Regulation of Na+-K+-2Cl- cotransport in turkey red cells: the role of oxygen tension and protein phosphorylation

Abstract

1. Na+-K+-2Cl- cotransport (NKCC) was studied in turkey red cells using Na+ dependence or bumetanide sensitivity of 86Rb+ influx to monitor activity of the transporter. 2. Deoxygenation was the major physiological stimulus for NKCC activity: oxygen tensions (PO2) over the physiological range modulated the transporter, with a PO2 for half-maximal activation of about 41 mmHg (n = 3). In air, activity of NKCC was also stimulated by shrinkage and isoproteronol (isoprenaline, 5 microgr;M). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or beta-adrenergic stimulation. 3. Calyculin A, a protein phosphatase inhibitor, stimulated cotransport with a lag of about 5 min. N-Ethylmaleimide (NEM) inhibited cotransport and also blocked the stimulatory effect of calyculin A if administered before calyculin A. Stimulation by calyculin A and deoxygenation were not additive. Staurosporine (2 microM) inhibited deoxygenated-stimulated K+ influxes, but not those stimulated by calyculin A. NEM added during calyculin A stimulation, i.e. during the 5 min lag, caused transport activity to be clamped at levels intermediate between maximal (calyculin A alone) and control. Cells treated with calyculin A alone or with calyculin A followed by NEM were no longer sensitive to volume, isoproteronol or PO2. 4. The results have characterized the interaction between deoxygenation and other stimuli of NKCC activity. They have also shown that it is possible to manipulate the transporter in a reciprocal way to that shown previously for K+-Cl- cotransport.

Figure 4. Effect of calyculin A on K⁺ uptake into oxygenated turkey red cells

Cells were suspended at low haematocrit (about 4%) in oxygenated saline and ⁸⁶Rb⁺ was added at time 0. At the indicated intervals thereafter, aliquots of cells were removed and processed to measure K⁺ uptake (mmol (l cells)⁻¹) against time. After 20 min, calyculin A (100 nM) was added. Ouabain (100 μm) was present in all cases. Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.

Figure 6. Effect of ‘clamping’ on K⁺ influx in turkey red cells

At time 0, aliquots of oxygenated turkey red cells were exposed to (i) calyculin A (Cal; 100 nM) only; (ii) calyculin A followed 3.5 min later by N-ethylmaleimide (NEM; 1 mM) and 4.5 min later by dithiothreitol (DTT; 2 mM); (iii) calyculin A followed 2.5 min later by NEM and 3.5 min later by DTT; or (iv) no drug (control cells). K⁺ influx (mmol (l cells h)⁻¹) was then measured at the indicated times in the presence of ouabain (100 μm). Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.

Figure 1. The effect of oxygen tension on K⁺ influx in turkey red cells

Cells were incubated in tonometers for 15 min at the requisite oxygen tension (P_O2). Aliquots of cells were then removed and diluted 10-fold into saline pre-equilibrated at the same gas tension for measurement of K⁺ influx (mmol (l cells h)⁻¹), in the presence (▵) and absence (▿) of bumetanide (Btn; 10 μm). Bumetanide-sensitive K⁺ influx is also shown (•). Ouabain (100 μm) was present in all cases. Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.

Figure 2. Volume sensitivity of K⁺ influx in oxygenated and deoxygenated turkey red cells

Cells were fully oxygenated (•) or deoxygenated (○) for 30 min in tonometers flushed with O₂ or N₂, respectively. Aliquots were then diluted into different salines, pre-equilibrated at the same gas tension as the tonometers, at the indicated final osmolalities (changed by addition of water or hypertonic sucrose) to alter cell volume anisotonically. K⁺ influx (mmol (l cells h)⁻¹) was then measured. Data are presented as influxes normalized to the values measured in isotonic saline, which were 2.04 ± 0.05 and 29.43 ± 0.39 mmol (l cells h)⁻¹ (means ±s.d., n = 3) in oxygenated and deoxygenated cells, respectively. Ouabain (100 μm) was present in all cases. Vertical dashed line indicates osmolality of isotonic saline. Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.

Figure 3. Isoproteronol sensitivity of K⁺ influx in oxygenated and deoxygenated turkey red cells

Cells were fully oxygenated or deoxygenated for 30 min in tonometers flushed with O₂ or N₂, respectively. Aliquots were then diluted into different salines, pre-equilibrated at the same gas tension as the tonometers, with or without addition of isoproteronol (5 μm). K⁺ influx (mmol (l cells h)⁻¹) was measured 10 min later. Ouabain (100 μm) was present in all cases. Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.

Figure 5. Effect of calyculin A and staurosporine on K⁺ influx in oxygenated and deoxygenated turkey red cells

K⁺ influx (mmol (l cells h)⁻¹) was measured in control cells, under both (i) oxygenated and (ii) deoxygenated conditions; and in deoxygenated cells treated with protein phosphatase and/or protein kinase inhibitors: (iii) calyculin A (Cal; 100 nM) alone, (iv) staurosporine (Stau; 2 μm) alone, and (v and vi) the two in combination with staurosporine added 10 min after calyculin A. Ouabain (100 μm) was present in all cases. Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.

Figure 7. Effect of ‘clamping’ on volume sensitivity of K⁺ influx in turkey red cells

Aliquots of oxygenated turkey red cells were treated in the same way as described in Fig. 6. The osmolality of their saline was then adjusted by addition of water or hypertonic sucrose to alter cell volume anisotonically before measurement of K⁺ influx (mmol (l cells h)⁻¹). Data are presented as influxes normalized to the values measured in isotonic saline - these were 1.10 ± 0.05, 37.75 ± 1.58 and 27.43 ± 0.82 mmol (l cells h)⁻¹ (means ±s.d., n = 3), respectively, for control cells and those treated with calyculin A alone or with calyculin A followed by NEM. Ouabain (100 μm) was present in all cases. Vertical dashed line indicates osmolality of isotonic saline. Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.

Figure 8. Effect of ‘clamping’ on sensitivity of K⁺ influx in turkey red cells to oxygen tension and isoproteronol

Aliquots of oxygenated cells were treated in the same way as described in Fig. 6. They were then either (i) kept in O₂, (ii) incubated in N₂ until fully deoxygenated, or (iii) kept in O₂ but treated with isoproteronol (5 μm) for 10 min, before measurement of K⁺ influx (mmol (l cells h)⁻¹). Data are presented as influxes normalized to the values measured in oxygenated saline - these were 0.8 ± 0.1, 36.5 ± 0.4 and 5.9 ± 0.3 mmol (l cells h)⁻¹ (means ±s.d., n = 3), respectively, for control cells and those treated with calyculin A alone or with calyculin A followed by NEM. Ouabain (100 μm) was present in all cases. Data represent means ±s.d. for triplicate determinations in a single experiment, representative of 3 others.