Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)

Abstract

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin-luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing-type chamber. We developed a LL pulse protocol to determine the rate of ATP release (R(ATP)) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure R(ATP) as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 microl of A6 cells released ATP at a rate of 66 +/- 8 fmol min(-1). A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H(2)O)(-1) elevated R(ATP) rapidly to a peak value of 1.89 +/- 0.11 pmol min(-1) (R(ATP)(peak)) followed by a plateau phase reaching 0.51 +/- 0.07 pmol min(-1) (R(ATP)(plat)). Both R(ATP)(peak) and R(ATP)(plat) values increased with the degree of dilution. The magnitude of R(ATP)(plat) remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 +/- 0.08 pmol min(-1) was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H(2)O)(-1). This R(ATP) value, induced in the absence of cell swelling, is comparable to R(ATP)(plat). Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose-response curves for peak [Ca(2+)](i) increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with R(ATP)(peak). The link between the ATP release and the fast [Ca(2+)](i) transient was also demonstrated by the depression of both phenomena by Cl(-) removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin-sensitive rise of [Ca(2+)](i) during the hyposmotic shock.

Figure 1. Schematic diagram of the experimental setup for measuring luminescence

The entire filter cup that supports the epithelium is mounted in a holder that separates the apical (Ap) and basolateral (Bl) compartments. Perfusions of each chamber half proceed through black light-tight tubing. The volume of the basolateral compartment was 1 ml. The diffusion of ATP into the basolateral bath through the Anopore filter was accelerated by vigorously mixing the solution with a magnetic stirring bar coupled to a rotating magnet. The volume of the solution in contact with the apical side of the epithelium was 2 ml. The photon counter tube protected by a shutter device was mounted on a support that could easily be lifted to install the filter cup with cells. Changing the perfusates was possible without interrupting photon counting.

Figure 2. Calibration of the ATP measurements

Calibration was performed using the experimental setup depicted in Fig. 1. The luminescence that originated from the standard solution in the basolateral compartment was recorded with a cell free culture cup. The LL reagent was added to the ATP containing solutions at 50 μl ml⁻¹. A, time course of the luminescence signal recorded with hyposmotic solutions (140 mosmol (kg H₂O)⁻¹). The basolateral bath was continuously perfused and flow was interrupted after 5 ml of an LL reagent-containing solution had passed the chamber. During the indicated periods, the increase in luminescence expressed as counts per second was recorded with different amounts of ATP (Q_ATP) ranging from 0.1 to 10 pmol, corresponding to concentrations between 0.1 and 10 nm. B, linear regression of luminescence (photon counts) versus ATP amount (Q_ATP) of the experiment depicted in A.

Figure 4. Pulse protocol

Example of an experiment where the basolateral osmolality (π) was reduced to 140 mosmol (kg H₂O)⁻¹. The basolateral compartment was continuously perfused at a rate of 5 ml min⁻¹ with isosmotic or hyposmotic solutions. The perfusion was interrupted during 90 s intervals after 5 ml of the LL-containing solutions had passed the basolateral bath. R_ATP was calculated from the slope of increase in luminescence and the calibration factor listed in Table 1. The sensitivity and high dynamic range of the photon counter enabled the determination of R_ATP in iso- as well as in hyposmotic conditions.

Figure 3. ATP accumulation in the basolateral compartment during hypotonicity

A, recording of ATP accumulation. Initially, the epithelium was exposed to isosmotic solutions (260 mosmol (kg H₂O)⁻¹) and osmolality was reduced to 140 mosmol (kg H₂O)⁻¹ by removal of NaCl. ATP accumulation was recorded during hypotonicity after addition of the LL reagent and interruption of the perfusion. The first recording was initiated 20 min after imposing the hyposmotic challenge. Accumulation of ATP was monitored for 20 and 30 min periods during the first and subsequent LL exposures, respectively. The rate of ATP release (R_ATP) was determined as the slope of the initial rise in ATP content (Q_ATP) in the chamber compartment. The rate constant of disappearance (D_ATP) was calculated by fitting an exponential function (eqn (4)) to the data. Mean values of R_ATP and D_ATP are listed in Table 2. B, consumption of ATP. The rate constant of ATP consumption ($D_{\text{ATP}}^{\text{C}}$) was determined in cell-free experiments using a Lucite cup to seal the basolateral compartment. Introducing a hyposmotic salt solution containing 2 pmol ATP and the LL reagent in the basolateral compartment rapidly increased luminescence. Subsequently the signal followed an exponential decay. The mean value of the time constant determined from five experiments was 58.0 ± 1.1 min⁻¹. C, model calculations of ATP accumulation based on eqn (4) with R_ATP + 3 pmol min⁻¹ and D_ATP + 0.25 min⁻¹. R_ATP is the slope of the rise in Q_ATP, and the plateau level represents the ratio between R_ATP and D_ATP.

Figure 5. Time course of R_ATP during and after hyposmotic shocks

A, dependence of ATP release on the size of the osmotic perturbation. Effect on R_ATP of reducing the osmolality (π) from 260 to 200, 170, 155 and 140 mosmol (kg H₂O)⁻¹ (symbols for each concentration are shown on the right). During the entire experiment, we perfused the apical side with solutions having an osmolality equal to the osmolality of the basolateral hyposmotic solution. The osmotic perturbation was induced by NaCl removal. Probing of ATP release was performed in isosmotic conditions prior to and after the hypotonic shock, and in hyposmotic conditions at 2, 6, 10, 14, 18 and 37 min after the initiation of the hypotonic shock. Data points are means ± s.e.m. (n = 5). B, time course of the decay of R_ATP after restoring isosmotoc conditions. During the post-hyposmotic period we recorded R_ATP with an higher time resolution. An exponential function was fitted to the data recorded in isosmotic conditions (time constant 10.6 min). The area under the exponential curve amounted to 1.16 pmol.

Figure 6. Changes in [Ca²⁺]_i caused by different degrees of osmotic dilution and exogenous ATP

A, measurement of [Ca²⁺]_i changes caused by a sudden reduction of the osmolality (Δπ). During the entire experiment, apical osmolality was hyposmotic, i. e. equal to the osmolality of the basolateral solution during the hyposmotic shock. Peak values of [Ca²⁺]_i (Δ[Ca²⁺]_i) recorded during the first transient phase associated with the release of Ca²⁺ from intracellular stores were recorded at different Δπ values. Averaged values ± s.e.m. were calculated from three experiments. The inset demonstrates the time course of [Ca²⁺]_i during an osmotic shock of Δπ + 120 mosmol (kg H₂O)⁻¹. The solid bar indicates the duration of the osmotic shock. B, measurement of [Ca²⁺]_i changes caused by adding ATP to the basolateral bath. Cells were incubated in isosmotic solutions and ATP was added to the basolateral perfusate. Exogenous ATP concentrations ([ATP]_exo), ranging from 0.01 to 1000 μm, were tested in different tissues. The inset illustrates a typical experiment demonstrating the time course of [Ca²⁺]_i caused by ATP stimulation. The solid bar marks the presence of ATP. Means ± s.e.m. were calculated from three experiments. C, relationship between the peak value of ATP release ($R_{\text{ATP}}^{\text{Peak}}$) during a hyposmotic shock and the exogenous concentration of ATP ([ATP]_exo) needed to reach the same increase in [Ca²⁺]_i as in response to the specified hyposmotic shock. Values of Δ[Ca²⁺]_i at 140, 155, 170 and 200 mosmol (kg H₂O)⁻¹ were obtained from the data in (A). The values for [ATP]_exo represent the exogenous concentration of ATP that was needed to obtain the same increase of [Ca²⁺]_i as obtained during an hyposmotic shock and could be deduced by interpolation of the data in B. $R_{\text{ATP}}^{\text{Peak}}$ values were obtained from Table 3.

Figure 7. Removal of extracellular Cl⁻ depresses R_ATP and the release of Ca²⁺ from intracellular stores during hypotonic shock

Effect of SO₄^2- for Cl⁻substitution in the apical and basolateral perfusates. In this series of experiments, the reduction of osmolality (π) from 260 to 140 mosmol (kg H₂O)⁻¹ was performed by sucrose removal. A, time courses of R_ATP recorded during hypotonic shock with Cl⁻ and SO₄^2- solutions. Data are means of five experiments. B, intracellular Ca²⁺ concentration recorded in the absence (Control) and presence (+Mg²⁺) of 2 mm Mg²⁺ in the basolateral perfusate. Experiments were performed with SO₄^2- solutions.

Figure 8. Steady ATP release during long term exposure to hyposmotic conditions

A, recording of R_ATP during a hyposmotic shock of 120 min duration. Every 30 min we recorded R_ATP with the pulse protocol as illustrated in Fig. 4 (n = 4). B, recording of R_ATP while cells were in hyposmotic solutions, conditions achieved by gradual reduction of the osmolality (π) at a rate of 1 mosmol (kg H₂O)⁻¹. Data points are means ± s.e.m. (n = 4).