Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells

Abstract

Elevations of cytoplasmic free calcium concentrations ([Ca(2+)](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl(-) through Ca(2+)-activated Cl(-) channels, while Cl(-) enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl(-) secretion, we analyzed carbachol (CCh)-activated Cl(-) currents in submandibular acinar cells using the "gramicidin-perforated patch recording configuration." Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl(-) currents in the gramicidin-perforated patch recording were carried by Cl(-) efflux via Cl(-) channels, dependent upon Cl(-) entry through Cl(-) transporters expressed in the acinar cells. CCh-evoked oscillatory Cl(-) currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl(-) currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO(3)(-) had significant effects, suggesting that the cotransporter rather than parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers is the primary Cl(-) uptake pathway. Pharmacological manipulation of the activities of the Ca(2+)-activated Cl(-) channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl(-) currents, while adjusting to the rate imposed by the Ca(2+)-activated Cl(-) channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl(-) movements may effectively provide a driving force for fluid secretion in intact acinar cells.

Figure 1.

CCh induces repetitive spikes of Cl⁻ currents associated with the oscillations of membrane potential. (A) CCh-induced Cl⁻ and K⁺ currents in the conventional whole-cell patch configuration were measured in submandibular acinar cells that were voltage clamped and stepped between −80 mV and 0 mV. CCh (500 nM) was added to the perfusate during the period indicated by the horizontal bar. (B) CCh-induced anion current and changes in membrane potential, recorded in the gramicidin-perforated patch configuration. The current and membrane potential were recorded under the voltage- and current-clamp modes, respectively. Thus, by repetitively switching those modes, the current and membrane potential were measured during the period indicated as −80 mV and 0 nA, respectively. CCh (500 nM) was added to the perfusate during the period indicated by the horizontal bar. (C) Representative time-resolved traces of CCh-induced repetitive spikes of inward current associated with the oscillations of membrane potential. (D) The effects of DPC on CCh-induced inward current and changes in membrane potential. CCh (500 nM) and DPC (500 μM) were added to the perfusate during the period indicated by the horizontal bars. (E) Representative traces of the anion current and membrane potential of isolated acinar cells during the application of CCh with the gluconate substitution. CCh (500 nM) was added to the perfusate, and 103 mM Cl⁻ in the extracellular solution was replaced by equimolar amounts of gluconate during the period indicated by the horizontal bars. (F) The effects of charybdotoxin and ouabain on the CCh-induced inward current in the gramicidin-perforated patch recording. CCh (500 nM), charybdotoxin (CTX, 100 nM), and ouabain (500 μM) were added to the perfusate during the period indicated by the horizontal bars. (G) Schematic illustration of acinar cells under the gramicidin-perforated patch configuration. The CCh-activated anion currents in the gramicidin-perforated patch recording are dependent on the Cl⁻ entry transporters, or the production of HCO₃ ⁻. The currently accepted model of the distribution of key anion transport proteins in submandibular acinar cells is also shown. Depicted in the apical membrane is the Ca²⁺-activated anion channel(s). Depicted in the basolateral membrane are a Na⁺-K⁺-2Cl⁻ cotransporter and a Cl⁻-HCO₃ ⁻ exchanger. Also shown is CO₂ diffusing across the basolateral membrane and reacting with H₂O in the presence of carbonic anhydrase (C.A.) to form H⁺ and HCO₃ ⁻.

Figure 2.

The effects of bumetanide and methazolamide on the CCh-induced anion current and oscillations of membrane potential in the gramicidin-perforated patch configuration. (A and C) Representative traces of the anion current and membrane potential of isolated acinar cells during the application of CCh (500 nM), bumetanide (Bum, 500 μM), and methazolamide (Met, 500 μM) added to the perfusate during the period indicated by the horizontal bars. (B and D) Comparison of the CCh-induced anion current and the average membrane potential in the presence of bumetanide and/or methazolamide. The average current and membrane potential in B and D were determined in the final 20 s before switching between the voltage and current clamp modes in the absence (control) or presence of CCh, followed by the sequential addition of bumetanide and/or methazolamide, as indicated in A and C, respectively (n = 3–5). (E) The effects of the addition of Na⁺ to the pipette on the CCh-induced anion current. The CCh-induced anion current was recorded using the pipette solution containing 145 mM KCl, 5 mM NaCl, and 10 mM HEPES (pH 7.4), and the effects of bumetanide (500 μM) and methazolamide (500 μM) on the current were determined. (F) The effect of the external HCO₃ ⁻ depletion on the CCh-induced anion current. CCh (500 nM) and the HCO₃ ⁻-free external solution, in which 25 mM HCO₃ ⁻ was replaced by Cl⁻ and CO₂ was removed, were applied during the period indicated by the horizontal bars. (G) The representative time-resolved traces of the CCh-induced repetitive spikes of inward current in the presence of methazolamide and bumetanide. The traces of the CCh-induced inward currents before (a, closed circles) and after (b, closed triangles) the application of methazolamide, followed by the simultaneous addition of methazolamide and bumetanide (c, open diamonds), were deduced from a single gramicidin-perforated patch recording.

Figure 3.

Comparison of the durations of the oscillatory spikes of CCh-induced inward currents in the presence or absence of bumetanide and methazolamide. (A) Schematic representation of the analyzed durations in the CCh-induced inward currents (in the top panel). The periods of an increasing phase (T_i) and a decreasing phase (T_d) were determined in the various experimental procedures. Also, the durations spent to reach one half of the peak amplitude in the increasing phase (T_i(1/2)) and the decreasing phase (T_d(1/2)) were calculated. The lower panels show the representative traces employed to analyze the oscillatory events of the CCh-induced inward current before and after the application of methazolamide (500 μM), followed by the simultaneous addition of methazolamide and bumetanide (500 μM). (B) Comparison of T_i(1/2), T_i, T_d(1/2), T_d, T_i(1/2)/T_i, T_d(1/2)/T_d, T_d/T_i, and T_d(1/2)/T_i(1/2) in the CCh-induced repetitive spikes of inward currents from the isolated acinar cells in the presence or absence of methazolamide and bumetanide. Results are presented as means ± SEM (n = 11).

Figure 4.

Effect of genistein on NKCC activity and the CCh-induced Cl⁻ currents. (A) Representative pH_i traces from isolated acinar cells challenged with NH₄Cl. CCh (500 nM) and NH₄Cl (30 mM) were added to the perfusate during the period indicated by the horizontal bars. The traces were from the acinar cells with (the thin line) or without (closed circles) bumetanide (Bum, 500 μM) added 20 s before the application of NH₄Cl. (B) Representative pH_i traces from isolated acinar cells preincubated with genistein and challenged with 30 mM NH₄Cl. CCh (500 nM), genistein (Gen, 100 μM), and NH₄Cl (30 mM) were added to the perfusate during the period indicated by the horizontal bars. The traces were from the acinar cells with (the thin line) or without (closed circles) bumetanide (500 μM) added 20 s before the application of NH₄Cl. (C) Comparison of the initial rates of pH_i recovery from an NH₄ ⁺-induced alkaline load, determined in CCh-pretreated acinar cells in the presence or absence of genistein. The initial recovery rates were calculated from pH_i traces indicated as A and B, as described in materials and methods. Results are presented as means ± SEM of the initial recovery rates from CCh-pretreated cells (n = 27), CCh- and Bum-pretreated cells (n = 8), CCh- and Gen-pretreated cells (n = 14), and CCh-, Gen-, and Bum-pretreated cells (n = 9). (D) The effect of genistein on the CCh-induced Cl⁻ and K⁺ currents in the conventional whole-cell patch configuration. (E) The effect of genistein on the CCh-induced anion current recorded under the gramicidin-perforated patch configuration. CCh (500 nM), genistein (Gen, 100 μM), and bumetanide (Bum, 500 μM) were added to the perfusate during the period indicated by the horizontal bars. (F) Comparison of the effect of genistein on the CCh-induced anion currents under either the conventional whole-cell patch or the gramicidin-perforated patch configurations. The relative currents before (black columns) and after (white columns) the addition of genistein were determined in the final 20 s before changing solutions. The relative currents are expressed as a percentage of the anion current induced by CCh alone under either the conventional whole-cell patch or the gramicidin-perforated patch configurations (n = 3).

Figure 5.

Effect of NPPB on the CCh-induced Cl⁻ currents. (A) Representative trace showing the effect of 4 μM NPPB on the CCh-induced Cl⁻ current in the conventional whole-cell patch configuration. (B) Representative trace showing the effect of 4 μM NPPB on the CCh-induced anion current recorded under the gramicidin-perforated patch configuration. CCh (500 nM), NPPB (4 μM), and bumetanide (Bum, 500 μM) were added to the perfusate during the period indicated by the horizontal bars. (C) Comparison of NPPB sensitivity of the CCh-induced anion currents under either the conventional whole-cell patch (black columns) or the gramicidin-perforated patch configurations (white columns). The relative currents after the addition of the various concentrations of NPPB were determined from the current traces as shown in A and B. The relative currents are expressed as a percentage of the anion current induced by CCh alone under either the conventional whole-cell patch or the gramicidin-perforated patch configurations (n = 3 or 4).