Caldesmon, calponin and alpha-smooth muscle actin expression in subcultured smooth muscle cells from human airways.

Calponin and caldesmon are two proteins considered to play a regulatory role in smooth muscle contraction, which have never previously been found to be expressed in subcultured cells. In the present study, immunocytochemistry and immunoblotting were performed to identify these proteins in smooth muscle cells (SMC) from human bronchi. It was found that human airway SMC, kept in a non-proliferative state, continued to express caldesmon and calponin at least until the 8th passage. The expression of alpha-smooth muscle actin studied under the same conditions was also shown to be preserved in subcultured bronchial SMC.

Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture.

Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against alpha-smooth muscle actin (alpha SMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[3H]inositol in order to label the phosphoinositide pool, and the various [3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IP1 isomers were separated in three peaks; IP2, IP3, IP4, IP5 and IP6 (phytic acid) were each eluted as single peaks. The identity of the [3H]peaks was verified with corresponding [3H]IP standards. The accumulation of [3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCl, with or without a bronchoconstrictor agent (carbachol, histamine, PGF2 alpha). Histamine, 10(-4) M, elicited a four times larger IP accumulation than carbachol, 10(-4) M, and than PGF2 alpha, 5 10(-5) M. Dose-response curves were established for histamine and carbachol in the range 10(-7)-10(-4) M. At 10(-7) M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways.

Coupling of active sodium transport to oxidative metabolism in the rabbit distal colon.

1. Active transepithelial Na+ transport (Ji) and O2 consumption (Jr) were measured simultaneously in rabbit distal colon, under standard (control) incubation conditions and after various manoeuvres, known to inhibit Na+ transport. 2. The determination of Jr was complicated by the presence of fluctuations of the PO2 in the incubation solution and by spontaneous variations of the tissue respiration, which usually declined slowly with time. 3. The control values of Ji and Jr after 2 h incubation were 55 +/- 4 nequiv min-1 cm-2 and 16 +/- 1 nmol O2 min-1 cm-2, respectively (n = 44). The electrical resistance was 386 +/- 23 omega cm2; it was stable over 6 h. 4. Ji was reduced to a very low level with either amiloride, ouabain or Na+ substitution with choline. In all instances, Jr decreased concomitantly by 15-30%. 5. A plot of the change in Jr versus the change in Ji gave a straight line for all situations, i.e. for the spontaneous decline of Na+ transport and respiration and for the effects of the inhibitors. 6. The linearity between Jr and Ji allows for the determination of a stoichiometric ratio. It is of similar magnitude, when calculated either with the data of spontaneous variations or with those obtained by the action of any inhibitor tested. It is 15-20 Na+ ions per O2 molecule, a value close to that reported previously for amphibian epithelia and also close to the maximum theoretical value of 18 Na+ ions per O2 molecule.

Oxygen consumption and active sodium and chloride transport in bovine tracheal epithelium.

The O2 consumption (Jr) and the short-circuit current (Ji) were measured simultaneously in bovine tracheal epithelium in vitro. In this tissue, Ji is the sum of two active transport processes, Cl- secretion and Na+ absorption. Jr was determined from the decrease of PO2 in the incubation solution, at 37 +/- 0.05 degrees C and at a PO2 around 600 torr. Microbial contamination and leaks of dissolved O2 from the solution never exceeded 4% of the rate of PO2 decrease due to the O2 consumption of the tissue. Ji and Jr were stable over 5 h of incubation under standard conditions. Ji was 106 +/- 4 nequiv min-1 cm-2 and Jr was 39.8 +/- 1.1 nmol O2 min-1 cm-2 (mean +/- S.E., n = 46). Ji was varied with several agents known to affect ion transport across the tracheal epithelium. Na+ absorption was inhibited partly with amiloride or completely following Na+ substitution with choline. Cl- secretion was selectively suppressed by furosemide. Ji was also reduced to a very low level, using ouabain or K+ suppression to inhibit the Na+-K+-ATPase. All these manoeuvres resulted in significant reductions of both Ji and Jr. Basal Jr was not affected when Ji was modified. A plot of the relative change in suprabasal Jr versus the relative change of Ji gave a straight line (r = 0.98, n = 60). A plot using absolute values yielded a stoichiometric ratio of 13.9 ions per O2 molecule, for Na+ as well as for Cl-. The stoichiometric ratio was also calculated for each experiment. Its mean value was 14.9 ions per O2 molecule. The population of the ratios was widely dispersed, but this was explained as a predictable statistical phenomenon.

Effects of chloropyramine, dimethindene and diphenhydramine on transepithelial ion transport: studies in bovine tracheal epithelium and frog skin.

The effects on transepithelial ion transports of chloropyramine, dimetindene and diphenhydramine, which are three antagonists of H1-receptors of histamine, were examined in bovine tracheal epithelium and in frog skin. The short-circuit current I0 across bovine tracheal epithelium is the sum of active secretion of Cl- and absorption of Na+. In this tissue, all three drugs induced a reversible, dose-related inhibition of I0, up to 100%. The concentrations giving 50% of maximal effect were 1.4 X 10(-4) M for chloropyramine, 2.0 X 10(-4) M for dimetindene and 2.5 X 10(-4) M for diphenhydramine. The effect was unrelated to the agonist binding site of H1-receptors of histamine, since it was not altered in the presence of 10(-3) M histamine. Experiments in which Na+ transport was selectively reduced by 5 X 10(-5) M amiloride, or in which Cl- transport was selectively abolished by 10(-3) M furosemide, 10(-4) M bumetanide or Cl- removal, indicated that Na+ and Cl- transports were equally affected by the drugs. The action of chloropyramine was composed of an early inhibition of Na+ and Cl- movements, followed by a slow recovery of Cl- secretion. In frog skin, each one of the three H1-antagonists modified the I0, following two main patterns of response, a stimulation at the lower concentrations tested, or an inhibition at higher concentrations. Dose-response relationships were obscured by a large variability in response of individual skins. These observations in bovine tracheal epithelium and frog skin suggest that H1-antagonists might alter the functioning of other epithelia as well.

Effects of harmaline on ion transport and oxygen consumption by the bovine tracheal epithelium.

The alkaloid harmaline is known to affect various membrane transport systems. This study examines the action of the drug on the short-circuit current (I0) and on the oxidative metabolism (Jr) in the tracheal epithelium of the cow. In this tissue I0 corresponds to the sum of two active transports: Na+ is absorbed and Cl- is secreted by a process based on the activity of the Na+ pump. A well defined relationship has been previously demonstrated between these active transports and the rate of O2 consumption (Schoenenweid et al., 1984 b). Low concentrations of harmaline (10(-6) to 5.10(-6) M) induced a small stimulation of I0. In contrast, larger concentrations (between 5.10(-5) and 10(-3) M) yielded a dose-related inhibition of I0, with an apparent concentration yielding 50% of maximal effect of 7.1.10(-4) M and maximal effect approaching 100%. The action was fully reversible after removal of the drug. The measurements of the fluxes of 22Na and 36Cl revealed that harmaline at a concentration of 8.10(-4) M, which decreased the I0 by 74 +/- 1% (n = 23), diminished both Na+ and Cl- transports, by 81 and 52%, respectively. The time course of I0 decay following the administration of harmaline was made of three components, with half-times of 0.34, 2.2 and 15.2 min. The time course was not appreciably modified when Cl- secretion was abolished with furosemide. Although harmaline, 10(-3)M, inhibited markedly I0, it did not modify Jr significantly. In contrast, when K+ in the incubation solution was omitted, both Ji and Jr were lowered.(ABSTRACT TRUNCATED AT 250 WORDS)

Current-induced volume flow across bovine tracheal epithelium: evidence for sodium-water coupling.

The passage of a constant current from lumen to serosa (Il-s), in the range 0.5-2.0 mA, across ouabain-treated bovine tracheal epithelium, induced a stable volume flow (Jv) toward the serosa, proportional to the current. No consistent Jv occurred when current was applied from serosa to lumen. When the standard K+ (6 mM) in the bathing solution was omitted or replaced by choline, Jv was in the same direction as, and proportional to, the current, both with Is-l and with Il-s. The electro-osmotic permeability beta was in the range of 10-15 microl h-1 cm-2 mA-1, i.e. 3-4 X 10(-6) cm s-1 mA-1. The fluxes of Na+, Cl- and mannitol were measured in current-clamp (1 mA, passed from serosa to lumen or lumen to serosa) or voltage-clamp (-20, 0 and +20 mV) conditions, with and without K+. Net transepithelial Na+ fluxes toward the cathode were either smaller than (with Is-l) or equal to (with Il-s) the net fluxes of Cl- toward the anode. The total transepithelial conductance (Gt) increased with the applied electrical gradient, both with Is-l and with Il-s, the change in Gt being larger with Il-s than with Is-l. This increase of Gt was less pronounced when K+ was omitted. The analyses of partial ionic conductances (GNa and GCl) and of the flux ratios indicate the existence of non-conductive diffusion for Cl- and also for Na+. The direction of the electrical gradient influenced the permeability ratio PNa/PCl. With Is-l, PNa/PCl was consistently lower than 0.7, i.e. the mobility ratio of Na+ and Cl- in solution. With Il-s, PNa/PCl was closer to 0.7. The highest Cl- selectivity of the epithelium was observed with Is-l in the presence of K+, i.e. under conditions which failed to induce any conspicuous Jv. The passage of current at 1 mA induced a net flux of mannitol toward the cathode, i.e. in the same direction as Na+ net flux and Jv. However, this mannitol flux was significant only in the absence of K+. These results indicate that Jv was predominantly coupled to the migration of Na+ along the electrical gradient, through a paracellular pathway.

Volume flow, hydraulic conductivity and electrical properties across bovine tracheal epithelium in vitro: effect of histamine.

Volume flow (Jv), potential difference (delta psi), short-circuit current (io) and electrical resistance (R) were measured simultaneously across bovine tracheal epithelium in vitro. Under basal conditions, with no applied hydrostatic or osmotic pressure gradients (delta P = 0, delta phi = 0), no spontaneous Jv was observed. delta psi was 31 +/- 2 mV (lumen negative ), io 161 +/- 8 microA cm-2 and R 202 +/- 9 omega cm2, n = 50. When a delta pi was applied, by adding 20 - 80 mM sucrose into the medium bathing either the luminal or the serosal side of the tissue, a linear relationship was found between delta pi and Jv toward the lumen or toward the serosa. The apparent hydraulic conductivity (apparent Lp) was 4.6 - 4.9 10(-6) cms-1 atm-1. Histamine 10(-4) M did not induce any spontaneous Jv under basal conditions and had no effect on io nor on R. However, histamine caused a 100% increase in Jv elicited by sucrose gradients. It was concluded that histamine exerts a selective action on the hydraulic conductivity of bovine tracheal epithelium. Experiments using H1-receptors antagonists (diphenhydramine, dimetindene, chloropyramine) and H2-antagonists (cimetidine, metiamide) or a H2-agonist (impromidine) showed that the increase of Lp induced by histamine was mediated via H2-receptors.

A comparative study of the effects of norepinephrine and vasopressin on Na transport and O2 consumption in frog skin.

Experiments were designed to compare the effects of two hormones-vasopressin and norepinephrine-on the energetics of Na transport in frog skin. Simultaneous measurements of O2 consumption and net Na flux were performed in the same skins by means of O2 cathodes and the short circuit current technique. The results showed that both hormones induced similar increments in Na transport. In contrast, there was a conspicuous difference in O2 consumption values, norepinephrine having a very small stimulatory effect compared to the one induced by vasopressin. Thus, despite the fact that both hormones increase Na permeability of frog skin by similar mechanisms and to a similar extent, they appear to exert very different effects on cell metabolism.