Tohoku
J. Exp.
Effect on
Med.,
1992, 166, 85-91
of Acute
Blood
Hypertensive
and
Pressure
Chronic
Treatment
of Tin
in Spontaneously
Rats
MICHAL LANIADO-SCHWARTZMAN, NADER G. ABRAHAM, DAVID SACERDOTI,BRUNO ESCALANTE and JOHN C. MCGIFF Department of Pharmacology and Medicine, New York Medical College, Valhalla, New York 10595, USA
LANIADO-SCHWARTZMAN, M., ABRAHAM, N.G., SACERDOTI, D., ESCALANTE, B. and MCGIFF, J.C. Effect of Acute and Chronic Treatment of Tin on Blood Pressure in Spontaneously Hypertensive Rats. Tohoku J. Exp. Med., 1992, 166 (1), 85-91 Cytochrome P450-dependent metabolites of arachidonic acid (AA) are increased in the kidneys of spontaneously hypertensive rats (SHR) as compared to control rats (WKY) in the period of rapid elevation of blood pressure (BP) from 5 to 13 weeks. We treated rats with stannous chloride (SnC12)(10 mg/ 100 g body weight/day for 4 days) to decrease selectively renal cytochrome P450 content through increasing renal heme oxygenase activity. A decrease in renal cytochrome P450-dependent AA metabolites was associated with decreased BP and increased urinary Na+ excretion in 7- but not in 20-week-old SHR rats. Chronic treatment with SnC12(10 mg/100 g body weight twice a week) from 5 to 20 weeks prevented the elevation of BP in SHR rats. Further, the antihypertensive effects of tin persisted for 7 weeks beyond its discontinuation. BP in WKY rats was unaffected by tin. Both the acute and chronic treatment with tin are the first studies to demonstrate amelioration of hypertension in SHR by an intervention which is targeted at a single enzyme system. arachidonic acid ; blood pressure ; cytochrome ; P450; heme oxygenase ; SHR
The mechanisms responsible for the elevation of blood pressure (BP) in essential hypertension are not understood. The animal model most frequently used to study essential hypertension is the spontaneously hypertensive rat (SHR). Between ages 5 to 13 weeks, BP increases most rapidly, e.g., systolic BP rises from 100 mmHg to 170 mmHg in the SHR whereas in the age-matched normotensive rat systolic BP rarely exceeds 130 mmHg. Elevation of BP in the SHR can be partially suppressed by renal transplant from a normotensive donor (Kawabe et al. 1978), suggesting that abnormalities in kidney function are responsible for the elevation of BP in SHR (Beierwaltes et al. 1982; Dilley et al. 1984). These abnormalities consist of reduced excretion of sodium and water as well as de-
York
Correspondence : Michal Laniado-Schwartzman, Medical College, Valhalla, NY 10595, USA. 85
Department
of Pharmacology,
New
86
M. Laniado-Schwartzman
et al.
creased renal blood flow and glomerular filtration rate.
They are evident only in
young (4-8-week-old) animals (Beierwaltes et al. 1982 ; Dilley et al. 1984), but are compensated for in the older SHR. The abnormalities of renal function in young SHR may be the functional expression of alterations in renal arachidonic acid (AA) metabolism through the cyclooxygenase pathway (Pace-Asciak 1976 ; Dunn 1976, 1978 ; Limas and Limas 1977 ; Ahnfelt-Ronne and Arrigoni-Martelli 1977; Shibouta et al. 1981), and through the cytochrome P450-dependent pathway (Sacerdoti et al. 1988). AA metabolites arising from these pathways can affect vascular tone and salt and water excretion (Schwartzman et al. 1985 ; Miller et al. 1986). Treatment of SHR with inhibitors of cyclooxygenase further increased BP (Lopez-Ovejero et al. 1978), while thromboxane synthase inhibitors decreased BP in young SHR, and delayed, but did not prevent, the onset of hypertension in SHR (Shibouta et al. 1985). We have recently reported that the kidneys of SHR and control Wistar-Kyoto (WKY) rats are able to metabolize AA through the cytochrome P450-dependent pathway (Sacerdoti et al. 1988). Metabolism of AA through this pathway was increased in kidneys of the SHR during the developmental phase of hypertension, but not in the established phase (Sacerdoti et al. 1988). Renal cytochrome P450 levels were always increased, both in the developmental and established phase (Sacerdoti et al. 1988). To understand the significant of increased cytochrome P450 levels in relation to altered renal synthesis of AA metabolites by the cytochrome P450 system in the pathophysiology of elevation of BP in SHR, we studied the effect of decreasing renal cytochrome P450 content on BP and cytochrome P450-dependent AA metabolites. Heavy metals are potent inducers of heme oxygenase (Maines and Kappas 1974) and thereby, cause a marked depletion of cytochrome P450 as heme oxygenase regulates the availability of heme for cytochrome P450. Among the heavy metals, tin is the most selective for the kidney, relative to its effect on heme oxygenase and cytochrome P450 (Kappas and Maines 1976). In this study, we depleted renal cytochrome P450 by treating the SHR with tin chloride (SnCl2 ), and then related changes in cytochrome P450 content and. AA metabolite formation to changes in BP.
METHODS AND MATERIALS Animal treatment. SHR and WKY were purchased from Charles River Laboratories, and fed and housed under identical conditions. Rats were injected subcutaneously with SnC12 (10 mg/100 g body weigh/day) for 4 days (acute study) or twice a week (chronic study). In some experiments the rats were injected with tin-protoporphyrin (4 mg/100 g body weigh/day) in addition to the treatment with tin. Blood pressure was monitored by the standard tail cuff method during the treatment without anesthesia. Body weight, urine volume, Na+ and K+ excretion were measured before sacrificing. Preparation of renal microsomes. Rats were anesthetized with sodium pentobarbital (100 mg/kg body weigh, i.p) and the abdominal cavity opened. The kidneys were flushed
Tin
and Blood
Pressure
Regulation
87
with 0.9% saline through the aorta, removed and the cortex was separated from the outer medulla. The microsomal fractions were obtained by homogenization of the tissue with Tris-HC1 buffer, pH 7.6, containing 0.25 M sucrose, and centrifuged at 2,000 X g for 10 min and 100,000x g for 60 min. The resulting pellets were resuspended in 0.1 M potassium phosphate buffer, pH 7.6, and microsomal protein was determined according to Lowry et al. (1951). Cytochrome P450-dependent AA metabolism. Cytochrome P450-dependent AA metabolites were determined by incubating the microsomes (0.3 mg protein) with [14C]-AA (0.2-0.4 Ci), with or without indomethacin (10 EM), NADPH-generating system (glucose 6-phosphate, 0.1 mM ; NADP 0.4 mM ; glucose 6-phosphate dehydrogenase, l unit), (or SKF 525A (200pM)) in a total volume of 1 ml, for 30 min at 3TC. The reaction was terminated by acidification to pH 4.0, and the metabolites were extracted with ethyl acetate and subjected to thin-layer chromatography (TLC) for separation of AA and oxygenated metabolites using the upper phase of ethyl-acetate : iso-octane : acetic acid : water (110: 50: 20: 100). The TLC plates were evaluated on a computerized Berthold 512B radiochromatogram scanner. Cytochrome P450 content. Cytochrome P450 content was determined in renal and hepatic microsomes by measuring the carbon monoxide difference spectra and using an extinction coefficient of 91 mM-1 cm-' as described by Omura and Sate (1964). Heme oxygenase activity. The activity of heme oxygenase in both hepatic and renal microsomes was measured by incubating the microsomes with heme in the presence of biliverdin reductase. Bilirubin formation was measured as described by Abraham et al. (1983).
RESULTSANDDISCUSSION SHR and WKY, ages 7 and 20 weeks, were injected with SnC12 (10 mg/100 g body weight s.c.) for 4 days. After treatment, the BP was measured as well as AA metabolism, cytochrome P450 level and heme oxygenase activity. As seen in Fig. 1, treatment with tin caused a 30% decrease in BP only in 7-week SHR while no effect was observed in 20-week WKY. As a consequence of treatment with tin, renal heme oxygenase was stimulated and renal cytochrome P450 content was decreased in SHR and WKY of 7 and 20 weeks (Table 1). In the liver, heme oxygenase and cytochrome P450 content in both 7- and 20-week-old SHR and WKY were not changed by treatment with tin (data not shown). Cytochrome P450- dependent metabolites of AA, defined as those which are dependent on the presence of NADPH and are inhibited by SKF-525A (Sacerdoti et al. 1988), were significantly decreased in the renal cortex only in 7-week-old SHR (Table 1). The same qualitative changes were observed for the outer medullary AA metabolites where a reduction of 40% was seen, from 954 + 58 to 534 + 69 ng AA converted/mg/30 min. When cytochrome P450-dependent metabolites were separated by reverse-phase HPLC, three peaks were obtained as previously reported (Sacerdoti et al. 1988). Only peak II, which has a retention time of 21 min and comigrates with 19- and 20-hydroxyeicosatetraenoic acids, was significantly decreased in 7-week-old SHR (control cortex 713+95 ng AA/mg/30' vs tin cortex 361 + 50 ng AA/mg/30', p
J. Exp.
Effect on
Med.,
1992, 166, 85-91
of Acute
Blood
Hypertensive
and
Pressure
Chronic
Treatment
of Tin
in Spontaneously
Rats
MICHAL LANIADO-SCHWARTZMAN, NADER G. ABRAHAM, DAVID SACERDOTI,BRUNO ESCALANTE and JOHN C. MCGIFF Department of Pharmacology and Medicine, New York Medical College, Valhalla, New York 10595, USA
LANIADO-SCHWARTZMAN, M., ABRAHAM, N.G., SACERDOTI, D., ESCALANTE, B. and MCGIFF, J.C. Effect of Acute and Chronic Treatment of Tin on Blood Pressure in Spontaneously Hypertensive Rats. Tohoku J. Exp. Med., 1992, 166 (1), 85-91 Cytochrome P450-dependent metabolites of arachidonic acid (AA) are increased in the kidneys of spontaneously hypertensive rats (SHR) as compared to control rats (WKY) in the period of rapid elevation of blood pressure (BP) from 5 to 13 weeks. We treated rats with stannous chloride (SnC12)(10 mg/ 100 g body weight/day for 4 days) to decrease selectively renal cytochrome P450 content through increasing renal heme oxygenase activity. A decrease in renal cytochrome P450-dependent AA metabolites was associated with decreased BP and increased urinary Na+ excretion in 7- but not in 20-week-old SHR rats. Chronic treatment with SnC12(10 mg/100 g body weight twice a week) from 5 to 20 weeks prevented the elevation of BP in SHR rats. Further, the antihypertensive effects of tin persisted for 7 weeks beyond its discontinuation. BP in WKY rats was unaffected by tin. Both the acute and chronic treatment with tin are the first studies to demonstrate amelioration of hypertension in SHR by an intervention which is targeted at a single enzyme system. arachidonic acid ; blood pressure ; cytochrome ; P450; heme oxygenase ; SHR
The mechanisms responsible for the elevation of blood pressure (BP) in essential hypertension are not understood. The animal model most frequently used to study essential hypertension is the spontaneously hypertensive rat (SHR). Between ages 5 to 13 weeks, BP increases most rapidly, e.g., systolic BP rises from 100 mmHg to 170 mmHg in the SHR whereas in the age-matched normotensive rat systolic BP rarely exceeds 130 mmHg. Elevation of BP in the SHR can be partially suppressed by renal transplant from a normotensive donor (Kawabe et al. 1978), suggesting that abnormalities in kidney function are responsible for the elevation of BP in SHR (Beierwaltes et al. 1982; Dilley et al. 1984). These abnormalities consist of reduced excretion of sodium and water as well as de-
York
Correspondence : Michal Laniado-Schwartzman, Medical College, Valhalla, NY 10595, USA. 85
Department
of Pharmacology,
New
86
M. Laniado-Schwartzman
et al.
creased renal blood flow and glomerular filtration rate.
They are evident only in
young (4-8-week-old) animals (Beierwaltes et al. 1982 ; Dilley et al. 1984), but are compensated for in the older SHR. The abnormalities of renal function in young SHR may be the functional expression of alterations in renal arachidonic acid (AA) metabolism through the cyclooxygenase pathway (Pace-Asciak 1976 ; Dunn 1976, 1978 ; Limas and Limas 1977 ; Ahnfelt-Ronne and Arrigoni-Martelli 1977; Shibouta et al. 1981), and through the cytochrome P450-dependent pathway (Sacerdoti et al. 1988). AA metabolites arising from these pathways can affect vascular tone and salt and water excretion (Schwartzman et al. 1985 ; Miller et al. 1986). Treatment of SHR with inhibitors of cyclooxygenase further increased BP (Lopez-Ovejero et al. 1978), while thromboxane synthase inhibitors decreased BP in young SHR, and delayed, but did not prevent, the onset of hypertension in SHR (Shibouta et al. 1985). We have recently reported that the kidneys of SHR and control Wistar-Kyoto (WKY) rats are able to metabolize AA through the cytochrome P450-dependent pathway (Sacerdoti et al. 1988). Metabolism of AA through this pathway was increased in kidneys of the SHR during the developmental phase of hypertension, but not in the established phase (Sacerdoti et al. 1988). Renal cytochrome P450 levels were always increased, both in the developmental and established phase (Sacerdoti et al. 1988). To understand the significant of increased cytochrome P450 levels in relation to altered renal synthesis of AA metabolites by the cytochrome P450 system in the pathophysiology of elevation of BP in SHR, we studied the effect of decreasing renal cytochrome P450 content on BP and cytochrome P450-dependent AA metabolites. Heavy metals are potent inducers of heme oxygenase (Maines and Kappas 1974) and thereby, cause a marked depletion of cytochrome P450 as heme oxygenase regulates the availability of heme for cytochrome P450. Among the heavy metals, tin is the most selective for the kidney, relative to its effect on heme oxygenase and cytochrome P450 (Kappas and Maines 1976). In this study, we depleted renal cytochrome P450 by treating the SHR with tin chloride (SnCl2 ), and then related changes in cytochrome P450 content and. AA metabolite formation to changes in BP.
METHODS AND MATERIALS Animal treatment. SHR and WKY were purchased from Charles River Laboratories, and fed and housed under identical conditions. Rats were injected subcutaneously with SnC12 (10 mg/100 g body weigh/day) for 4 days (acute study) or twice a week (chronic study). In some experiments the rats were injected with tin-protoporphyrin (4 mg/100 g body weigh/day) in addition to the treatment with tin. Blood pressure was monitored by the standard tail cuff method during the treatment without anesthesia. Body weight, urine volume, Na+ and K+ excretion were measured before sacrificing. Preparation of renal microsomes. Rats were anesthetized with sodium pentobarbital (100 mg/kg body weigh, i.p) and the abdominal cavity opened. The kidneys were flushed
Tin
and Blood
Pressure
Regulation
87
with 0.9% saline through the aorta, removed and the cortex was separated from the outer medulla. The microsomal fractions were obtained by homogenization of the tissue with Tris-HC1 buffer, pH 7.6, containing 0.25 M sucrose, and centrifuged at 2,000 X g for 10 min and 100,000x g for 60 min. The resulting pellets were resuspended in 0.1 M potassium phosphate buffer, pH 7.6, and microsomal protein was determined according to Lowry et al. (1951). Cytochrome P450-dependent AA metabolism. Cytochrome P450-dependent AA metabolites were determined by incubating the microsomes (0.3 mg protein) with [14C]-AA (0.2-0.4 Ci), with or without indomethacin (10 EM), NADPH-generating system (glucose 6-phosphate, 0.1 mM ; NADP 0.4 mM ; glucose 6-phosphate dehydrogenase, l unit), (or SKF 525A (200pM)) in a total volume of 1 ml, for 30 min at 3TC. The reaction was terminated by acidification to pH 4.0, and the metabolites were extracted with ethyl acetate and subjected to thin-layer chromatography (TLC) for separation of AA and oxygenated metabolites using the upper phase of ethyl-acetate : iso-octane : acetic acid : water (110: 50: 20: 100). The TLC plates were evaluated on a computerized Berthold 512B radiochromatogram scanner. Cytochrome P450 content. Cytochrome P450 content was determined in renal and hepatic microsomes by measuring the carbon monoxide difference spectra and using an extinction coefficient of 91 mM-1 cm-' as described by Omura and Sate (1964). Heme oxygenase activity. The activity of heme oxygenase in both hepatic and renal microsomes was measured by incubating the microsomes with heme in the presence of biliverdin reductase. Bilirubin formation was measured as described by Abraham et al. (1983).
RESULTSANDDISCUSSION SHR and WKY, ages 7 and 20 weeks, were injected with SnC12 (10 mg/100 g body weight s.c.) for 4 days. After treatment, the BP was measured as well as AA metabolism, cytochrome P450 level and heme oxygenase activity. As seen in Fig. 1, treatment with tin caused a 30% decrease in BP only in 7-week SHR while no effect was observed in 20-week WKY. As a consequence of treatment with tin, renal heme oxygenase was stimulated and renal cytochrome P450 content was decreased in SHR and WKY of 7 and 20 weeks (Table 1). In the liver, heme oxygenase and cytochrome P450 content in both 7- and 20-week-old SHR and WKY were not changed by treatment with tin (data not shown). Cytochrome P450- dependent metabolites of AA, defined as those which are dependent on the presence of NADPH and are inhibited by SKF-525A (Sacerdoti et al. 1988), were significantly decreased in the renal cortex only in 7-week-old SHR (Table 1). The same qualitative changes were observed for the outer medullary AA metabolites where a reduction of 40% was seen, from 954 + 58 to 534 + 69 ng AA converted/mg/30 min. When cytochrome P450-dependent metabolites were separated by reverse-phase HPLC, three peaks were obtained as previously reported (Sacerdoti et al. 1988). Only peak II, which has a retention time of 21 min and comigrates with 19- and 20-hydroxyeicosatetraenoic acids, was significantly decreased in 7-week-old SHR (control cortex 713+95 ng AA/mg/30' vs tin cortex 361 + 50 ng AA/mg/30', p
