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The effect of metabolic acidosis and alkalosis on the H+-ATPase of rat cerebral microvessels
Life Sciences,
Vol. 61, No. 22, pp. 2247-2253,
Copyright 01997 Elswier Printed
in the USA.
THE
1997 Inc.
All tights reserved
cum-3205/97
ELSEVIER
Science $17.00
+ .ofJ
PI1SOOZ43205(97)00927-2
EFFECT OF METABOLIC ACIDOSIS AND ALKALOSIS H+-ATPASE OF RAT CEREBRAL MICROVESSELS
ON THE
Arshag D. Mooradian', Bahar Bastanib St. Louis V.A. Medical Center' and the Divisions of Endocrinology' and Nephrologyb, Department of Internal Medicine, St. Louis University School of Medicine, St. Louis, MO 63104 (U.S.A.) (Received in final form September 8,
1997)
Summary To
determine the role of the protontranslocating adenosine triphosphatase (H+-ATPase) of the blood-brain barrier, the density of the 31 Kd subunit of the vacuolar type H+-ATPase was quantitated in isolated rat cerebral microvessels with immunoblotting techniques. To establish the tissue specificity of the findings, synaptosomal membranes were Metabolic acidosis was induced with 1.5% also studied. ammonium chloride in drinking water for five days. Metabolic alkalosis was induced with 2.35% NaHCO, in drinking water and daily injections of 10 mg/Kg furosemide intraperitoneally for 5 days. The quantity of the 31 Kd subunit (in arbitrary units) in cerebral microvessels was significantly increased in acidosis (3.98 f 0.45) (~~0.05) and was significantly decreased in metabolic alkalosis (0.49 + 0.16) (p
Key Words: H+-ATPase, blood-brain barrier, metabolic acidosis,alkalosis
In a previous study we had identified vacuolar type H+-ATPase in microvessels immunoblotting and rat cerebral with immunocytochemical techniques as well as with N-ethylmaleimide (NEM) sensitivity of the ATPase activity.(l) We had concluded that Address Correspondence to: Arshag D. Mooradian, M.D., Division of Endocrinology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 Phone: (314) 577-8458, Fax: (314) 773-4567
2248
H+-ATPase
in Cerebral Microvessels
Vol. 61, No. 22, 1997
the vacuolar type H+-ATPase and not the gastric H, K - ATPase is present in cerebral tissue including cerebral microvessels. A potential role for this H+-ATPase activity in the physiology of acid-base balance of the central nervous system (CNS) was suspected but not documented. Neuronal membranes possess a Na+/H+ and a HCO,-/Clexchanger which transports H+ actively out of cells. The role of vacuolar H+-transporter in these cells is not known. In addition, the function of H+-ATPase in cerebral microvessels remains ill defined. In this tissue, H+-ATPase may have a role in actively transporting H+ out of the CNS. To further probe into the functional role of the H+-ATPase in the blood-brain barrier (HHH), we investigated changes in the quantity of the 31 Kd subunit of the vacuolar H+-ATPase activity in response to 5 days of acid or alkali loads. Synaptosomal membranes were also studied to validate the specificity of changes seen in cerebral microvessels. Materials and Methods Experimental Animals: Male Fischer 344 rats at 3 months of age were obtained from the Harlan Industries (Indiana). A group of control rats (n=70) maintained on laboratory chow and tap water ad libitum was compared to rats subjected to metabolic acidosis (n=70) or metabolic alkalosis (n=70). Metabolic acidosis was induced with 1.5% NH&l (280 mM) and 1% sucrose in drinking water for 5 days. The large number of animals was required for the studies on isolated microvessels. Metabolic alkalosis was induced with 2.35% NaHCO, (280 mM)and 1% sucrose in drinking water and daily intraperitoneal injection of furosemide (Sigma Co. St. Louis) 10 On the day of the experiment, the arterial mg/kg for 5 days. blood pH and bicarbonate content were measured. The blood was obtained from the aorta while the rats were under light pentobarbital anesthesia (40 mg/Kg intraperitoneally). Cerebral microvessel and synaptosomal membrane preparations: The cerebralmicrovessels were isolated as described previously(24). The purity of the microvessels was documented with more than 20-fold enrichment of gamma glutamyl transpeptidase activity, a biochemical marker of cerebral microvessels. Microscopic studies indicate that the percentage of contaminating cells in these preparations is less than 2%. The yield and purity data of rat cerebral have been microvessels reported in previous publications(2-4). The synaptosomal membrane fractions were prepared by the method of DeRobertis(5) using discontinuous sucrose gradient as previously described(6). The recovered membrane fraction from the sucrose gradient was washed in Tris buffer by centrifugation at 20,000 x g for 20 min twice. The enrichment of Na+-K+-ATPase activity in this fraction compared to total cerebral homogenates was approximately three-fold. There were no differences in the purity or yield of cerebral microvessels or synaptosomal preparations of the three groups of rats studied.
H+-ATPase inCerebral Microvessels
Vol. 61, No.22,1997
Gel electrophoresis
2249
and immunoblotting:
Rat cerebral microvessel and synaptosomal proteins (15 pg of each) were resolved on 11.25% sodium dodecyl sulfate polyacrylamide gels membrane by transferred to nitrocellulose (SDS-PAGE) and Membranes were probed with Eli, a mouse electroblotting. monoclonal antibody to the 31 Kd subunit of the vacuolar H+ATPase(7). (gift from Stephen Gluck, M.D., Washington University, St. Louis, MO). Enhanced chemiluminescence (ECL) Western blotting technique (ECL Kit) was used as described in the manufacturer's protocol (Amersham Co., Arlington Heights, IL). The density of the 31 Kd band was quantitated with densitometry. The linearity between the density of this band and the amount of proteins applied to the gel was established (data not shown). The quantitation of the 31 Kd band in arbitrary units of density was done with densitometer and the results of each band were normalized against internal control included within each gel. Statistical
Analysis:
All values are expressed as mean + SEM. Analysis of variance (one way ANOVA) was carried out with Bonferroni's correction for the statistical analysis of differences in study groups. pcO.05 was considered statistically significant.
Results On the day the rats were killed, the arterial blood pH in the acidotic group was 7.34 -+ 0.03, and plasma bicarbonate HC03content was 16.5 + 0.5 mM and the arterial blood pH in the alkalotic group was 7.48 +0.02 and the HCO, was 27.3 f 0.6 mM compared to control group with an arterial pH of 7.43 f 0.01 and plasma HCO; of 26.0 + 0.65 mM. The effect of metabolic acidosis or alkalosis on H+-ATPase activity was more demonstrable in immunoblotting studies. Figure 1A shows the immunoblot using the E,, monoclonal antibody. A prominent 31 Kd band of rat kidney cortical microvesicle protein is shown next to somewhat fainter bands of the same size in rat cerebral microvessels. The increased intensity of the band in acidotic animals (Lanes 3 and 4) compared to control (Lanes 1 and 2) or alkalotic rats (Lanes 5 and 6) is evident. The results of the quantitation of the density of the 31 Kd band are summarized in Figure 1B. The density of the 31 Kd band in alkalotic rats (0.49 + 0.16) was significantly reduced compared to control rats (1.77 + 0.73) p c 0.01, while in acidotic rats the density of the 31 Kd band was significantly increased (3.98 + 0.45) p < 0.01. The immunoblot of synaptosomalmembranes probed with E,,antibody is The shown in Figure 2A. A prominent 31 Kd band is also seen. intensity of the band in alkalotic rats (Lanes l-3) was reduced compared to control (Lanes 7-10) or acidotic rats (Lanes 4-6).
2250
H+-ATPasein Cerebral Microvessels
Vol. 61,No.22,1997
M. Wt. K
..
1
3
4
5
6
,s
0:
5 .
4
4
E
2
3
3
2
1 ‘j g
1
Control
Acidosis
Alkalosis
Fig. la Immunoblot of cerebral microvessels and renal cortical microvesicles (K) using E,, monoclonal antibody specific for the 31 Kd subunit of the vacuolar H+-ATPase pump. A prominent band at 31 Kd is seen. K, renal cortical microsomal protein, Lanes l-2 are control rats, Lanes 3-4 are acidotic rats and Lanes 5-6 are alkalotic rats. Fig. lb The mean f SEM density of 31 Kd subunit of vacuolar H+ microvessels. ATPase in cerebral n=7 in each group. *p
H+-ATPase in Cerebral Microvessels
Vol. 61, No.22,1997
2251
M. 1
2
3
4
Alkalosis
5
6
7
Acidosis
6
9
10
wt.
(Kd)
Control
Fig. 2a Immunoblot of synaptosomal membrane preparations using E,, monoclonal antibody specific for the 31 Kd subunit of vacuolar H+-ATPase pump. Lanes l-3 are from alkalotic rats, Lanes 4-6 are from acidotic rats, and Lanes 7-10 are from control rats. Fig. 2b The mean + SEM density of 31 Kd subunit of vacuolar H+ ATPase in synoptosomal membrane preparations. n=8 in each group. *p
2252
H+-ATPase
in
CerebralMicrovessels
Vol.61, No.22,1997
The results of the quantitation of the density of the 31 Kd band in synaptosomal membranes are summarized in Figure 2B. Metabolic alkalosis resulted in a significant decrease in 31 Kd band (0.62 _+ 0.12 vs 0.92 + 0.01; p<0.01) while metabolic acidosis was associated with insignificant increase in 31 Kd band of synaptosomal membranes (1.11 + 0.03). Discussion The results of the immunoblotting studies clearly indicate that the proton pump of the cerebral microvessels is modulated by the Thus it appears that systemic changes in acid-base balance. metabolic acidosis is associated with up regulation while metabolic alkalosis is associated with down regulation of the vacuolar H+Changes in synaptosomal membranes were similar, albeit ATPaSe. less robust compared to the changes seen in cerebral microvessels. It is noteworthy that the changes in H+-ATPase mass could be appreciated in a relatively short period of time (5 days) and in response to modest changes in acid-base balance. This is in contrast to the changes in H+-ATPase of renal tubular cells which in response to similar conditions do not demonstrate modulations of vacuolar H+-ATPase mass but rather the distribution of this enzyme within the cell is altered(8). Thus the mechanisms of the response of vacuolar proton pump in the blood-brain barrier or synaptosomal membranes are probably different from those operating in the kidney. Of concern is that the state of metabolic alkalosis could not be documented with significant changes in the bicarbonate content of the arterial blood on the day of the experiment, although the arterial blood pH was in alkalemic range. Despite this lack of documented change in bicarbonate, significant changes could be found which were attributed to alkalosis. The precise cause of this apparent discrepancy is not known. It is possible that the small changes in pH or high bicarbonate load in the drinking water per se may have been sufficient to alter H+-ATPase content. In this respect it is noteworthy that rats treated with a similar regimen show significant changes in H+-ATPase distribution in the kidney despite insignificant increase in the arterial blood pH(8). The responsiveness of the vacuolar H'-ATPase in the BBB to changes in acid-base balance suggests that this enzyme has a functional role. The literature on H+ transport across the BBB is somewhat controversial. Although previous studies have suggested that distribution of hydrogen ion (H+) or bicarbonate (HCO,-)between the cerebrospinal fluid (CSF) and blood is a passive process(9-12), some have found that neither H+ or HCO, in CSF were in electrochemical equilibrium during hypocapnic alkalosis(13). These observations suggest that these ions may cross the BBB by an active transport process(l3). However, the validity of these conclusions was subsequently questioned(9,14). Our findings of H+-ATPase in the BBB and its modulation by systemic changes in acid-base balance support the notion that an active transport component may contribute to the physiology of acid-base balance in the central nervous system. The precise localization of this enzyme on endothelial cell membranes is not known nor the direction of H+ transported by this pump is clear. It is noteworthy that our
Vol. 61, No. 22, 1997
H ’ -ATF’ase in Cerebral Microvessels
2253
previous work with immunohistological localization of H+-ATPase indicated intense activity at the choroid plexus(l). Future work should address the effect of systemic changes in pH on the H+In addition, studies ATPase activity of the choroid plexus. correlating the pH changes in the cerebrospinal fluid with ATPase levels in the microvessels will be helpful in clarifying the role of this Ht pump in the blood-brain barrier. Acknowledgments This work is supported by the medical research of the Department of Veterans Affairs (ADM). The authors thank Patricia Schneiderjohn and Liyeng Yang for excellent technical assistance. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
A.D. MOORADIAN and B. BASTANI, Brain Res 629 128-132 (1993). A.D. MOORADIAN, A. MORIN, L. CLIP, and H. HASPEL, Brain Res 551 145-149 (1990). A.D. MOORADIAN and P.J. SCARPACE, Brain Res 583 155-160 (1992). A.D. MOORADIAN and T.L. SMITH, Neurochem Res 17 233-237 (1992). E. DEROBERTIS, Science 156 907-914 (1967). A.D. MOORADIAN, F. DICKERSON, and T.L. SMITH, Neurochem Res 15 981-985 (1990). P.H. HEMKEN, G. XIAO-LI, W. ZHI-QIANG, 2. KUN, and S. GLUCK, J Biol Chem 267 9948-9957 (1992). B. BASTANI, H. PURCELL, P.H. HEMKEN, D. TRIGG, and S. GLUCK, J Clin Invest 88 126-136 (1991). T.F. HORNBEIN and E.G. PAVLIN, Am J Physiol 228 1145-1148 (1975). T.F. -HORNBEIN and E.G. PAVLIN, Am J Physiol 228 1149-1154 (1975). E.G. PAVLIN and T.F. HORNBEIN, Am J Physiol 228 1134-1140 (1975). E.G. PAVLIN and T.F. HORNBEIN, Am J Physiol 228 1145-1148 (1975). J.w. SEVERINGHAUS, R.A. MITCHELL, B.W. RICHARDSON, and M.M. SINGER, J Appl Physiol 18 1155-1166 (1963). L. GRANHOLM and B.X. SIESJO, Acta Physiol Scan 81 307-314 (1971).
Vol. 61, No. 22, pp. 2247-2253,
Copyright 01997 Elswier Printed
in the USA.
THE
1997 Inc.
All tights reserved
cum-3205/97
ELSEVIER
Science $17.00
+ .ofJ
PI1SOOZ43205(97)00927-2
EFFECT OF METABOLIC ACIDOSIS AND ALKALOSIS H+-ATPASE OF RAT CEREBRAL MICROVESSELS
ON THE
Arshag D. Mooradian', Bahar Bastanib St. Louis V.A. Medical Center' and the Divisions of Endocrinology' and Nephrologyb, Department of Internal Medicine, St. Louis University School of Medicine, St. Louis, MO 63104 (U.S.A.) (Received in final form September 8,
1997)
Summary To
determine the role of the protontranslocating adenosine triphosphatase (H+-ATPase) of the blood-brain barrier, the density of the 31 Kd subunit of the vacuolar type H+-ATPase was quantitated in isolated rat cerebral microvessels with immunoblotting techniques. To establish the tissue specificity of the findings, synaptosomal membranes were Metabolic acidosis was induced with 1.5% also studied. ammonium chloride in drinking water for five days. Metabolic alkalosis was induced with 2.35% NaHCO, in drinking water and daily injections of 10 mg/Kg furosemide intraperitoneally for 5 days. The quantity of the 31 Kd subunit (in arbitrary units) in cerebral microvessels was significantly increased in acidosis (3.98 f 0.45) (~~0.05) and was significantly decreased in metabolic alkalosis (0.49 + 0.16) (p
Key Words: H+-ATPase, blood-brain barrier, metabolic acidosis,alkalosis
In a previous study we had identified vacuolar type H+-ATPase in microvessels immunoblotting and rat cerebral with immunocytochemical techniques as well as with N-ethylmaleimide (NEM) sensitivity of the ATPase activity.(l) We had concluded that Address Correspondence to: Arshag D. Mooradian, M.D., Division of Endocrinology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 Phone: (314) 577-8458, Fax: (314) 773-4567
2248
H+-ATPase
in Cerebral Microvessels
Vol. 61, No. 22, 1997
the vacuolar type H+-ATPase and not the gastric H, K - ATPase is present in cerebral tissue including cerebral microvessels. A potential role for this H+-ATPase activity in the physiology of acid-base balance of the central nervous system (CNS) was suspected but not documented. Neuronal membranes possess a Na+/H+ and a HCO,-/Clexchanger which transports H+ actively out of cells. The role of vacuolar H+-transporter in these cells is not known. In addition, the function of H+-ATPase in cerebral microvessels remains ill defined. In this tissue, H+-ATPase may have a role in actively transporting H+ out of the CNS. To further probe into the functional role of the H+-ATPase in the blood-brain barrier (HHH), we investigated changes in the quantity of the 31 Kd subunit of the vacuolar H+-ATPase activity in response to 5 days of acid or alkali loads. Synaptosomal membranes were also studied to validate the specificity of changes seen in cerebral microvessels. Materials and Methods Experimental Animals: Male Fischer 344 rats at 3 months of age were obtained from the Harlan Industries (Indiana). A group of control rats (n=70) maintained on laboratory chow and tap water ad libitum was compared to rats subjected to metabolic acidosis (n=70) or metabolic alkalosis (n=70). Metabolic acidosis was induced with 1.5% NH&l (280 mM) and 1% sucrose in drinking water for 5 days. The large number of animals was required for the studies on isolated microvessels. Metabolic alkalosis was induced with 2.35% NaHCO, (280 mM)and 1% sucrose in drinking water and daily intraperitoneal injection of furosemide (Sigma Co. St. Louis) 10 On the day of the experiment, the arterial mg/kg for 5 days. blood pH and bicarbonate content were measured. The blood was obtained from the aorta while the rats were under light pentobarbital anesthesia (40 mg/Kg intraperitoneally). Cerebral microvessel and synaptosomal membrane preparations: The cerebralmicrovessels were isolated as described previously(24). The purity of the microvessels was documented with more than 20-fold enrichment of gamma glutamyl transpeptidase activity, a biochemical marker of cerebral microvessels. Microscopic studies indicate that the percentage of contaminating cells in these preparations is less than 2%. The yield and purity data of rat cerebral have been microvessels reported in previous publications(2-4). The synaptosomal membrane fractions were prepared by the method of DeRobertis(5) using discontinuous sucrose gradient as previously described(6). The recovered membrane fraction from the sucrose gradient was washed in Tris buffer by centrifugation at 20,000 x g for 20 min twice. The enrichment of Na+-K+-ATPase activity in this fraction compared to total cerebral homogenates was approximately three-fold. There were no differences in the purity or yield of cerebral microvessels or synaptosomal preparations of the three groups of rats studied.
H+-ATPase inCerebral Microvessels
Vol. 61, No.22,1997
Gel electrophoresis
2249
and immunoblotting:
Rat cerebral microvessel and synaptosomal proteins (15 pg of each) were resolved on 11.25% sodium dodecyl sulfate polyacrylamide gels membrane by transferred to nitrocellulose (SDS-PAGE) and Membranes were probed with Eli, a mouse electroblotting. monoclonal antibody to the 31 Kd subunit of the vacuolar H+ATPase(7). (gift from Stephen Gluck, M.D., Washington University, St. Louis, MO). Enhanced chemiluminescence (ECL) Western blotting technique (ECL Kit) was used as described in the manufacturer's protocol (Amersham Co., Arlington Heights, IL). The density of the 31 Kd band was quantitated with densitometry. The linearity between the density of this band and the amount of proteins applied to the gel was established (data not shown). The quantitation of the 31 Kd band in arbitrary units of density was done with densitometer and the results of each band were normalized against internal control included within each gel. Statistical
Analysis:
All values are expressed as mean + SEM. Analysis of variance (one way ANOVA) was carried out with Bonferroni's correction for the statistical analysis of differences in study groups. pcO.05 was considered statistically significant.
Results On the day the rats were killed, the arterial blood pH in the acidotic group was 7.34 -+ 0.03, and plasma bicarbonate HC03content was 16.5 + 0.5 mM and the arterial blood pH in the alkalotic group was 7.48 +0.02 and the HCO, was 27.3 f 0.6 mM compared to control group with an arterial pH of 7.43 f 0.01 and plasma HCO; of 26.0 + 0.65 mM. The effect of metabolic acidosis or alkalosis on H+-ATPase activity was more demonstrable in immunoblotting studies. Figure 1A shows the immunoblot using the E,, monoclonal antibody. A prominent 31 Kd band of rat kidney cortical microvesicle protein is shown next to somewhat fainter bands of the same size in rat cerebral microvessels. The increased intensity of the band in acidotic animals (Lanes 3 and 4) compared to control (Lanes 1 and 2) or alkalotic rats (Lanes 5 and 6) is evident. The results of the quantitation of the density of the 31 Kd band are summarized in Figure 1B. The density of the 31 Kd band in alkalotic rats (0.49 + 0.16) was significantly reduced compared to control rats (1.77 + 0.73) p c 0.01, while in acidotic rats the density of the 31 Kd band was significantly increased (3.98 + 0.45) p < 0.01. The immunoblot of synaptosomalmembranes probed with E,,antibody is The shown in Figure 2A. A prominent 31 Kd band is also seen. intensity of the band in alkalotic rats (Lanes l-3) was reduced compared to control (Lanes 7-10) or acidotic rats (Lanes 4-6).
2250
H+-ATPasein Cerebral Microvessels
Vol. 61,No.22,1997
M. Wt. K
..
1
3
4
5
6
,s
0:
5 .
4
4
E
2
3
3
2
1 ‘j g
1
Control
Acidosis
Alkalosis
Fig. la Immunoblot of cerebral microvessels and renal cortical microvesicles (K) using E,, monoclonal antibody specific for the 31 Kd subunit of the vacuolar H+-ATPase pump. A prominent band at 31 Kd is seen. K, renal cortical microsomal protein, Lanes l-2 are control rats, Lanes 3-4 are acidotic rats and Lanes 5-6 are alkalotic rats. Fig. lb The mean f SEM density of 31 Kd subunit of vacuolar H+ microvessels. ATPase in cerebral n=7 in each group. *p
H+-ATPase in Cerebral Microvessels
Vol. 61, No.22,1997
2251
M. 1
2
3
4
Alkalosis
5
6
7
Acidosis
6
9
10
wt.
(Kd)
Control
Fig. 2a Immunoblot of synaptosomal membrane preparations using E,, monoclonal antibody specific for the 31 Kd subunit of vacuolar H+-ATPase pump. Lanes l-3 are from alkalotic rats, Lanes 4-6 are from acidotic rats, and Lanes 7-10 are from control rats. Fig. 2b The mean + SEM density of 31 Kd subunit of vacuolar H+ ATPase in synoptosomal membrane preparations. n=8 in each group. *p
2252
H+-ATPase
in
CerebralMicrovessels
Vol.61, No.22,1997
The results of the quantitation of the density of the 31 Kd band in synaptosomal membranes are summarized in Figure 2B. Metabolic alkalosis resulted in a significant decrease in 31 Kd band (0.62 _+ 0.12 vs 0.92 + 0.01; p<0.01) while metabolic acidosis was associated with insignificant increase in 31 Kd band of synaptosomal membranes (1.11 + 0.03). Discussion The results of the immunoblotting studies clearly indicate that the proton pump of the cerebral microvessels is modulated by the Thus it appears that systemic changes in acid-base balance. metabolic acidosis is associated with up regulation while metabolic alkalosis is associated with down regulation of the vacuolar H+Changes in synaptosomal membranes were similar, albeit ATPaSe. less robust compared to the changes seen in cerebral microvessels. It is noteworthy that the changes in H+-ATPase mass could be appreciated in a relatively short period of time (5 days) and in response to modest changes in acid-base balance. This is in contrast to the changes in H+-ATPase of renal tubular cells which in response to similar conditions do not demonstrate modulations of vacuolar H+-ATPase mass but rather the distribution of this enzyme within the cell is altered(8). Thus the mechanisms of the response of vacuolar proton pump in the blood-brain barrier or synaptosomal membranes are probably different from those operating in the kidney. Of concern is that the state of metabolic alkalosis could not be documented with significant changes in the bicarbonate content of the arterial blood on the day of the experiment, although the arterial blood pH was in alkalemic range. Despite this lack of documented change in bicarbonate, significant changes could be found which were attributed to alkalosis. The precise cause of this apparent discrepancy is not known. It is possible that the small changes in pH or high bicarbonate load in the drinking water per se may have been sufficient to alter H+-ATPase content. In this respect it is noteworthy that rats treated with a similar regimen show significant changes in H+-ATPase distribution in the kidney despite insignificant increase in the arterial blood pH(8). The responsiveness of the vacuolar H'-ATPase in the BBB to changes in acid-base balance suggests that this enzyme has a functional role. The literature on H+ transport across the BBB is somewhat controversial. Although previous studies have suggested that distribution of hydrogen ion (H+) or bicarbonate (HCO,-)between the cerebrospinal fluid (CSF) and blood is a passive process(9-12), some have found that neither H+ or HCO, in CSF were in electrochemical equilibrium during hypocapnic alkalosis(13). These observations suggest that these ions may cross the BBB by an active transport process(l3). However, the validity of these conclusions was subsequently questioned(9,14). Our findings of H+-ATPase in the BBB and its modulation by systemic changes in acid-base balance support the notion that an active transport component may contribute to the physiology of acid-base balance in the central nervous system. The precise localization of this enzyme on endothelial cell membranes is not known nor the direction of H+ transported by this pump is clear. It is noteworthy that our
Vol. 61, No. 22, 1997
H ’ -ATF’ase in Cerebral Microvessels
2253
previous work with immunohistological localization of H+-ATPase indicated intense activity at the choroid plexus(l). Future work should address the effect of systemic changes in pH on the H+In addition, studies ATPase activity of the choroid plexus. correlating the pH changes in the cerebrospinal fluid with ATPase levels in the microvessels will be helpful in clarifying the role of this Ht pump in the blood-brain barrier. Acknowledgments This work is supported by the medical research of the Department of Veterans Affairs (ADM). The authors thank Patricia Schneiderjohn and Liyeng Yang for excellent technical assistance. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
A.D. MOORADIAN and B. BASTANI, Brain Res 629 128-132 (1993). A.D. MOORADIAN, A. MORIN, L. CLIP, and H. HASPEL, Brain Res 551 145-149 (1990). A.D. MOORADIAN and P.J. SCARPACE, Brain Res 583 155-160 (1992). A.D. MOORADIAN and T.L. SMITH, Neurochem Res 17 233-237 (1992). E. DEROBERTIS, Science 156 907-914 (1967). A.D. MOORADIAN, F. DICKERSON, and T.L. SMITH, Neurochem Res 15 981-985 (1990). P.H. HEMKEN, G. XIAO-LI, W. ZHI-QIANG, 2. KUN, and S. GLUCK, J Biol Chem 267 9948-9957 (1992). B. BASTANI, H. PURCELL, P.H. HEMKEN, D. TRIGG, and S. GLUCK, J Clin Invest 88 126-136 (1991). T.F. HORNBEIN and E.G. PAVLIN, Am J Physiol 228 1145-1148 (1975). T.F. -HORNBEIN and E.G. PAVLIN, Am J Physiol 228 1149-1154 (1975). E.G. PAVLIN and T.F. HORNBEIN, Am J Physiol 228 1134-1140 (1975). E.G. PAVLIN and T.F. HORNBEIN, Am J Physiol 228 1145-1148 (1975). J.w. SEVERINGHAUS, R.A. MITCHELL, B.W. RICHARDSON, and M.M. SINGER, J Appl Physiol 18 1155-1166 (1963). L. GRANHOLM and B.X. SIESJO, Acta Physiol Scan 81 307-314 (1971).