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Fast effects of aldosterone on electrolytes in human lymphocytes are mediated by the sodium-proton-exchanger of the cell membrane
J Mel
Cell
Cardiol
22
(Supplement
I) (1990)
P52
Na+ LOADING AND EFFLUX IN SINGLE RAT VENTRICULAR CELLS: ASSESSMENT WITH SBFI Steven Borzak, Martin Reers, Thomas W. Smith, James D. Marsh. Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115. We used the fluorescent, Nat-sensitive dye SBFI to examine mechanisms of Nat entry and efflux in dissociated rat ventricular myocytes. Quiescent cells were loaded with SBFI-AM and cellular Nat activity at 37' was monitored continuously and nondestructively a the rati (R) of fluorescence emission 1500 nm) at 340/380 nm Et Bt excitation. In Ca - and Mg -free buffer, R (ref$+ ecting a 1 rose 13+6% (mean&SD; n=7) by 3 min. Net entry was likely via L-type Ca channe r s as it was abolished by verapamil (loM). In contrast, in Kt-free buffer R rose 5+1% (n=3) by 3 min with the appearance of rapid, spontaneous beating and diastoli$+shortening; these effects were abolished by Na/Ca exchange blockade with 1 mM Nl . Net efflux of Nat after divalent-free exposure occurred with a T of 76 set and increased somewhat to 108 set in the presence of 1 mM Nl'*+, blZ effl ux in Kt-free buffer was almost abolished and hypercontracture rapidly developed. Thus, SBFI permits study of Nat entry and efflux in single cells without impalement. Net efflux of Nat from a single myocyte follow!ng Nat loading in divalent cation-free buffer is mediated predominantly by the Nh+ pump; when this pathway is inhibited, Na/Ca exchange results in cellular Ca overload.
P53
FAST DIATED
EFFECTS OF ALDOSTERONE ON BY THE SODIUM-PROTON-EXCHANGER
ELECTROLYTES OF
THE
IN HUMAN LYMPHOCYTES CELL MEMBRANE.
ARE
ME-
Martin Wehling, Johannes KLmayr, Karl Theisen. Medizinische Klinik Innenstadt, Universitat MOnchen, 8000 Munich 2, West-Germany Recently, we demonstrated in vitro effects of aldosterone (8) on intracellular sodium, potassium and calcium concentrations and cell volume in human mononuclear leukocytes (HML). As an early event of the cell activation, the effect of A on the Na+/H+-exchanger of the cell membrane was investigated by the Coulter@ counter determination of cell volume in isotonic Na+-propionate. In Na+-propionate alone, HML swell from 7.4 to 7.8 pm in diameter within 30 min. In a dose-dependent manner, A in the incubation medium significantly stimulates this process, HML reached a diameter of 8.0 urn after 30 minutes. From 2 minutes, all differences were significant for 1.4 nM A (n = 28). The Kd-value for this process is 0.025 nM thus being almost two decades below that for the receptor binding of A. This effect had to be attributed to an increased activity of the Na+/H+-exchanger since 400 pM amiloride blocked it completely. These data are the first to demonstrate the Na+/H+-exchanger as a primary target of the mineralocorticoid action in HML and identify amiloride as an antagonist for the primary effects of A. If true also in cardiovascular tissues, the results would explain known vasodilatory effects of newer analogues of amilorlde ( e.g N-ethyl-isopropyl-amiloride).
P54
ROLE OF ATRIAL NATEIURETIC PEPTIDE IN TER MDDULATIOIJ OF KIDMgY (CA2+ + HG2+)ATPase IN CEROHIC DIABgTKS. Dept. Animesh Sahai. Pallab K. Ganguly. of Anatomy, Div. of Cardiovascular Sciences, St. Boniface Get-r. Hosp. Res. Centre. Univ. of Manitoba, Canada R2H 2A6. Atria1 natriuretic peptide (ANP) is known to have an influence on cation transport in various tissues. In an effort to understand whether or not ANP alters the activity of membrane-bound enzyme responsible for cation movement in diabetes, i.v.) and studied 2 and 6 months rats were injected with streptozotocin (65 mg/kg. This was thereafter. Plasma ANP was increased in 2 month diabetic rats. accompanied by increased ANP binding and (Ca2+ t Mg2+)-ATPase activity in renal cortical plasma membranes. In contrast, 6 month diabetic rats showed a decreased level of plasma ANP; ANP binding and (Ca2+ Mg2+)-ATPase were also decreased. MgZt)-ATPase activity in both control and In the presence of ANP, (Ca2+ diabetic membranes was increased in a concentration dependent manner (10-1210-gM). Only in membranes isolated from 6 month old diabetic rats such response Our results indicate that ANP may serve as an important modulator of was absent. Since (Ca2+ t Mg2+)-ATPase serves ( Ca2+ t Mg2+)-ATPase in renal membranes. as a Ca*+ pump maintaining Ca2+ homeostasis in the cell, a decreased activity at a later stage may precipitate diabetic nephropathy. (Supported by Canadian Diabetes Association).
S.18
Cell
Cardiol
22
(Supplement
I) (1990)
P52
Na+ LOADING AND EFFLUX IN SINGLE RAT VENTRICULAR CELLS: ASSESSMENT WITH SBFI Steven Borzak, Martin Reers, Thomas W. Smith, James D. Marsh. Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115. We used the fluorescent, Nat-sensitive dye SBFI to examine mechanisms of Nat entry and efflux in dissociated rat ventricular myocytes. Quiescent cells were loaded with SBFI-AM and cellular Nat activity at 37' was monitored continuously and nondestructively a the rati (R) of fluorescence emission 1500 nm) at 340/380 nm Et Bt excitation. In Ca - and Mg -free buffer, R (ref$+ ecting a 1 rose 13+6% (mean&SD; n=7) by 3 min. Net entry was likely via L-type Ca channe r s as it was abolished by verapamil (loM). In contrast, in Kt-free buffer R rose 5+1% (n=3) by 3 min with the appearance of rapid, spontaneous beating and diastoli$+shortening; these effects were abolished by Na/Ca exchange blockade with 1 mM Nl . Net efflux of Nat after divalent-free exposure occurred with a T of 76 set and increased somewhat to 108 set in the presence of 1 mM Nl'*+, blZ effl ux in Kt-free buffer was almost abolished and hypercontracture rapidly developed. Thus, SBFI permits study of Nat entry and efflux in single cells without impalement. Net efflux of Nat from a single myocyte follow!ng Nat loading in divalent cation-free buffer is mediated predominantly by the Nh+ pump; when this pathway is inhibited, Na/Ca exchange results in cellular Ca overload.
P53
FAST DIATED
EFFECTS OF ALDOSTERONE ON BY THE SODIUM-PROTON-EXCHANGER
ELECTROLYTES OF
THE
IN HUMAN LYMPHOCYTES CELL MEMBRANE.
ARE
ME-
Martin Wehling, Johannes KLmayr, Karl Theisen. Medizinische Klinik Innenstadt, Universitat MOnchen, 8000 Munich 2, West-Germany Recently, we demonstrated in vitro effects of aldosterone (8) on intracellular sodium, potassium and calcium concentrations and cell volume in human mononuclear leukocytes (HML). As an early event of the cell activation, the effect of A on the Na+/H+-exchanger of the cell membrane was investigated by the Coulter@ counter determination of cell volume in isotonic Na+-propionate. In Na+-propionate alone, HML swell from 7.4 to 7.8 pm in diameter within 30 min. In a dose-dependent manner, A in the incubation medium significantly stimulates this process, HML reached a diameter of 8.0 urn after 30 minutes. From 2 minutes, all differences were significant for 1.4 nM A (n = 28). The Kd-value for this process is 0.025 nM thus being almost two decades below that for the receptor binding of A. This effect had to be attributed to an increased activity of the Na+/H+-exchanger since 400 pM amiloride blocked it completely. These data are the first to demonstrate the Na+/H+-exchanger as a primary target of the mineralocorticoid action in HML and identify amiloride as an antagonist for the primary effects of A. If true also in cardiovascular tissues, the results would explain known vasodilatory effects of newer analogues of amilorlde ( e.g N-ethyl-isopropyl-amiloride).
P54
ROLE OF ATRIAL NATEIURETIC PEPTIDE IN TER MDDULATIOIJ OF KIDMgY (CA2+ + HG2+)ATPase IN CEROHIC DIABgTKS. Dept. Animesh Sahai. Pallab K. Ganguly. of Anatomy, Div. of Cardiovascular Sciences, St. Boniface Get-r. Hosp. Res. Centre. Univ. of Manitoba, Canada R2H 2A6. Atria1 natriuretic peptide (ANP) is known to have an influence on cation transport in various tissues. In an effort to understand whether or not ANP alters the activity of membrane-bound enzyme responsible for cation movement in diabetes, i.v.) and studied 2 and 6 months rats were injected with streptozotocin (65 mg/kg. This was thereafter. Plasma ANP was increased in 2 month diabetic rats. accompanied by increased ANP binding and (Ca2+ t Mg2+)-ATPase activity in renal cortical plasma membranes. In contrast, 6 month diabetic rats showed a decreased level of plasma ANP; ANP binding and (Ca2+ Mg2+)-ATPase were also decreased. MgZt)-ATPase activity in both control and In the presence of ANP, (Ca2+ diabetic membranes was increased in a concentration dependent manner (10-1210-gM). Only in membranes isolated from 6 month old diabetic rats such response Our results indicate that ANP may serve as an important modulator of was absent. Since (Ca2+ t Mg2+)-ATPase serves ( Ca2+ t Mg2+)-ATPase in renal membranes. as a Ca*+ pump maintaining Ca2+ homeostasis in the cell, a decreased activity at a later stage may precipitate diabetic nephropathy. (Supported by Canadian Diabetes Association).
S.18