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Angiotensin increases microsomal (Na+−K+-ATPase activity in several tissues
BIOCHIMICAET BIOPHYSICAACTA
401
BBA Report BBA 21335
Angiotensin increases microsomal (Na+-K +)-ATPase activity in several tissues Y. GUTMAN,Y. SHAMIR, D. GLUSHEVITZKYand S. HOCHMAN Department o f Pharmacology, The Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(Received May 24th, 1972)
SUMMARY
Angiotensin increased microsomal (Na +-K+)-activated ATPase from rat hypothalamus, mucosa of colon and bovine adrenal cortex but not brain cortex. Angiotensin did not change significantly Mg/+-ATPase in these tissues. Enhancement by angiotensin was evident at concentrations of 10- 8 - 1 0 -12 M. In bovine adrenal cortex angiotensin increased ATPase activity in the outer and not in the inner layer. Angiotensin increased ATPase activity with Na+ above 20 mM and K + above 5 mM present simultaneously, but not with either ion alone.
Angiotensin exerts both a pressor vascular effect I and an increase of aldosterone secretion by the adrenal glands 2. The basic mechanism underlying these effects, however, has not been elucidated. Recently, several other functions have been ascribed to the renin-angiotensin system. Thirst evoked by hypovolemia or by isoprenaline injection has been shown to be mediated by renin released from the kidney 3-8. Angiotensin exerts a direct effect on the hypothalamus resulting in increased drinking 9 and increased release of antidiuretic hormone 1°' 11. Angiotensin has also been shown to au~nent net transport of Na+ and fluid across the rat intestinal mucosa, particularly the colon 12. Corticosterone production in the adrenal is enhanced by angiotensin 13. On the other hand, corticosterone production in calf adrenal is suppressed by ouabain which inhibits (Na+-K+)-activated ATPase 14. A possible relation of angiotensin to Na + transport has been reported by Ttirker et al. ~s, viz. increased Na+ efflux from isolated carotid arteries and from uterus in the presence of angiotensin. Levin has found increased (Na+-K+)-ATPase activity in kidney cortex microsomes in the presence of angiotensin (at a concentration of 10-6 M) 16. It seemed of interest, therefore, to study the effect of angiotensin on microsomal Biochim. Biophys. Acta, 273 (1972) 401-405
C L~
--2
Bovine adrenal cortex (N = 13)
(N = 5)
Rat colon
( N = 7)
Rat brain cortex
Rat hypothalamus (N= 7)
Tissue
5.5+-0.62
23.4*0.9
1.45±0.30
7.3+_0.6
43.3±3.3
22.2*4.5
17.6±l.8
15.9*2.3
(~#
('Ya + K+j-activated A TPase
MgZ+-A TPase
10-"
10-' 2
1(I ~
10 -~
A ttgiotensiJ, cone
Activit)' {~moles Pi/mg protein per h)
(1' < 0.01)
0.78*0.2 t
• IP < 0.05)
3.3±0.9
(n.s,I
0.3+-1.0
6.85' 1.85 (P = 0.01 )
Ulnole~ t i/mg prou
)
.
per h
53.7
45.2
0.7
30.8
.
~:
Increase o f (.\'a + -K+)-actirated A 7"Pase
N = number of experiments;P values: for the difference between (Na+-K+)-ATPase activity in the presence and absence of angiotensin, n.s. = difference not statistically significant. No significant difference in Mg2+-ATPase activity was observed in the presence and absence of angiotensin.
EFFECT OF ANGIOTENSIN ON MICROSOMAL ATPase ACTIVITY
TABLE I
©
b~
BBA REPORT
403
(Na+-K+)-activated ATPase in several tissues which show physiologically measurable effects of this peptide. Rat hypothalanms, rat brain cortex and mucosal scrapings of rat colon were taken from animals nephrectomized 24 h previously. Bovine adrenals were obtained from the slaughterhouse, placed immediately in ice, and brought 1o the laboratory where cortex and medulla were separated. The various tissues were homogenized as described previously ~v in 10 vol. of an ice-cold medium of 0.25 M sucrose with 2 mM EDTA and centrifuged at 1000 × g for 10 rain. The supernatant was centrifuged at 10 000 X g for 10 rain, and the supernatant of this centrifugation was spun for 30 rain at 105 000 X g. The pellet obtained was resuspended in 0.25 M sucrose and served as microsoma! enzyme suspension. Assay of ATPase was carried out by incubating 0.2 ml of the enzyme suspension (containing approximately 0.5 nrg protein) in 3 ml of medium, consisting of 100 mM NaCI, 10 mM KC1, 4 mM MgCI2,4 mM ATP and either with or without ouabain (final concentration 10 - 3 M)at 37 °C for 20 rain. Inorganic phosphate (Pi) liberated was measured according to the method of Baginski and Zak ~s and protein according to the method of Lowry et al. ~9. Angiotensin (Val-5-hypertensin ll-Asp-/3 amide, obtained from CIBA) was added as indicated below. Tire difference in activity in the presence and absence of ouabain is taken as (Na + K+)-activated ATPase while the activity persisting in the presence of ouabain is expressed as Mg2+-ATPase. Table I shows that microsomal (Na + K +)-activated ATPase from rat hypothalamus was markedly enhanced in the presence of angiotensin, whereas no mthancement was seen in rat cortex following addition of angiotensin. A significant increase of (Na + K+)-ATPase activity was also observed in rat colon microsomes upon addition of angiotensin (Table I). Microsomal (Na + K+)-ATPase activity from bovine adrenal cortex was also enhanced in the presence of angiotensin (Table 11). The enhancement by angiotensin was
TABLE II I:FFECTOFANGIOTENSINON MICROSOMAL(Na+ K+)-ATPaseACTIVITYOFBOVINF ADRENAL CORTEX Fresh adrenal glands were cut to separate medulla from cortex. The adrenal cortex was then divided into an outer and an inner layer. The two portions were homogenized, prepared and assayed as described in the text (see also ref. 17). Angiotensin was added at a final concentration of 10 9 M. A"= number of experiments. P: for difference between activity in the presence and absence of angiotensin, n.s. = difference not statistically significant. l;*c'rease of (Na + K+)-ATPase activity hy angiotensin (#moles Piling protein per h) Outer layer of adrenal cortex (N = 24)
0.74_+0.26
(P < 0.01 )
Inner layer of adrenal cortex (N = 24)
0.29_+0.27 (n.s.) Biochim. Biophys. Acta, 273 (1972) 401 -405
404
BBA REPORT
specific for the outer portion of the adrenal cortex, while microsomes from the inner layer of the cortex o f the adrenal showed no activation o f (Na + K+)-ATPase in the presence of angiotensin, as seen in Table I1. F u r t h e r m o r e , the increase of (Na + - K + ) - A T P a s e activity by angiotensin was evident only in the presence of Na + c o n c e n t r a t i o n s above 20 mM and with K + c o n c e n t r a t i o n s higher than 5 mM. In the presence of either Na + or K + alone, even at I00 mM, angiotensin showed no significant a u g m e n t a t i o n of ( N a + - K + ) - A T P a s e activity (Fig. 1 ).
0.50 o
I,,..
1:3. Cn
E
i
o~
I
l
I
5
10
20
[K'I
1.50 -
13... Ul
1.00
o
E
0.50
1
I
1
25
50
I [Na÷] 100
mM Fig 1. Effect of angiotensin on microsomal (Na + K+)-ATPase activity of bovine adrenal cortex at different concentrations of Na+ and K +. Ordinate: increase of (Na+-K+)-ATPase activity in presence o f 10 - 9 M angiotensin. Vertical bars: S.E. Lower part: incubation at [K +] = 100 mM and varying [Na +] ; N = 20. At [Na +] = 50 mM, P < 0.02 ; for [ Na+] = 100 raM, P < 0.01. Upper part: incubation at [Na +] = 100 mM and varying [K+] ;N = 15. For [K +] = 10 mM and 20 mM,P < 0.01. At all other concentrations of Na + or K+ the increase of (Na + K+)-ATPase in the presence of angiotensin was not statistically significant.
These experiments demonstrate that angiotensin can enhance significantly the activity of microsomal (Na + K+)-ATPase from three different tissues: rat h y p o t h a l a m u s , rat colon and bovine adrenal cortex. It is of interest that in rat brain cortex, where n o physiological effect o f angiotensin has been d e m o n s t r a t e d , there was also no e n h a n c e m e n t of ( N a + - K + ) - A T P a s e activity in the presence of angiotensin (Table 1). F u r t h e r m o r e , it is n o t e w o r t h y that the c o n c e n t r a t i o n of angiotensin affecting (Na + K+)-ATPase activity was rather low. In the case of rat colon, the e n h a n c e m e n t of fluid transport across the Biochim. Biophys. Acta, 273 (1972) 401-405
BBA REPORT
405
mucosa reported by Davis et al. ~ 2, was obtained at similar angiotensin concentrations ( 1 0 - 1 ° - 1 0 -12 M). In the adrenal cortex the effect o f angiotensin on (Na+-K+)-ATPase activity could be demonstrated only in the outer layer of the adrenal, which is the site of angiotensin enhancement o f aldosterone production and release 2°, while the inner layer of the adrenal, synthesizing mainly glucocorticoids, was unaffected by angiotensin (Table II). The effect of angiotensin was dependent on the simultaneous presence of Na + and K +, which corroborates the suggestion that angiotensin affects membrane ( N a + - K + ) activated ATPase. Cautiousness as to the implications of our findings is certainly advisable at the present stage; however, when adding the report of Levin on the enhancement of kidney cortex (Na +-K+)-ATPase by angiotensin 16 it seems plausible to consider the possibility of microsomal (Na+-K+)-ATPase being part of a receptor oritarget of angiotensin action. This work was supported by Ford Foundation, Grant 6D-I.
REFERENCES 1 Douglas, W.W. (1970) in The Pharmacological Basis of Therapeutics (Goodman, L.S. and Gilman, A., eds), 4th edn, p. 663, Macmillan, New York 2 Gross, F. (1968) Acta Endocrinol. Suppl. 124, 41 3 Fitzsimons, J.T. (1966)J. Physiol. (London) 186, 130P 4 Fitzsimons, J.T. and Simons, B.J. (1969)J. Physiol. (London) 203, 45 5 Gutman, Y., Benzakein, F. and Chaimovitz, M. (1967)Israel J. Med. Sci. 3,910 6 Gutman, Y. and Benzakein, F. (1969) Israel J. Med. Sci. 5, 411 7 Houpt, A. and Epstein, A.N. (1969)Physiologist 12,257 8 Gutman, Y., Benzakein, F. and Livneh, P. (1971)Eur. J. Pharmacol. 16,380 9 Epstein, A.N., Fitzsimons, J.T. and Simons, B.J. (1970)J. Physiol. (London) 210,457 10 Bonjour, J.P. and Malvin, R.L. (1970)Am. J. Physiol. 218, 1555 11 Andersson, B. and Westbye, O. (1970) Life Sci. Part 1 Physiol. Pharmacol. 9,601 12 Davis, N.T., Munday, K.A. and Parsons, B.J. (1970)Z Endocrinol. 48, 39 13 Davis, J.O., Howards, S.S., Johnston, C.I. and Wright, F.S. (1968) Proc. Soc. Exp. Biol. Med. 127, 164 14 Wellen, J.J. and Benraad, T.J. (1969) Biochim. Biophys. Acta 183, 110 15 Tiirker, K.R., Page, I.H. and Khairallah, P.A. (1967)Arch. lnt. Pharmacodvn. 165,394 16 Levin, K. (1970) Acta Physiol. Scand. 79, 37 17 Gutman, Y. and Katzper-Shamir, Y. (1971) Biochim. Biophys. Acta 233,133 18 Baginski, E. and Zak, B. (1960) Clin. Chim. Acta 5,834 19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951)J. Biol. Chem. 183,265 20 Davis, J.O. (1971) in Kidney Hormones (Fisher, J.W., ed.), p. 173, Academic Press, New York
Biochim. Biophys. Acta, 273 (1972) 401-405
401
BBA Report BBA 21335
Angiotensin increases microsomal (Na+-K +)-ATPase activity in several tissues Y. GUTMAN,Y. SHAMIR, D. GLUSHEVITZKYand S. HOCHMAN Department o f Pharmacology, The Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(Received May 24th, 1972)
SUMMARY
Angiotensin increased microsomal (Na +-K+)-activated ATPase from rat hypothalamus, mucosa of colon and bovine adrenal cortex but not brain cortex. Angiotensin did not change significantly Mg/+-ATPase in these tissues. Enhancement by angiotensin was evident at concentrations of 10- 8 - 1 0 -12 M. In bovine adrenal cortex angiotensin increased ATPase activity in the outer and not in the inner layer. Angiotensin increased ATPase activity with Na+ above 20 mM and K + above 5 mM present simultaneously, but not with either ion alone.
Angiotensin exerts both a pressor vascular effect I and an increase of aldosterone secretion by the adrenal glands 2. The basic mechanism underlying these effects, however, has not been elucidated. Recently, several other functions have been ascribed to the renin-angiotensin system. Thirst evoked by hypovolemia or by isoprenaline injection has been shown to be mediated by renin released from the kidney 3-8. Angiotensin exerts a direct effect on the hypothalamus resulting in increased drinking 9 and increased release of antidiuretic hormone 1°' 11. Angiotensin has also been shown to au~nent net transport of Na+ and fluid across the rat intestinal mucosa, particularly the colon 12. Corticosterone production in the adrenal is enhanced by angiotensin 13. On the other hand, corticosterone production in calf adrenal is suppressed by ouabain which inhibits (Na+-K+)-activated ATPase 14. A possible relation of angiotensin to Na + transport has been reported by Ttirker et al. ~s, viz. increased Na+ efflux from isolated carotid arteries and from uterus in the presence of angiotensin. Levin has found increased (Na+-K+)-ATPase activity in kidney cortex microsomes in the presence of angiotensin (at a concentration of 10-6 M) 16. It seemed of interest, therefore, to study the effect of angiotensin on microsomal Biochim. Biophys. Acta, 273 (1972) 401-405
C L~
--2
Bovine adrenal cortex (N = 13)
(N = 5)
Rat colon
( N = 7)
Rat brain cortex
Rat hypothalamus (N= 7)
Tissue
5.5+-0.62
23.4*0.9
1.45±0.30
7.3+_0.6
43.3±3.3
22.2*4.5
17.6±l.8
15.9*2.3
(~#
('Ya + K+j-activated A TPase
MgZ+-A TPase
10-"
10-' 2
1(I ~
10 -~
A ttgiotensiJ, cone
Activit)' {~moles Pi/mg protein per h)
(1' < 0.01)
0.78*0.2 t
• IP < 0.05)
3.3±0.9
(n.s,I
0.3+-1.0
6.85' 1.85 (P = 0.01 )
Ulnole~ t i/mg prou
)
.
per h
53.7
45.2
0.7
30.8
.
~:
Increase o f (.\'a + -K+)-actirated A 7"Pase
N = number of experiments;P values: for the difference between (Na+-K+)-ATPase activity in the presence and absence of angiotensin, n.s. = difference not statistically significant. No significant difference in Mg2+-ATPase activity was observed in the presence and absence of angiotensin.
EFFECT OF ANGIOTENSIN ON MICROSOMAL ATPase ACTIVITY
TABLE I
©
b~
BBA REPORT
403
(Na+-K+)-activated ATPase in several tissues which show physiologically measurable effects of this peptide. Rat hypothalanms, rat brain cortex and mucosal scrapings of rat colon were taken from animals nephrectomized 24 h previously. Bovine adrenals were obtained from the slaughterhouse, placed immediately in ice, and brought 1o the laboratory where cortex and medulla were separated. The various tissues were homogenized as described previously ~v in 10 vol. of an ice-cold medium of 0.25 M sucrose with 2 mM EDTA and centrifuged at 1000 × g for 10 rain. The supernatant was centrifuged at 10 000 X g for 10 rain, and the supernatant of this centrifugation was spun for 30 rain at 105 000 X g. The pellet obtained was resuspended in 0.25 M sucrose and served as microsoma! enzyme suspension. Assay of ATPase was carried out by incubating 0.2 ml of the enzyme suspension (containing approximately 0.5 nrg protein) in 3 ml of medium, consisting of 100 mM NaCI, 10 mM KC1, 4 mM MgCI2,4 mM ATP and either with or without ouabain (final concentration 10 - 3 M)at 37 °C for 20 rain. Inorganic phosphate (Pi) liberated was measured according to the method of Baginski and Zak ~s and protein according to the method of Lowry et al. ~9. Angiotensin (Val-5-hypertensin ll-Asp-/3 amide, obtained from CIBA) was added as indicated below. Tire difference in activity in the presence and absence of ouabain is taken as (Na + K+)-activated ATPase while the activity persisting in the presence of ouabain is expressed as Mg2+-ATPase. Table I shows that microsomal (Na + K +)-activated ATPase from rat hypothalamus was markedly enhanced in the presence of angiotensin, whereas no mthancement was seen in rat cortex following addition of angiotensin. A significant increase of (Na + K+)-ATPase activity was also observed in rat colon microsomes upon addition of angiotensin (Table I). Microsomal (Na + K+)-ATPase activity from bovine adrenal cortex was also enhanced in the presence of angiotensin (Table 11). The enhancement by angiotensin was
TABLE II I:FFECTOFANGIOTENSINON MICROSOMAL(Na+ K+)-ATPaseACTIVITYOFBOVINF ADRENAL CORTEX Fresh adrenal glands were cut to separate medulla from cortex. The adrenal cortex was then divided into an outer and an inner layer. The two portions were homogenized, prepared and assayed as described in the text (see also ref. 17). Angiotensin was added at a final concentration of 10 9 M. A"= number of experiments. P: for difference between activity in the presence and absence of angiotensin, n.s. = difference not statistically significant. l;*c'rease of (Na + K+)-ATPase activity hy angiotensin (#moles Piling protein per h) Outer layer of adrenal cortex (N = 24)
0.74_+0.26
(P < 0.01 )
Inner layer of adrenal cortex (N = 24)
0.29_+0.27 (n.s.) Biochim. Biophys. Acta, 273 (1972) 401 -405
404
BBA REPORT
specific for the outer portion of the adrenal cortex, while microsomes from the inner layer of the cortex o f the adrenal showed no activation o f (Na + K+)-ATPase in the presence of angiotensin, as seen in Table I1. F u r t h e r m o r e , the increase of (Na + - K + ) - A T P a s e activity by angiotensin was evident only in the presence of Na + c o n c e n t r a t i o n s above 20 mM and with K + c o n c e n t r a t i o n s higher than 5 mM. In the presence of either Na + or K + alone, even at I00 mM, angiotensin showed no significant a u g m e n t a t i o n of ( N a + - K + ) - A T P a s e activity (Fig. 1 ).
0.50 o
I,,..
1:3. Cn
E
i
o~
I
l
I
5
10
20
[K'I
1.50 -
13... Ul
1.00
o
E
0.50
1
I
1
25
50
I [Na÷] 100
mM Fig 1. Effect of angiotensin on microsomal (Na + K+)-ATPase activity of bovine adrenal cortex at different concentrations of Na+ and K +. Ordinate: increase of (Na+-K+)-ATPase activity in presence o f 10 - 9 M angiotensin. Vertical bars: S.E. Lower part: incubation at [K +] = 100 mM and varying [Na +] ; N = 20. At [Na +] = 50 mM, P < 0.02 ; for [ Na+] = 100 raM, P < 0.01. Upper part: incubation at [Na +] = 100 mM and varying [K+] ;N = 15. For [K +] = 10 mM and 20 mM,P < 0.01. At all other concentrations of Na + or K+ the increase of (Na + K+)-ATPase in the presence of angiotensin was not statistically significant.
These experiments demonstrate that angiotensin can enhance significantly the activity of microsomal (Na + K+)-ATPase from three different tissues: rat h y p o t h a l a m u s , rat colon and bovine adrenal cortex. It is of interest that in rat brain cortex, where n o physiological effect o f angiotensin has been d e m o n s t r a t e d , there was also no e n h a n c e m e n t of ( N a + - K + ) - A T P a s e activity in the presence of angiotensin (Table 1). F u r t h e r m o r e , it is n o t e w o r t h y that the c o n c e n t r a t i o n of angiotensin affecting (Na + K+)-ATPase activity was rather low. In the case of rat colon, the e n h a n c e m e n t of fluid transport across the Biochim. Biophys. Acta, 273 (1972) 401-405
BBA REPORT
405
mucosa reported by Davis et al. ~ 2, was obtained at similar angiotensin concentrations ( 1 0 - 1 ° - 1 0 -12 M). In the adrenal cortex the effect o f angiotensin on (Na+-K+)-ATPase activity could be demonstrated only in the outer layer of the adrenal, which is the site of angiotensin enhancement o f aldosterone production and release 2°, while the inner layer of the adrenal, synthesizing mainly glucocorticoids, was unaffected by angiotensin (Table II). The effect of angiotensin was dependent on the simultaneous presence of Na + and K +, which corroborates the suggestion that angiotensin affects membrane ( N a + - K + ) activated ATPase. Cautiousness as to the implications of our findings is certainly advisable at the present stage; however, when adding the report of Levin on the enhancement of kidney cortex (Na +-K+)-ATPase by angiotensin 16 it seems plausible to consider the possibility of microsomal (Na+-K+)-ATPase being part of a receptor oritarget of angiotensin action. This work was supported by Ford Foundation, Grant 6D-I.
REFERENCES 1 Douglas, W.W. (1970) in The Pharmacological Basis of Therapeutics (Goodman, L.S. and Gilman, A., eds), 4th edn, p. 663, Macmillan, New York 2 Gross, F. (1968) Acta Endocrinol. Suppl. 124, 41 3 Fitzsimons, J.T. (1966)J. Physiol. (London) 186, 130P 4 Fitzsimons, J.T. and Simons, B.J. (1969)J. Physiol. (London) 203, 45 5 Gutman, Y., Benzakein, F. and Chaimovitz, M. (1967)Israel J. Med. Sci. 3,910 6 Gutman, Y. and Benzakein, F. (1969) Israel J. Med. Sci. 5, 411 7 Houpt, A. and Epstein, A.N. (1969)Physiologist 12,257 8 Gutman, Y., Benzakein, F. and Livneh, P. (1971)Eur. J. Pharmacol. 16,380 9 Epstein, A.N., Fitzsimons, J.T. and Simons, B.J. (1970)J. Physiol. (London) 210,457 10 Bonjour, J.P. and Malvin, R.L. (1970)Am. J. Physiol. 218, 1555 11 Andersson, B. and Westbye, O. (1970) Life Sci. Part 1 Physiol. Pharmacol. 9,601 12 Davis, N.T., Munday, K.A. and Parsons, B.J. (1970)Z Endocrinol. 48, 39 13 Davis, J.O., Howards, S.S., Johnston, C.I. and Wright, F.S. (1968) Proc. Soc. Exp. Biol. Med. 127, 164 14 Wellen, J.J. and Benraad, T.J. (1969) Biochim. Biophys. Acta 183, 110 15 Tiirker, K.R., Page, I.H. and Khairallah, P.A. (1967)Arch. lnt. Pharmacodvn. 165,394 16 Levin, K. (1970) Acta Physiol. Scand. 79, 37 17 Gutman, Y. and Katzper-Shamir, Y. (1971) Biochim. Biophys. Acta 233,133 18 Baginski, E. and Zak, B. (1960) Clin. Chim. Acta 5,834 19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951)J. Biol. Chem. 183,265 20 Davis, J.O. (1971) in Kidney Hormones (Fisher, J.W., ed.), p. 173, Academic Press, New York
Biochim. Biophys. Acta, 273 (1972) 401-405