold the incorporation of [32P]orthophosphate into two phosphoproteins (which we term germ cell-dependent phosphoproteins 1 and 2 or GC1 and GC2) of Sertoli cells. Increased phosphorylation of GC1 and GC2 was rapid, germ cell dose dependent, and calcium dependent. The increased phosphorylation of GC1 appears to involve calmodulin-dependent protein kinase, while phosphorylation of GC2 appears to involve the activation of calcium/phospholipid-dependent protein kinase (protein kinase C). Medium conditioned by germ cells was capable of eliciting the same response in Sertoli cells. Germ cells had no effect on the phosphorylation of proteins in Chinese hamster ovarian cells, but did result in increased phosphorylation of a protein in TR-ST cells, which migrated similarly to GC1 of Sertoli cells. Neither Chinese hamster ovarian nor TR-ST cells had any effect on Sertoli cell protein phosphorylation. These results indicate that germ cells may be directly involved in the local regulation of Sertoli cell function within the seminiferous epithelium. The results further suggest that the mechanism of germ cell-Sertoli cell interaction involves the mobilization of intracellular calcium, activation of Ca2+/calmodulin-dependent protein kinase, and protein kinase C. We infer from these results that germ cell-Sertoli cell interaction may operate via hydrolysis of Sertoli cell membrane phophatidylinositols.

Animals↗
PubMed1986

Chronic amphetamine treatment increases striatal calmodulin in rats.

A radioimmunoassay was developed to measure calmodulin in striatum from rats treated with one dose or repeated injections of amphetamine. Chronic, but not acute, amphetamine treatment resulted in a significant increase in total calmodulin levels in striatal homogenates. This effect may be linked to the behavioral sensitization which develops after chronic amphetamine treatments.

Amphetamine↗
PubMed1986

An apical-membrane chloride channel in human tracheal epithelium.

The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

Anthracenes↗
PubMed1986

Effect of lead acetate on Sertoli cell lactate production and protein synthesis in vitro.

The effects of lead acetate on protein synthesis and lactate production by cultures of rat Sertoli cells in vitro were studied. Sertoli cell cultures prepared from 20 day old Sprague-Dawley rats were exposed to 0.01, 0.05 and 0.10 mM lead acetate. Lactate production was significantly elevated by all concentrations of lead after 3, 6, 9 and 12 hours of exposure. Protein biosynthesis as measured by [3H]-leucine incorporation was significantly depressed by 0.05 and 0.10 mM lead acetate after 2 hours of exposure. These results support the hypothesis that lead acetate may inhibit spermatogenesis by a disturbance of the metabolic activities of the Sertoli cells.

Animals↗
PubMed1986

Single apical membrane anion channels in primary cultures of canine tracheal epithelium.

To investigate the cellular mechanism responsible for the apical membrane Cl conductance in airway epithelia, we used the patch-clamp technique to study single ion channels in primary cultures of canine tracheal epithelium. The cells contained an anion channel that had a single channel conductance of approximately 29 pS at negative voltages in symmetrical 145 mmol/l Cl solutions. In symmetrical Cl solutions the excised single-channel current-voltage relation was nonlinear, with conductance increasing at depolarizing voltages. The channel was inhibited by diphenylamine-2-carboxylate and anthracene-9-carboxylic acid at concentrations similar to those required to inhibit transepithelial Cl secretion. The channel was found in freshly isolated cells, isolated cells in culture, and in the apical membrane of confluent areas of cells. When attached to the cell the channel was activated by addition of isoproterenol in some, but not all cases. Often it was not observed to open until the patch was excised from the cell. The channel was not strongly voltage-gated, nor was it acutely regulated by internal Ca in the excised, inside-out configuration. These results suggest that this channel may be responsible for the apical Cl conductance in canine tracheal epithelium.

Animals↗
PubMed1986

Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: cellular electrophysiology.

We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage psi a and decreased the fractional resistance of the apical membrane fR. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized psi a and increased fR, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, psi a depolarized and fR decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of alpha adrenergic receptors because it was reproduced by addition of the alpha agonist phenylephrine, and blocked by the alpha antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of alpha 1 adrenergic receptors. The acute alpha adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both beta and alpha adrenergic agents mediate the ion transport processes in canine tracheal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Calcium-activated potassium channels in canine airway smooth muscle.

Airway smooth muscle cells from canine trachealis muscle were dispersed by treatment with collagenase and elastase. Cells were identified as smooth muscle by their binding of anti-smooth muscle gamma-isoactin monoclonal antibodies and by their contraction in response to acetylcholine. The patch-clamp technique was used to study single channel currents in cell-attached and isolated patches of membrane. The most common single channel currents had a conductance of 266 +/- 12 pS (mean +/- S.D., n = 7) in symmetrical 135 mM-K solutions. The reversal potential of the channel was unaltered by large chemical gradients for Cl, Na and Ca and was determined exclusively by the chemical K gradient. Thus, the channel is highly selective for K. In both cell-attached and isolated patches of membrane, depolarization increased the frequency of channel opening and the duration of the open state. In isolated patches of membrane, increasing [Ca] on the cytoplasmic side of the membrane from 10(-8) to 10(-6) M increased both the frequency of channel opening and the duration of the open state. Tetraethylammonium, tetramethylammonium, or Cs (10 mM) on the cytoplasmic side of the membrane caused a voltage-dependent decrease in conductance of the open channel while having no obvious effect on channel kinetics. These blocks were completely reversible. Ba (10 mM) on the cytoplasmic side of the membrane slightly decreased inward currents and completely blocked outward currents through the channel. External Ba (10 mM) caused a voltage-dependent decrease in inward current. External tetraethylammonium (10 mM) completely blocked single channel currents.

Action Potentials↗
PubMed1986

Variability of functional characteristics of MDCK cells.

We measured several functional parameters of MDCK cells cultured as monolayers in order to more fully characterize their ion transport properties. Most of the present studies were completed with five groups (A-E) of MDCK cells studied from passage 62 to 78. Each group represents the same subline of MDCK cells after having been frozen, stored, and thawed at passage 62 or 64. The median transmonolayer resistances of the groups were 507, 149, 284, 72, and 126 omega X cm2. Addition of amphotericin B to the apical solution induced a oubain-sensitive transepithelial current. The apical membrane voltage and fractional resistance exhibited a wide range of values in two of the groups studied, with mean values of -32 mV and 0.68 in group B and -40 mV and 0.78 in group E. Neither apical nor basolateral membrane displayed significant Na+ conductance. K+ conductance was present in the basolateral but not in the apical membrane. Acidification or alkalinization of the apical solution was dependent on the conditions used to study the cells. The 4,4'-diisothyocyano-2,2'-disulfonic stilbene inhibited acidification (or induced alkalinization), whereas increasing ambient HCO-3 concentration induced alkalinization. The results of these studies indicate qualitatively similar behavior between five groups of MDCK cells but significant quantitative differences between the groups. Analysis of the variability of the measured parameters indicated that there were no differences as a function of passage number within a group. The factors responsible for functional differences between groups are not known but may be related to the cell storage process.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗