Acetylcholine-activated chloride current in the T-84 colonic cell line.

The stimulation of epithelial chloride secretion by hormones and neurotransmitters involves the activation of apical membrane chloride channels. The regulation of chloride current by acetylcholine in the T-84 colonic cell line was investigated using single-channel patch-clamp techniques. Treatment with carbachol resulted in the stimulation of transient chloride currents in 18 of 32 previously quiescent patches. Lack of resolvable single-channel openings suggests that single-channel conductance is less than 5-pS. Of 18 responsive patches, 4 showed multiple current oscillations. Treatment of the cells with AlF4- activated sustained chloride currents, suggesting that G proteins are involved. In excised patches, chloride current was markedly sensitive to free Ca2+ concentrations between 500 and 1000 nM. Time-dependent activation and inactivation of chloride current occurred at +60 and -60 mV. These results indicate that the chloride channels responsible for cholinergic activation of chloride conductance in the T-84 colonic cell line are members of the very low conductance family of chloride channels.

Directed case study method for teaching human anatomy and physiology.

A mastery of human anatomy and physiology requires a familiarity with a vast number of details about the human body. A directed method of case analysis is described that helps students deepen and solidify their understanding of anatomical and physiological facts, concepts, and principles. The successful case had four distinctive features as follows: clear learning objectives, a concise and informative scenario, straightforward and didactic questions, and an emphasis on information readily available to the student. A directed case study is presented, and its salient features are described. A procedure for integrating case analyses into an undergraduate anatomy and physiology course is outlined. Student response to this type of case study suggests that this method improves the ease of learning, the depth of learning, and an appreciation of the relevance of and a curiosity about anatomy and physiology. The addition of case analyses to a two-semester integrated course in anatomy and physiology was also associated with an improvement in exam performance. The regular use of directed case analysis is a valuable addition to the traditional methods of lecture, textbook reading, and laboratory for the teaching of human anatomy and physiology.

cAMP-activated Cl channels in CFTR-transfected cystic fibrosis pancreatic epithelial cells.

Retrovirus-mediated transfection of cDNA for the cystic fibrosis (CF) gene into the CF pancreatic cell line, CFPAC-1, confers adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of Cl conductance. We used patch-clamp techniques to identify the single-channel basis of this conductance pathway and to study its properties. Forskolin or cAMP activated Cl channels with a conductance of 9 +/- 1 pS in 26 of 62 cell-attached patches of cystic fibrosis transmembrane conductance regulator (CFTR)-transfected CFPAC-1 cells. The current-voltage (I-V) relation showed slight outward rectification (chord conductance of 10 +/- 2 pS at +80 mV vs. 7 +/- 1 pS at -80mV) with high Cl concentrations (170 mM) in the pipette solution. Channel kinetics were voltage sensitive, with longer openings at positive clamp voltages. Channel properties were unaffected by the substitution of N-methyl-D-glucamine for pipette Na or by the addition of disulfonic stilbenes (100 microM DNDS or DIDS) to the pipette. The channels usually inactivated within seconds of patch excision, but in three of nine patches, activity could be maintained by addition of the catalytic subunit of protein kinase A and ATP. With equal Cl concentrations on both membrane surfaces, the single-channel I-V relation was linear, suggesting that the outward rectification of the cell-attached channel is due to a pipette-to-cell Cl gradient. Anion substitution on the extracellular side of the membrane indicates a halide permselectivity of Br approximately Cl greater than I.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretory renal proximal tubules in seawater- and freshwater-adapted killifish.

A population of proximal tubules when isolated from the glomerular kidneys of seawater-adapted (SW) and freshwater-adapted (FW) killifish (Fundulus heteroclitus) spontaneously secrete fluid. Regardless of SW or FW adaptation, Na and Cl are the dominant electrolytes in secreted fluid. Mg concentrations in fluid secreted by both tubules are significantly greater than those in the peritubular bath, and Mg concentrations are inversely related to Na concentrations. Proximal tubules from either SW or FW fish exhibit low transepithelial voltage (-1 to -2 mV) and low transepithelial resistances (20-30 omega.cm2) typical of other vertebrate proximal tubules. Transepithelial diffusion potentials for Na, Cl, Mg, and SO4 suggest that the paracellular pathway is Na selective and impermeable to divalent ions. Consideration of transepithelial electrochemical potential differences for Na, Cl, Mg, and SO4 suggests active transport of Mg, SO4, and Cl in proximal tubules isolated from SW- and FW-adapted fish. The similarities in the functional properties of secretory proximal tubules isolated from SW- and FW-adapted killifish are striking and raise questions about the in vivo role of these tubules in the renal adaptations to seawater and freshwater.

Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer.

We have used retrovirus-mediated gene transfer to demonstrate complementation of the cystic fibrosis (CF) defect in vitro. Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into CFPAC-1, a pancreatic adenocarcinoma cell line derived from a patient with CF that stably expresses the chloride transport abnormalities characteristic of CF. CFPAC-1 cells were exposed to control virus (PLJ) and CFTR-expressing virus (PLJ-CFTR); viral-transduced clones were isolated and subjected to molecular and physiologic analysis. RNA analysis detected a viral-derived CFTR transcript in all of the PLJ-CFTR clones that contained unrearranged proviral sequences. Agents that increase intracellular cAMP stimulated 125I efflux in PLJ-CFTR clones but not PLJ clones. Whole-cell patch-clamp performed on three responding clones showed that the anion efflux responses were due to cAMP stimulation of Cl conductance. Our findings indicate that expression of the normal CFTR gene confers cAMP-dependent Cl channel regulation on CF epithelial cells.

Separate Cl- conductances activated by cAMP and Ca2+ in Cl(-)-secreting epithelial cells.

We studied the cAMP- and Ca2(+)-activated secretory Cl- conductances in the Cl(-)-secreting colonic epithelial cell line T84 using the whole-cell patch-clamp technique. Cl- and K+ currents were measured under voltage clamp. Forskolin or cAMP increased Cl- current 2-15 times with no change in K+ current. The current-voltage relation for cAMP-activated Cl- current was linear from -100 to +100 mV and showed no time-dependent changes in current during voltage pulses. Ca2+ ionophores or increased pipette Ca2+ increased both Cl- and K+ currents 2-30 times. The Ca2(+)-activated Cl- current was outwardly rectified, activated during depolarizing voltage pulses, and inactivated during hyperpolarizing voltage pulses. Addition of ionophore after forskolin further increased Cl- conductance 1.5-5 times, and the current took on the time-dependent characteristics of that stimulated by Ca2+. Thus, cAMP and Ca2+ activate Cl- conductances with different properties, implying that these second messengers activate different Cl- channels or that they induce different conductive and kinetic states in the same Cl- channel.

A volume-sensitive chloride conductance in human colonic cell line T84.

The chloride-secreting colonic cell line, T84, was studied under whole cell patch clamp with Cl as the permeant ion in pipette and bath solutions. Transmembrane current was initially small (approximately 50 pA at +100 mV) but increased steadily to average values of 1-3 nA within 5-10 min. The development of this current was associated with visible cell swelling, either without a shape change or with membrane blebbing. Basal, preswelling current levels were restored by the addition of 50-75 mM sucrose to the bath or when pipette osmolality was reduced by an equivalent amount. These findings suggest that an isosmotic pipette filling solution behaves as if it is hypertonic by approximately 60 mosmol/kgH2O to the bath. Currents traversing the swelling-induced conductance were outwardly rectified and showed activation at hyperpolarizing voltages and inactivation at depolarizing voltages. They were Cl selective because the reversal potential for current flow approached the Cl equilibrium potential when bath [Cl] was varied. Under nonswelling conditions (bath solution, 300 mosmol/kgH2O; pipette solution, 240 mosmol/kgH2O), single-channel steps (approximately 9 pA at +100 mV) could be resolved. The single-channel characteristics were similar to the macroscopic currents recorded from swollen cells, showing inactivation at positive voltages and an outwardly rectified current-voltage relation. Summation of these single-channel events yielded currents that were similar to those from swollen cells, implying that activation of multiple channels with these properties is the basis of the swelling-induced Cl conductance. This volume-sensitive Cl conductance would contribute to a regulatory volume decrease when T84 cells swell. Its relation to the secretory Cl conductance in these cells is unknown.

Fluid secretion in glomerular renal proximal tubules of freshwater-adapted fish.

Tubular secretion by renal proximal tubules, as a mechanism for delivering fluid and electrolytes to the urine, has received little attention in modern conceptions of renal function in vertebrates even though it is the mechanism for urine production in aglomerular fish. This report demonstrates that some proximal tubules of glomerular kidneys of freshwater-adapted euryhaline fish spontaneously secrete fluid. The fluid consists primarily of Na (138 mM) and Cl (160 mM). NaCl and fluid secretion can be stimulated by adenosine 3',5-cyclic monophosphate, suggesting that tubular fluid secretion is under hormonal control. Fluid secretion driven by NaCl secretion in glomerular proximal tubules of fish that already filter NaCl and water suggests that secretion of fluid and NaCl may play a fundamental role in vertebrate renal function beyond a preadaptation for aglomerular urine formation.

Renal proximal tubule of flounder. I. Physiological properties.

The proximal segment of the winter flounder, Pseudopleuronectes americanus, was investigated. Isolated tubules net secrete fluid, although at low rates, 37 pl X min-1 X mm-1. The dominant ions in secreted fluid are Na and Cl, with [Cl] significantly higher than in the bath. Mg and SO4 concentrations in secreted fluid are more than 10-fold greater than in the bath. The transepithelial voltage (-1.9 mV) and resistance (26 omega X cm2) indicate an electrically leaky epithelium, and transepithelial diffusion potentials suggest the Na selectivity of the paracellular pathway. Transepithelial electrochemical potentials point to active transport of Mg, SO4, and probably also Cl and to transepithelial equilibrium of Na. Failure to observe any secretory transport in tubules perfused in vitro at the commonly used perfusion rates illustrates the necessity of low, preferably subnanoliter, perfusion rates in detecting and studying low-capacity epithelial transport systems by the method of Burg.

Renal proximal tubule of flounder II. Transepithelial Mg secretion.

The kinetics of transepithelial Mg secretion were studied in isolated perfused proximal tubule II of the flounder, Pseudopleuronectes americanus. To detect measurable changes of luminal [Mg] as a function of bath [Mg], the lumen had to be perfused at subnanoliter rates. Transepithelial Mg secretion did not obey first-order reaction kinetics; hence values for half saturation of transport (0.22 mM) and transport maximum (1.5 pmol X min-1 X mm-1) are apparent and suggest a high-affinity low-capacity transport system. Because all experiments were done in the absence of bath SO4, the independence of Mg transport from SO4 transport is established. In the absence of perfusion, when tubules secrete fluid spontaneously, secreted fluid contained Cl (156 +/- 3 mM), Na (130 +/- 6 mM), and Mg (27 +/- 5 mM), all significantly different from the bath. Rates of Cl, Na, and Mg secretion were all positively correlated with fluid secretion, but Na and Mg concentrations in secreted fluid were inversely proportional. The results indicate that NaCl secretion provides basal rates of fluid secretion, and when MgCl2 is secreted in addition, fluid secretion increases with the effect of generating inverse relationships between luminal Na and Mg concentrations.