Time course of sodium-induced Na+-K+-ATPase in rabbit cortical collecting tubule N. COUTRY,
M. BLOT-CHABAUD,
P. MATEO,
J. P. BONVALET,
recruitment
AND N. FARMAN
Institut National de la Santk et de la Recherche Mkdicale Unit6 246, Unit6 d%nseignement et de Recherche Xavier Bichat, 75877 Paris Cedex 18, France Coutry, N., M. Blot-Chabaud, P. Mateo, J. P. Bonvalet, and N. Farman. Time course of sodium-induced Na+-K+ATPase recruitment in rabbit cortical collecting tubule. Am. J. Physiol. 263 (Cell Physiol. 32): C61-C68, 1992.-In cortical collecting tubules (CCD) of aldosterone-repleted rabbit kidney, an increase in intracellular sodium concentration (NaJ induces the recruitment and/or activation of latent Na+-K+-ATPase pumps (Blot-Chabaud et al., J. Biol. Chem. 265: 11676-11681, 1990). The present study was addressed to determine the time course of this Nai-dependent pump recruitment and to examine some of the factors possibly involved in this phenomenon. CCD from adrenalectomized rabbits complemented with aldosterone and dexamethasone were incubated at 4°C either in a K+-free saline solution (Na+-loaded CCD) or in a sucrose solution (control CCD) and then rewarmed for various time periods to allow pump recruitment to occur. The number of pumps in the membrane was determined by specific [3H]ouabain binding; Nai was measured using 22Na. A rise in Na; induced a threefold increase in the number of basolateral pumps, which was fully achieved within l-2 min. This pump recruitment was reversible within 15 min after restoration of low Nai. It was unaffected by inhibitors of cytoskeleton and Ca2+ ionophore A 23187. The blocker of the Na+-H+ antiporter, amiloride, did not prevent it. The protein kinase C activator, phorbol12-myristate 13-acetate, did not induce it in the absence of Na+. We conclude that Nai is a major determinant of pump recruitment and/or activation, which occurs over a very short period of time. It may constitute a rapid adaptative response to an increase in the cell Na+ load. aldosterone; ouabain binding; intracellular sodium concentration; cytoskeleton; phorbol 12-myristate 13-acetate; amiloride; A23187 A MAJOR ROLE in Na+ reabsorption and K+ secretion by the cortical collecting tubules (CCD) of the mammalian kidney is played by Na+-K+-ATPase pumps (22). Aldosterone stimulates Na+ reabsorption in these tubular segments (36). On one hand, it increases cell Na+ entry across the apical membrane via amiloride-sensitive Na+ channels (31). On the other hand, it stimulates the neosynthesis of Na+-K+-ATPase pumps (18), which extrude Na+ across the basolateral membrane. Recently, we (4) and others (2) demonstrated that, in the presence of aldosterone, a rise in cell Na+ induces the recruitment and/or activation of latent pumps at the basolateral membrane of rabbit and rat CCD cells. The aim of the present study was to establish precisely the time course of this pump recruitment. For this purpose, the number of pumps inserted in the basolatera1 membrane, as determined by specific [3H]-labeled ouabain binding, was determined as a function of intracellular Na+ concentration (Nai). Measurements were done after various periods of pump recruitment in previously Na+-loaded CCD of rabbit kidney. In addition, the reversibility of this phenomenon and the effects of various factors likely to influence pump recruitment were examined. 0363-6143/92
$2.00
Copyright
Na+-induced recruitment and/or activation of latent pumps occurred within l-2 min, suggesting that this phenomenon may constitute a rapid adaptative response to an increase in cell Na+ load. Pump recruitment was rapidly reversed after cell Na+ lowering. It was unaffected by inhibitors of cytoskeleton, stimulation of the protein kinase C (PKC), or an increase in cell Ca2+ concentration. Furthermore, the inhibition of Na+-H+ antiporter did not prevent it. MATERIALS AND METHODS Experiments were performed on bilaterally adrenalectomized female New Zealand White rabbits (1.0-1.2 kg body wt) complemented with both aldosterone and dexamethasone using osmotic minipumps (Alzet 2001, Alza, Palo Alto, CA). Adrenalectomy was done under anesthesia (80% nitrogen peroxide-20% oxygen in halothane) through an abdominal incision. After surgery, rabbits were continuously infused either with aldosterone (3 pg. 100 g body wt-l *day-l) and dexamethasone (1 ,ug*100 g body wt-l *day+) or with dexamethasone only. They were fed a standard laboratory diet and had free access to tap water. Experiments were performed 5-7 days after surgery. Microdissection
of CCD
Microdissection was performed aspreviously described(12). Animals were injected with heparin (250 U iv) 5 min before death. They were killed by a blow behind the neck. Blood (5 ml) was collected from the aorta to determine plasma aldosterone concentration (Radioimmunoassay, Oris Industrie, Saclay, France). The left kidney was first perfused with an ice-cold rinsing solution containing (in mM) 137 NaCl, 5 KCl, 0.8 MgS04, 0.33 Na,HPO,, 0.44 KH2P04, 1 MgC12,1 CaCl,, 5 D-ghcase, and 10 tris(hydroxymethyl)aminomethane (Tris) HCl, pH 7.4, via the renal artery. It then wasperfusedwith a collagenasesolution, which was identical to the rinsing solution except that 0.1% collagenase(0.85 U/mg; Serva, Heidelberg, Germany) was added,under pressureuntil rupture of the renal capsule.Thin pyramid pieces were cut and incubated in the presenceof 0.05% collagenaseat 30°C for 1 h. Pyramids were rinsed in ice-cold microdissectionsolution. Dissection of CCD was performed at 4°C in either saline solution without potassium [in mM: 139 NaCl, 10 NaHC03, 0.8 MgS04, 5 D-glucose, 1 MgC12, 1 CaCl,, 1 alanine, and 20 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-Tris, pH 7.41or sucrosesolution (similar to the previous one except that NaCl and NaHC03 were replaced by 250 mM sucrose) complemented with 0.1% bovine serumalbumin (BSA; Sigma,St. Louis, MO). Thesetwo solutionswill be referred to asK+-free salinesolution and sucrosesolution, respectively, throughout the text. l
Protocol 1: Time Course of Total pH]Ouabain at 37OC in Presence of Vanadate
Binding
We have previously shownthat the time required to obtain a plateau value of [3H]ouabain binding is - IO min at 20°C in the presenceof 2 x 10V5 M [3H]ouabain (4). To study the time dependenceof Nai-dependent pump recruitment within time
0 1992 the American
Physiological
Society
C61
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 11, 2019.
C62
RAPID
RECRUITMENT
OF NA+-K+-ATPASE
frames
M. BLOT-CHABAUD,
P. MATEO,
J. P. BONVALET,
recruitment
AND N. FARMAN
Institut National de la Santk et de la Recherche Mkdicale Unit6 246, Unit6 d%nseignement et de Recherche Xavier Bichat, 75877 Paris Cedex 18, France Coutry, N., M. Blot-Chabaud, P. Mateo, J. P. Bonvalet, and N. Farman. Time course of sodium-induced Na+-K+ATPase recruitment in rabbit cortical collecting tubule. Am. J. Physiol. 263 (Cell Physiol. 32): C61-C68, 1992.-In cortical collecting tubules (CCD) of aldosterone-repleted rabbit kidney, an increase in intracellular sodium concentration (NaJ induces the recruitment and/or activation of latent Na+-K+-ATPase pumps (Blot-Chabaud et al., J. Biol. Chem. 265: 11676-11681, 1990). The present study was addressed to determine the time course of this Nai-dependent pump recruitment and to examine some of the factors possibly involved in this phenomenon. CCD from adrenalectomized rabbits complemented with aldosterone and dexamethasone were incubated at 4°C either in a K+-free saline solution (Na+-loaded CCD) or in a sucrose solution (control CCD) and then rewarmed for various time periods to allow pump recruitment to occur. The number of pumps in the membrane was determined by specific [3H]ouabain binding; Nai was measured using 22Na. A rise in Na; induced a threefold increase in the number of basolateral pumps, which was fully achieved within l-2 min. This pump recruitment was reversible within 15 min after restoration of low Nai. It was unaffected by inhibitors of cytoskeleton and Ca2+ ionophore A 23187. The blocker of the Na+-H+ antiporter, amiloride, did not prevent it. The protein kinase C activator, phorbol12-myristate 13-acetate, did not induce it in the absence of Na+. We conclude that Nai is a major determinant of pump recruitment and/or activation, which occurs over a very short period of time. It may constitute a rapid adaptative response to an increase in the cell Na+ load. aldosterone; ouabain binding; intracellular sodium concentration; cytoskeleton; phorbol 12-myristate 13-acetate; amiloride; A23187 A MAJOR ROLE in Na+ reabsorption and K+ secretion by the cortical collecting tubules (CCD) of the mammalian kidney is played by Na+-K+-ATPase pumps (22). Aldosterone stimulates Na+ reabsorption in these tubular segments (36). On one hand, it increases cell Na+ entry across the apical membrane via amiloride-sensitive Na+ channels (31). On the other hand, it stimulates the neosynthesis of Na+-K+-ATPase pumps (18), which extrude Na+ across the basolateral membrane. Recently, we (4) and others (2) demonstrated that, in the presence of aldosterone, a rise in cell Na+ induces the recruitment and/or activation of latent pumps at the basolateral membrane of rabbit and rat CCD cells. The aim of the present study was to establish precisely the time course of this pump recruitment. For this purpose, the number of pumps inserted in the basolatera1 membrane, as determined by specific [3H]-labeled ouabain binding, was determined as a function of intracellular Na+ concentration (Nai). Measurements were done after various periods of pump recruitment in previously Na+-loaded CCD of rabbit kidney. In addition, the reversibility of this phenomenon and the effects of various factors likely to influence pump recruitment were examined. 0363-6143/92
$2.00
Copyright
Na+-induced recruitment and/or activation of latent pumps occurred within l-2 min, suggesting that this phenomenon may constitute a rapid adaptative response to an increase in cell Na+ load. Pump recruitment was rapidly reversed after cell Na+ lowering. It was unaffected by inhibitors of cytoskeleton, stimulation of the protein kinase C (PKC), or an increase in cell Ca2+ concentration. Furthermore, the inhibition of Na+-H+ antiporter did not prevent it. MATERIALS AND METHODS Experiments were performed on bilaterally adrenalectomized female New Zealand White rabbits (1.0-1.2 kg body wt) complemented with both aldosterone and dexamethasone using osmotic minipumps (Alzet 2001, Alza, Palo Alto, CA). Adrenalectomy was done under anesthesia (80% nitrogen peroxide-20% oxygen in halothane) through an abdominal incision. After surgery, rabbits were continuously infused either with aldosterone (3 pg. 100 g body wt-l *day-l) and dexamethasone (1 ,ug*100 g body wt-l *day+) or with dexamethasone only. They were fed a standard laboratory diet and had free access to tap water. Experiments were performed 5-7 days after surgery. Microdissection
of CCD
Microdissection was performed aspreviously described(12). Animals were injected with heparin (250 U iv) 5 min before death. They were killed by a blow behind the neck. Blood (5 ml) was collected from the aorta to determine plasma aldosterone concentration (Radioimmunoassay, Oris Industrie, Saclay, France). The left kidney was first perfused with an ice-cold rinsing solution containing (in mM) 137 NaCl, 5 KCl, 0.8 MgS04, 0.33 Na,HPO,, 0.44 KH2P04, 1 MgC12,1 CaCl,, 5 D-ghcase, and 10 tris(hydroxymethyl)aminomethane (Tris) HCl, pH 7.4, via the renal artery. It then wasperfusedwith a collagenasesolution, which was identical to the rinsing solution except that 0.1% collagenase(0.85 U/mg; Serva, Heidelberg, Germany) was added,under pressureuntil rupture of the renal capsule.Thin pyramid pieces were cut and incubated in the presenceof 0.05% collagenaseat 30°C for 1 h. Pyramids were rinsed in ice-cold microdissectionsolution. Dissection of CCD was performed at 4°C in either saline solution without potassium [in mM: 139 NaCl, 10 NaHC03, 0.8 MgS04, 5 D-glucose, 1 MgC12, 1 CaCl,, 1 alanine, and 20 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-Tris, pH 7.41or sucrosesolution (similar to the previous one except that NaCl and NaHC03 were replaced by 250 mM sucrose) complemented with 0.1% bovine serumalbumin (BSA; Sigma,St. Louis, MO). Thesetwo solutionswill be referred to asK+-free salinesolution and sucrosesolution, respectively, throughout the text. l
Protocol 1: Time Course of Total pH]Ouabain at 37OC in Presence of Vanadate
Binding
We have previously shownthat the time required to obtain a plateau value of [3H]ouabain binding is - IO min at 20°C in the presenceof 2 x 10V5 M [3H]ouabain (4). To study the time dependenceof Nai-dependent pump recruitment within time
0 1992 the American
Physiological
Society
C61
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 11, 2019.
C62
RAPID
RECRUITMENT
OF NA+-K+-ATPASE
frames
