Osmosensitive C1- currents and their relevance to regulatory volume decrease in human intestinal T84 cells: outwardly vs. inwardly rectifying currents

Abstract

1. The swelling-activated outwardly rectifying Cl- current (ICl(swell)) recorded in T84 human intestinal cells was completely blocked by 10 microM tamoxifen, while 300 microM Cd2+ had no effect. 2. A ClC-2-like, inwardly rectifying Cl- current was activated after strong hyperpolarization in T84 cells. This current was completely inhibited by 300 microM Cd2+, unaffected by 10 microM tamoxifen, and its magnitude increased slightly in response to cell swelling under hyposmotic conditions. However, the swelling-dependent modulation occurred only after prior activation by hyperpolarizing voltages. 3. T84 cells behaved initially close to perfect osmometers in response to changes in external osmolalities between +20 and -30 %. The cells underwent full regulatory volume decrease (RVD) within 16 min when exposed to 30 or 10 % hyposmotic shocks. 4. Pharmacological tools were used to determine the anionic pathway(s) involved in RVD in T84 cells. Tamoxifen (10 microM), 1,9-dideoxyforskolin (DDFSK; 100 microM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 microM) blocked RVD while 300 microM Cd2+ had no effect upon RVD following a 30 % hyposmotic shock. The RVD response was similarly unaffected by Cd2+ when cells were exposed to a smaller (10 %) hyposmotic shock. 5. In conclusion, these data show that the anionic pathway primarily activated by cell swelling and relevant to RVD in T84 cells is the tamoxifen-, DDFSK- and DIDS-sensitive ICl(swell) and not the hyperpolarization-activated, Cd2+-sensitive Cl- current associated with the ClC-2 Cl- channel.

Figure 1. Cd²⁺ and tamoxifen sensitivity of I_Cl(swell) in T₈₄ cells

A, Cl⁻ currents recorded under isosmotic (Iso) conditions. B, swelling-activated outwardly rectifying Cl⁻ currents appeared after exposure to a 20 % hyposmotic (Hypo) bathing solution. Currents were measured after 3 min in hyposmotic solution. C, addition of 300 μm CdCl₂ to the bath had no effect upon I_Cl(swell) (measured after 3 min in CdCl₂). However, addition of 10 μm tamoxifen (D) immediately blocked this current (measured 1 min after addition of tamoxifen). Current traces were generated by holding the potential at 0 mV and pulsing from -80 to +80 mV in 40 mV steps, each step lasting 500 ms.

Figure 2. Cd²⁺ and tamoxifen sensitivity of ClC-2 currents in T₈₄ cells

A, current traces generated by holding the cell at 0 mV and then pulsing from -100 to +40 mV in 20 mV steps (10 s duration) every 20 s. B, hyperpolarization-activated inwardly rectifying Cl⁻ currents activated after holding the cell for 30 s at -120 mV. C, addition of 300 μm CdCl₂ to the bathing solution caused an immediate block of this current, which could be reversed upon removal. Current measured within 3 min in CdCl₂. D, addition of 10 μm tamoxifen to the bath had no effect upon these currents (measured within 3 min in tamoxifen). Intracellular solution as described in Methods but without ATP.

Figure 3. Voltage-dependent activation of ClC-2 currents under mild hyperosmotic gradient

A, currents recorded immediately after breaking into the whole-cell configuration. B and C, development of ClC-2 currents after repetitive hyperpolarization to -120 mV (every 35 s). The osmolality of the extracellular solution was raised to 325 mosmol kg⁻¹ by the addition of mannitol.

Figure 4. Osmosensitivity of ClC-2 currents in T₈₄ cells

A, Cl⁻ currents recorded immediately after breaking into the whole-cell configuration in a T₈₄ cell. B, ClC-2 currents were activated by the repetitive application of voltage pulses to -120 mV every 20 s. C, maximal activation of ClC-2 current was obtained within 6 min. D, ClC-2 current measured at -80 mV increased upon exposure to hyposmotic conditions. E, ClC-2 currents in D shown on an expanded time scale covering the first 600 ms of the current. Experiments were carried out with ATP-free intracellular solution and bathing solution containing 100 μm DDFSK. Currents were measured in response to 5 s pulses to -80 and +40 mV.

Figure 5. Relative contribution of I_Cl(swell) and ClC-2 currents to the total swelling-activated Cl⁻ current in T₈₄ cells

ClC-2 currents were negligibly low immediately after breaking into the whole-cell configuration (A) but were activated by repetitive application of the voltage pulse shown (B and C). D, addition of a hyposmotic bathing solution. E, superfusion with a hyposmotic solution containing 5 μm tamoxifen.

Figure 6. Osmotic behaviour of T₈₄ cells in response to anisosmotic challenges

Phase-contrast images of an individual T₈₄ cell in isosmotic solution (A), 1 min after exposure to a 30 % hyposmotic bathing solution (B) and after 16 min in hyposmotic solution (C). Scale bar, 6.5 μm. D, relationship between peak changes in the relative volume (V_T/V₀) of the cell and the reciprocal of the relative external osmotic pressure (π₀/π_T) is shown for the various osmotic solutions used (20 % hyperosmotic, n= 3; 10 % hyposmotic, n= 5; 30 % hyposmotic, n= 20). The dashed line represents the predicted behaviour of a perfect osmometer. E, relative changes in cell volume of individual T₈₄ cells were measured before and after replacement (at 0 min) of the isosmotic bathing solution with a 30 % hyposmotic bathing solution (n= 24). Values are means ±s.e.m.

Figure 7. Effects of tamoxifen, DDFSK, DIDS and Cd²⁺ on RVD after a 30 % hyposmotic shock

The indicated Cl⁻ channel blocker was added 3 min prior to the addition (at 0 min) of the hyposmotic bathing solution which also contained the appropriate blocker. A, 10 μm tamoxifen (n= 6); B, 100 μM DDFSK (n= 8); C, 100 μm DIDS (n= 4); and D, 300 μM CdCl₂ (n= 9). Values are means ±s.e.m. P < 0.05 at 6 and 16 min for tamoxifen, DDFSK and DIDS with respect to the control values plotted in Fig. 6E. All values in the presence of tamoxifen, DDFSK and DIDS were significantly different from the corresponding controls. For Cd²⁺, P= 0.3 and 0.35 at 6 and 16 min, respectively.

Figure 8. Effect of Cd²⁺ on RVD after a 10 % hyposmotic shock

A, relative changes in cell volume measured before and after the replacement (at 0 min) of the isosmotic bathing solution with a 10 % hyposmotic solution (n= 5). B, cells were pretreated with 300 μm CdCl₂ before replacement with a hyposmotic solution also containing CdCl₂ (n= 3). Values are means ±s.e.m.