320
Brain Research. 529 {19901 320-323 Elsevier
BRES 24308
Comparison of angiotensin metabolism by brain membranes from SHR and WKY rats Vickie I. Cook 1, John W. Wright t'2, Shonna A. Wright 1 and Joseph W. Harding 1'2 Departments of 1Veterinary and Comparative Anatomy, Pharmacology and Physiology, and 2psychology, Washington State University, Pullman, WA 99164-6520 (U.S.A. )
(Accepted 19 June 1990) Key words: Metabolism; Brain; Spontaneously hypertensive rat; Angiotensin II; Angiotensin III; Membrane
The ability of membrane-associated peptidases from the brains of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats to metabolize iodinated angiotensin (~25I-Ang II) and ~25I-AngIII was compared. ~25I-AngII was metabolized to 125I-Ang III and other fragments exclusively by membrane-associated peptidases. In contrast to ~25I-Ang III which was effectively degradated by both membrane-associated and residual cytosolic peptidases, ~25I-Ang II was unaltered by contaminating cytosolic enzymes. The ability of SHR-derived membranes to metabolize ]2SI-Ang II and produce 125I-Ang III was enhanced when compared to membranes from WKY rats. No difference was observed in the ability of membrane or cytosolic enzymes from SHR and WKY rats to degrade 125I-Ang III. These data are consistent with an increased availability of Ang III in the brains of SHRs.
The central angiotensin system plays a critical role in the regulation of cardiovascular function and body water balance. This role has been documented by the ability of centrally applied angiotensins to raise blood pressure and stimulate drinking behavior s'2°'2t. Elevations in blood pressure and water consumption can also be seen following the inhibition of endogenous angiotensin metabolism in the brain using peptidase inhibitors ]4'z4'25"27. Further, several studies have demonstrated the ability of intracerebroventricularly injected angiotensin antagonists to lower blood pressure in both hypertensive and normotensive rats 13`17'ts. Abnormalities of the central angiotensin system have been implicated in the etiology of the hypertension seen in spontaneously hypertensive rats (SHRs), an often used model of human essential hypertension 3"4a5. SHRs evidence a heightened sensitivity to intracerebroventricularly administered angiotensin with regard to this peptide's ability to elevate blood pressure 5"It and initiate drinking behavior 29. Several theories have been articulated to explain this enhanced angiotensin responsiveness including: (1) an increase in angiotensin turnover or release3"26; (2) increased receptor-transducer sensitivity due to alterations in receptor number, affinity, or coupling6"12; and (3) a decreased rate of angiotensin degradation 2s. Although an increased turnover of angiotensins is indicated by several studies, an alteration in
release rate would not be expected to play a role in the response to exogenously applied angiotensins. A more plausible explanation is that the n u m b e r of angiotensin receptors is increased in the brain of SHRs as compared to normotensive W i s t a r - K y o t o ( W K Y ) controls. Support for this idea has been mixed with some studies showing increased receptors 6'lzm, others demonstrating no difference in receptor numbers 2, and still others indicating a decrease in receptors =. The third possibility suggested by our laboratory is that SHRs possess a defect in the peptidases responsible for degrading active angiotelrsins. This notion was first supported by a study which examined the rate of ]2SI-Ang II and 125I-Ang III metabolism in the cerebroventricles of S H R , WKY, and S p r a g u e - D a w l e y rats 28. The data indicated a dramatic decrease in cerebroventricular angiotensin metabolism in SHRs. In concert with this finding, cultured neurons from SHRs metabolize angiotensin at a reduced rate 1°. The intent of the present study was to expand on these previous observations by directly assessing the ability of angiotensinases from the brain p a r e n c h y m a to metabolize Ang II and Ang II1. The metabolism was monitored using a brain tissue block containing the hypothalamusthalamus-septum-anteroventral third ventricle ( H T S A ) , areas known to be rich in angiotensin receptors 19. The results of this study, however, did not mirror the outcome of the previously mentioned cerebroventricular study but
Correspondence: J.W. Harding, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, U.S.A. 0006-8993/90/$03.50 © 19911 Elsevier Science Publishers B V. (Biomedical Division)
321 instead, demonstrated a selective increase in Ang II metabolism and its conversion to Ang III by plasma membrane aminopeptidases. Male SHR and WKY rats (3-4 months old, derived from stock purchased from Taconic Farms, Inc.) were housed at 21-22 °C, on a 12:12 h light-dark cycle initiated at 07.00 h, and provided with Purina laboratory pellets and water ad libitum. Rats were killed by decapitation and their brains were quickly removed and dissected over ice. The dissected tissue block contained the hypothalamus, thalamus, septum and anteroventral third ventricle. The tissue was then weighed and homogenized (Brinkman polytron setting -+ 8, 1 s/mi) with the addition of 40 volumes of hypotonic buffer (pH 7.4 at 4 °C, 50 mM Tris) and centrifuged for 10 min at 2000 rpm and 4 °C. The supernatant was recovered and the remaining pellet was resuspended and spun again as previously indicated. The collected supernatants were then combined and centrifuged for 10 min at 40,000 g and 4 °C. The resulting supernatant was discarded and the pellet was resuspended in isotonic buffer (pH 7.4 at 23 °C, 110 mM NaCl, 5 mM KCI, 2mM CaCl 2, l m M MgC12, 25 mM NaPO4) at 40 volumes and recentrifuged for 10 min at 40,000 g and 4 °C. The supernatant was discarded and the pellet resuspended in isotonic buffer at 40 mg of initial wet weight/ml. Half of the homogenate was centrifuged at 40,000 g for 10 min and the supernatant was collected. The final protein content of the tissue homogenate for SHR and WKY rats averaged 69.55/~g/100/A and was within 5 pg/100/~l of each other. The supernatant fraction averaged 1 pg/100/A for both WKY and SHRs. Protein concentration was determined by the method described by Lowry et al. 16 One hundred ~l of each tissue preparation (whole homogenate or supernatant) from each rat strain were incubated with 300 pl of isotonic (Krebs) buffer and 100 pl of either lzSI-Ang II or 125I-Ang III at a concentration of 0.1 nM resulting in a final volume of 0.5 ml. Incubations were carried out at room temperature for varying times up to 60 rain, at which time 0.5 ml of acetonitrile (ACN) was added to terminate metabolism. After 4 h the precipitated proteins were removed by centrifugation, the supernatant centrifuged through C TM mini-columns (Baker) in an Eppendorf microfuge, the eluant dried in a Savant vacuum concentrator, and the prepared sample stored at -20 °C. H P L C separations were performed on a C18 reverse phase column with a mobile phase that consisted of T E A P (250 mN phosphoric acid, titrated with triethylamine to pH 3) and 13.5% ACN. Intact peptide and fragments were separated under isocratic conditions at a flow rate of 6 ml/min. Standards (125I-Ang II, 125I-Ang
100
Brain Research. 529 {19901 320-323 Elsevier
BRES 24308
Comparison of angiotensin metabolism by brain membranes from SHR and WKY rats Vickie I. Cook 1, John W. Wright t'2, Shonna A. Wright 1 and Joseph W. Harding 1'2 Departments of 1Veterinary and Comparative Anatomy, Pharmacology and Physiology, and 2psychology, Washington State University, Pullman, WA 99164-6520 (U.S.A. )
(Accepted 19 June 1990) Key words: Metabolism; Brain; Spontaneously hypertensive rat; Angiotensin II; Angiotensin III; Membrane
The ability of membrane-associated peptidases from the brains of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats to metabolize iodinated angiotensin (~25I-Ang II) and ~25I-AngIII was compared. ~25I-AngII was metabolized to 125I-Ang III and other fragments exclusively by membrane-associated peptidases. In contrast to ~25I-Ang III which was effectively degradated by both membrane-associated and residual cytosolic peptidases, ~25I-Ang II was unaltered by contaminating cytosolic enzymes. The ability of SHR-derived membranes to metabolize ]2SI-Ang II and produce 125I-Ang III was enhanced when compared to membranes from WKY rats. No difference was observed in the ability of membrane or cytosolic enzymes from SHR and WKY rats to degrade 125I-Ang III. These data are consistent with an increased availability of Ang III in the brains of SHRs.
The central angiotensin system plays a critical role in the regulation of cardiovascular function and body water balance. This role has been documented by the ability of centrally applied angiotensins to raise blood pressure and stimulate drinking behavior s'2°'2t. Elevations in blood pressure and water consumption can also be seen following the inhibition of endogenous angiotensin metabolism in the brain using peptidase inhibitors ]4'z4'25"27. Further, several studies have demonstrated the ability of intracerebroventricularly injected angiotensin antagonists to lower blood pressure in both hypertensive and normotensive rats 13`17'ts. Abnormalities of the central angiotensin system have been implicated in the etiology of the hypertension seen in spontaneously hypertensive rats (SHRs), an often used model of human essential hypertension 3"4a5. SHRs evidence a heightened sensitivity to intracerebroventricularly administered angiotensin with regard to this peptide's ability to elevate blood pressure 5"It and initiate drinking behavior 29. Several theories have been articulated to explain this enhanced angiotensin responsiveness including: (1) an increase in angiotensin turnover or release3"26; (2) increased receptor-transducer sensitivity due to alterations in receptor number, affinity, or coupling6"12; and (3) a decreased rate of angiotensin degradation 2s. Although an increased turnover of angiotensins is indicated by several studies, an alteration in
release rate would not be expected to play a role in the response to exogenously applied angiotensins. A more plausible explanation is that the n u m b e r of angiotensin receptors is increased in the brain of SHRs as compared to normotensive W i s t a r - K y o t o ( W K Y ) controls. Support for this idea has been mixed with some studies showing increased receptors 6'lzm, others demonstrating no difference in receptor numbers 2, and still others indicating a decrease in receptors =. The third possibility suggested by our laboratory is that SHRs possess a defect in the peptidases responsible for degrading active angiotelrsins. This notion was first supported by a study which examined the rate of ]2SI-Ang II and 125I-Ang III metabolism in the cerebroventricles of S H R , WKY, and S p r a g u e - D a w l e y rats 28. The data indicated a dramatic decrease in cerebroventricular angiotensin metabolism in SHRs. In concert with this finding, cultured neurons from SHRs metabolize angiotensin at a reduced rate 1°. The intent of the present study was to expand on these previous observations by directly assessing the ability of angiotensinases from the brain p a r e n c h y m a to metabolize Ang II and Ang II1. The metabolism was monitored using a brain tissue block containing the hypothalamusthalamus-septum-anteroventral third ventricle ( H T S A ) , areas known to be rich in angiotensin receptors 19. The results of this study, however, did not mirror the outcome of the previously mentioned cerebroventricular study but
Correspondence: J.W. Harding, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, U.S.A. 0006-8993/90/$03.50 © 19911 Elsevier Science Publishers B V. (Biomedical Division)
321 instead, demonstrated a selective increase in Ang II metabolism and its conversion to Ang III by plasma membrane aminopeptidases. Male SHR and WKY rats (3-4 months old, derived from stock purchased from Taconic Farms, Inc.) were housed at 21-22 °C, on a 12:12 h light-dark cycle initiated at 07.00 h, and provided with Purina laboratory pellets and water ad libitum. Rats were killed by decapitation and their brains were quickly removed and dissected over ice. The dissected tissue block contained the hypothalamus, thalamus, septum and anteroventral third ventricle. The tissue was then weighed and homogenized (Brinkman polytron setting -+ 8, 1 s/mi) with the addition of 40 volumes of hypotonic buffer (pH 7.4 at 4 °C, 50 mM Tris) and centrifuged for 10 min at 2000 rpm and 4 °C. The supernatant was recovered and the remaining pellet was resuspended and spun again as previously indicated. The collected supernatants were then combined and centrifuged for 10 min at 40,000 g and 4 °C. The resulting supernatant was discarded and the pellet was resuspended in isotonic buffer (pH 7.4 at 23 °C, 110 mM NaCl, 5 mM KCI, 2mM CaCl 2, l m M MgC12, 25 mM NaPO4) at 40 volumes and recentrifuged for 10 min at 40,000 g and 4 °C. The supernatant was discarded and the pellet resuspended in isotonic buffer at 40 mg of initial wet weight/ml. Half of the homogenate was centrifuged at 40,000 g for 10 min and the supernatant was collected. The final protein content of the tissue homogenate for SHR and WKY rats averaged 69.55/~g/100/A and was within 5 pg/100/~l of each other. The supernatant fraction averaged 1 pg/100/A for both WKY and SHRs. Protein concentration was determined by the method described by Lowry et al. 16 One hundred ~l of each tissue preparation (whole homogenate or supernatant) from each rat strain were incubated with 300 pl of isotonic (Krebs) buffer and 100 pl of either lzSI-Ang II or 125I-Ang III at a concentration of 0.1 nM resulting in a final volume of 0.5 ml. Incubations were carried out at room temperature for varying times up to 60 rain, at which time 0.5 ml of acetonitrile (ACN) was added to terminate metabolism. After 4 h the precipitated proteins were removed by centrifugation, the supernatant centrifuged through C TM mini-columns (Baker) in an Eppendorf microfuge, the eluant dried in a Savant vacuum concentrator, and the prepared sample stored at -20 °C. H P L C separations were performed on a C18 reverse phase column with a mobile phase that consisted of T E A P (250 mN phosphoric acid, titrated with triethylamine to pH 3) and 13.5% ACN. Intact peptide and fragments were separated under isocratic conditions at a flow rate of 6 ml/min. Standards (125I-Ang II, 125I-Ang
100
