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J W Hanrahan

Publications and source records attributed to J W Hanrahan.

65 records · Page 4Linked to original sources
PubMed1990

Low-conductance chloride channel activated by cAMP in the epithelial cell line T84.

We have studied the modulation and pharmacological properties of two anion channels in T84 cells by recording single channel and transepithelial currents. One channel had an outwardly rectifying current-voltage I/V curve, was rarely active in cell-attached patches, and was unaffected by cAMP. The other channel had lower conductance (8.7 pS at 37 degrees C) and a more ohmic I/V relationship. Exposure to cAMP increased the probability of observing low-conductance channel activity in cell-attached patches greater than 6-fold. Extracellular DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) or [IAA-94 (an indanyloxyacetic acid) inhibited the outward rectifier but did not affect the low-conductance channel or cAMP-stimulated transepithelial current. These results suggest the low-conductance Cl channel may contribute to apical membrane conductance during cAMP-stimulated secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers.

The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studied using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris and bis-Tris at millimolar concentrations. HEPES (pK alpha = 7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6 mM HEPES on the cytoplasmic side (HEPESi) and this effect increased with [HEPESi] so that conductance at the reversal potential was diminished approximately 25% with 10 mM HEPEsi and approximately 70% at very high [HEPESi]. HEPESi block was relieved by applying positive voltage but positive currents were not consistent with a Woodhull-type blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPESi concentration at constant [HEPES-] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK alpha = 7.54), substituted glycine buffers (pK alpha = 8.1-8.4) and bis-Tris (pK alpha = 6.46) had no measurable effect on conductance and appear suitable for use with this channel.

Buffers↗
PubMed1990

Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals.

We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori-. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMP-dependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.

Cell Division↗
PubMed1989

Bicarbonate permeability of the outwardly rectifying anion channel.

Single anion-selective channels have been studied in cultured human epithelial cells using the patch-clamp technique. Three cell types were used as models for different anion transport systems: (i) PANC-1, a cell line derived from the pancreatic duct, (ii) T84, a Cl-secreting colonic cell line, and (iii) primary cultures of sweat duct epithelium. Outwardly rectifying anion-selective channels were observed in all three preparations and were indistinguishable with respect to conductance, selectivity and gating. Striking similarities between HCO3- and Cl-secreting epithelia, and the high density of outward rectifiers in pancreatic cells prompted us to study HCO3 permeation through this channel. HCO3 permeability was significant when channels were bathed in symmetrical 150 mM HCO3 solutions, Cl-HCO3 mixtures, and under bi-ionic conditions with outwardly and inwardly directed HCO3 gradients. Permeability ratios (PHCO3/PCl) estimated from bi-ionic reversal potentials ranged from 0.50 to 0.64, although conductance ratios greater than 1.2 were observed with high extracellular pH. Chloride did not inhibit HCO3 permeation noticeably but rather had a small stimulatory effect when present on the opposite side of the membrane. The prevalence of outward rectifiers in PANC-1 and their permeability to bicarbonate suggests the channel may have a dual role in HCO3 secretion; to allow Cl recycling at the apical membrane and to mediate some of the HCO3 flux. Defective modulation of this channel in cystic fibrosis might provide a common basis for dysfunction in epithelia having very different anion transport properties (e.g., HCO3 secretion, Cl secretion and Cl absorption.

Bicarbonates↗
PubMed1986

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium.

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.

Animals↗
PubMed1985

Apical and basolateral membrane ionic channels in rabbit urinary bladder epithelium.

This paper reviews the properties and regulation of single amiloride-sensitive Na+ channels in the apical membrane, and Cl- and K+ channels in the basolateral membrane of rabbit urinary bladder. According to fluctuation analysis, there is an average of one amiloride-sensitive Na+ channel for every 40 micron2 of apical membrane. Each Na+ channel passes 0.7 pA of current under normal, short-circuit conditions. Apical channels are hydrolysed by the endogenous enzyme urokinase, which is released into the urine by the kidney. After exposure to urokinase, the Na+ channel loses its amiloride sensitivity, and eventually becomes unstable in the membrane. The selectivity and kinetic properties of single anion and K+ channels in the basolateral membrane were also studied using the patch clamp technique. The properties of these channels are discussed in terms of the regulation of transepithelial Na+ transport.

Amiloride↗
PubMed1985

Single anion-selective channels in basolateral membrane of a mammalian tight epithelium.

Basolateral membrane chloride permeability of surface cells from rabbit urinary bladder epithelium was studied using the patch-clamp technique. Two types of anion-selective channel were observed. One channel type showed inward rectification and had a conductance of 64 pS at-50 mV when bathed symmetrically by saline solution containing 150 mM chloride; the other resembled high-conductance voltage-dependent anion channels (VDACs). Both channels had the selectivity sequence Cl-approximately equal to Br-approximately equal to I- approximately equal to SCN- approximately equal to NO3- greater than F- greater than acetate greater than gluconate greater than Na+ approximately equal to K+ and were sensitive to the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Basolateral chloride conductance in urinary bladder is apparently due to the 64 pS anion channel, which is active at physiological potentials. Imperfect selectivity of this channel against cations might also account for the low, but finite, sodium permeability of the basolateral membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Cellular mechanisms and control of KCl absorption in insect hindgut.

The hindgut of the desert locust possesses an unusual chloride transport system. The isolated locust rectum absorbs chloride from the mucosal (lumen) to the serosal (haemolymph) side at a rate which is equal to the short-circuit current (Isc). Net chloride transport (JClnet) persists in nominally Na-free or HCO3(CO2)-free saline, is insensitive to normal inhibitors of NaCl co-transport and anion exchange, and is independent of the net electrochemical gradient for sodium across the apical membrane. However, active chloride transport is strongly dependent on mucosal potassium (Ka = 5.3 mM-K). Chloride entry across the apical membrane is active, whereas the net electrochemical gradient across the apical membrane is active, whereas the net electrochemical gradient across the basal membrane favours passive Cl exit from the cell. Although mucosal potassium directly stimulates 'uphill' chloride entry, there is no evidence for coupled KCl co-transport, nor would co-entry with potassium be advantageous energetically. Net chloride absorption and Isc are stimulated by a peptide hormone from the central nervous system which acts via cyclic-AMP. Cyclic-AMP increases Isc and JClnet approximately 1000% and transepithelial conductance (Gt) approximately 100%. Approximately half of the delta Gt during stimulation results from increased Cl conductance at the basal cell border. This increase is also reflected in a shift of the basal membrane e.m.f. towards the Nernst potential for chloride. The remainder of the cAMP-induced delta Gt is due to an elevation of apical membrane K conductance, which causes a 400% increase in transepithelial potassium permeability as estimated by radiotracer diffusion. Because of this stimulation of K conductance, potassium serves as the principal counterion for active chloride transport under open-circuit conditions. Very high luminal levels of K oppose the stimulatory actions of cAMP on active Cl transport and K conductance. These and other results have been incorporated into a cellular model for KCl absorption across this insect epithelium.

Animals↗
PubMed1982

Electrogenic, K+-dependent chloride transport in locust hindgut.

Potassium chloride is the major salt recycled in most insect secretory systems. Ion and water reabsorption occur in the rectum by active transport of Cl- and largely passive movement of K+. Both these processes are stimulated several fold by a neuropeptide hormone acting via cyclic AMP (cAMP). This Cl- transport process was investigated by using intracellular ion-sensitive microelectrodes, radiotracer flux measurements, voltage clamping, ion substitutions and inhibitors. the mucosal entry step for Cl- is energy-requiring and highly-selective, and is stimulated directly by cAMP and luminal K+. Under some experimental conditions, measured electrochemical potentials for cations across the mucosal membrane are too small to drive C;- entry by NaCl or KCl cotransport mechanisms; moreover, net 36Cl- flux is independent of the apical Na+ potential. Similarly no evidence for a HCO3- -Cl- exchange was obtained. We conclude that Cl- transport in locust gut is different from mechanisms currently proposed for vertebrate tissues.

Animals↗