- Email: [email protected]
Insulin action on brain microvessels; Effect on alkaline phosphatase
BIOCHEMICAL
Vol. 150, No. 2, 1988 January 29, 1988
INSULIN
R.E.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 583-590
ACTION ON BRAIN MICROVESSELS; ALKALINE PHOSPHATASE Catalan'*. B.G.
A.M. Miguel'
EFFECT ON
Martfnez2. M.D. Aragones' and A. Robles 2
1 Departamento
de Bioquimica y Biologia Molecular Centro de Biologia Molecular (CSIC-UAM) Universidad Aut6noma de Madrid, E-28049, Madrid, Spain and 2Departamento de Bioquimica, Facultad de Ciencias Universidad Complutense, E-28040. Madrid, Spain Received December 4, 1987
SUMMARY: The effects of insulin on brain alkaline phosphatase activity have been examined. Insulin inhibited the activity of alkaline phosphatase on brain microvessels . . vitro experiments. The inhibition observed was of the ni:-cA:petitive type. These observations indicate that the hormone is able to induce neurochemical modifications revealed in this case as chanses in the phosphate transfer enzymes in brain microvessels. 0 1988
Academic
Press.
Inc.
Until recently, the central nervous system has not been generally considered to be a target tissue for insulin action and research on the effects of the hormone on nervous tissue has been a matter of controversy. The demonstration of the presence of insulin and specific insulin receptors in the brain have reevaluated the role played by insulin in the central nervous system (1). In this regard, the abundant receptors for insulin in brain microvessels that make up the blood-brain barrier have a capacity for binding insulin rivaling that of the liver plasma membrane (2); and, although the exact role of the microvessel insulin receptor remains unknown, it is quite logical to imagine a possible relationship between insulin and the physiology of the blood-brain barrier. Several studies have led to the suggestion that insulin may function as a neuromodulator and/or * To whom correspondence
should
be addressed 0006-291X/88
583
$1.50
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
neurotransmitter (3).Likewise a very recent study has demonstrated that neural cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions, a fact that strongly qualifies insulin as a neurotransmitter (4). Some reports have indicated the possibility of a direct action of insulin on ornithine decarboxylase (5,6) and acetylcholinesterase (7) from rat brain adding new data on the neuromodufator action of insulin. On the other hand, it has been reported that alkaline phosphatase activity is one of the main enzyme activities found in the brain microvessels (8). The function of the enzyme in the walls of blood vessels is unknown although the suggestion has been made that it may be involved with transendothelial transport and vascular permeability (8). Accordingly, the present studies were undertaken to determine if insulin is able to affect alkaline phosphatase in brain microvessels.
MATERIALS Preparation
AND METHODS
of microvesselq
Routinely, the bovine brains were obtained from a local slaughterhouse for each experiment and were used to prepare microvesssels, were used immediately. For optimum which preservation of metabolic activity the brains were transported to the laboratory in cold oxiqenated buffer (Krebs-Ringerminimize the effects of glucose (10 mM1 buffer, pH 7.2) to previously anoxia. The method was a slight modification of described methods (9.10). Pial vessels were carefully removed and discarded: the pieces of cerebral cortical qray matter were homogenized by hand-driven pestle (10 strokes 1 in 1 vol of ice-cold Krebs-Ringer-bicarbonate buffer, pH 7.2, containing 10 mM glucose, 1% bovine serum albumin and 15 mM HEPES. The homogenate was poured onto a 150 urn pore-size nylon sieve. The pellet consisted of two layers, the upper one containing finely divided material and the lower one with not entirely disrupted neuronic material. After disposal of supernatant fluid, the upper layer was dispersed in a small volume of the last buffer and removed with a Pasteur pipet. The lower layer was homogenized in 3 vol of fresh medium (10 strokes by hand) and centrifuqed as above: divided the upper layer of finely particles was removed and added to the remaining the first; granular bottom layer was dispersed in 1 vol medium by homogenization (10 This was added to the combined strokes). particle suspension from the two former centrifugations. The overall mixture was further disrupted by homogenization (10 strokes) and centrifuged at 1000 x g for 10 min. The resulting pellet was homogenized in 2 vol of sucrose 0.25 M. This suspension was centrifuged at 15000 x g for 10 min; the pellet was resuspended in 0.25 M sucrose and layered on a sucrose 584
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
density gradient consisting of 1 M and 1.5 M of sucrose (3 ml each) and centrifuged at 13500 x g for 10 min. The final pellet (capillary-enriched fraction) was homogenized (1:30) in a Krebs-Ringer-glucose buffer, pH 7.4 and the homogenate was utilized for determining the alkaline phosphatase activity.
Alkaline phosphatase using disodium activity was measured phenylphosphate as substrate. 0.4 ml of homogenate (180 ug protein) were incubated in a medium containing: 10 mM sodium bicarbonate buffer, pH 10, 5 mM substrate in the presence or the absence of insulin or vanadate in a total volume of 6.8 ml. The reaction was started by the addition of the substrate, disodium phenylphosphate. The mixture was incubated at 37OC for 30 min. Then, the reaction was stopped by addition of Folin reagent (11). After centrifugation at 3000 x g for 5 min, supernatants were treated with 20% Na CO and the phenol released was measured spectrophotometricJll? at 680 nm. Control lacking the enzyme as a blank was used. Protein concentration was measured by the method of Lowry et al. (12) with bovine serum albumin as the standard. A unit of alkaline phosphatase activity was defined as the formation of ug of phenol per min per ug protein.
Student's t-test was used to test the significance differences between means. Differences with a P value of than 0.05 were considered statisticallv siqnificant. RESULTS
of less
AND DISCUSSION
The availability of isolated brain microvessels provides the opportunity to study the structural and enzymatic properties of the blood-brain barrier at a biochemical level; in this regard it can be pointed out that the purity of brain microvesels used in these experiments was quite satisfactory for both morphological and biochemical criteria (Fig. 1). The effect of insulin on alkaline phosphatase activity is illustrated in Fig. 2 which shows a dose-response curve; it can be seen that a significant inhibition of the enzyme takes place at all the concentrations of insulin tested. In Fig. 3 the time-course is shown; the maximum inhibition was observed a 30 min incubation. This time is coincident with some data on the binding of insulin in bovine cerebral microvessels because in this case, binding is more rapid at 30X and reaches a maximum within 30 min (2). A slight decrease in the amount of specific binding was observed between 45 and 75 min at 3OOC. This phenomenon has also been reported for insulin binding to liver plasma membranes (13). This data could explain why our results indicate a lesser inhibition after 30 min incubation. 585
Vol. 150, No. 2, 1988
Fig.
BIOCHEMICAL
1. Photomicroqraph of isolated bovine microvessels consisting primarily Magnification x 400.
Taking
into
account
hat; been described addition, an in reported
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(IC;),
that
one of
the
brain cortical capillaries. of
main effects
(311the alkaline phosphatase (14,15) insulin-like effect of vanadate
a comparison
was carried
out.
The
of vanadate and that, has been comparative
-/:+y& i l “%,,,, * 1
,
-8
10
I -7
10
10
I
1o-5
10
“***+
-4
10
-3
(MI
Concentration
Fig.
,
-0
phosphatase 2. Dose dependent inhibition of alkaline Alkaline phoephatase activity is activity by insulin. shown as a function of insulin ( l ) or vanadate ( o ) indicated. deviations are concentration. Standard Control value: 3.6 + 0.1 units. 586
Vol.
150,
No.
2, 1988
BIOCHEMICAL
5
AND
15
30 Time
Fig.
BIOPHYSICAL
RESEARCH
45
COMMUNICATIONS
60
(min)
3. Time course of the action of insulin on alkaline phosphatase activity. Microvessels were incubated $ the absence or the presence of insulin 110 M concentration). Standard deviations are indicated.
results
are
takes
place in In order
shown
experiments these results designed alkaline
in
Fig.
2 and demonstrate
a similar fashion. to elucidate
were carried possible.
out Fig.
this
that
an
inhibition
inhibition
phenomenon, to make a better understanding of 4 shows the results of experiments
to test how insulin alters phosphatase. A non-competitive
the kinetic inhibition
parameters of was observed
A
-1
12
34
56
78910
12345678910
SUBSTRATE
Fig.
M-l
4. Comparison of the double-reciprocal plots of alkaline phosphatase activity from insulin-treated (A) or vanadate-treated (Bi brain microvessels. Alkaline phosphatase activity was assayed at different substrate concentrations and at different insulin or vanadate concentrations (Hf. Control = saline. 587
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the case of insulin (part A) while a competitive scheme of variation was observed in the case of vanadate (part B). In this regard, the effect of vanadate on alkaline phosphatase has already been described in some tissues. Likewise a competitive inhibition effect has been described in intestinal segments and matrix vesicle-enriched fractions from chicken ewphyseal cartilage (14,15) in agreement with our results herein described. For a long time the function of selective transport of substance from the blood to the brain has been attributed to cerebral microvessels (17-191. Since some metabolites appear to be transported across the blood-brain barrier, metabolic studies of microvessels isolated from brain lead to the may understanding of the factors that regulate permeability of the blood-brain barrier. Some authors (20) have demonstrated that brain microvessels have extremely high levels of butyrylcholinesterase, y-glutamyl transpeptidase and alkaline phosphatase compared with other enzymes, indicating this fact roles in the blood-brain that these enzymes may play important As has been indicated earlier, barrier functioning (20). alkaline phosphatase has been demonstrated to occur in cerebral capillaries but scarce data available (fundamentally the histochemical localization) do not provide enough support to for the enzyme in the blood-brain adscribe a specific function barrier: the suggestion has been made that it may be involved with transendothelial transport and vascular permeability. alkaline Moreover, we must emphasize that, in general, although phosphatase was described many years ago there is no clear Alkaline phosphatase is evidence for its physiological function. known to catalyze the hydrolysis of various phosphate compounds. phosphate transferase activity Hydrolysis of phosphoesters (21), (22) and transport of phosphate (23) have been suggested as The specific effects of functions of alkaline phosphatase. insulin on alkaline phosphatase activity have not been described (although not in detail and some reports in the literature in agreement with our closely related to our experiments) are slightly results because insulin decreased alkaline phosphatase in in vitro experiments in rat hepatoma cells (24). It is important to point out that alkaline phospbatase inactivation of enzymes be involved in the activation or in the metabolism dephosphorylation and may have a role 588
may by of
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Since Qyridoxal phosphate is a (25). as glutamate decarboxylase and cofactor such involved in the metabolism of glutamate enzymes is quite logical to imagine a possible neural tissue, it relation between alkaline Qhosphatase and neural enzymes that Pyridoxal can utilize pyridoxal phosphate as a cofactor. phosphate Qhosphatase activity has been demonstrated in purified sheeQ brain alkaline phosphatase (16). Likewise, in our case it has been detected that in brain capillaries pyridoxal phosphate is a good substrate for alkaline phosphatase, the affinity value of which is approximately five times higher than in the case of phenylphosphate utilized as a substrate (not shown). via In view of these facts we can speculate that insulin, alkaline Qhosphatase can modulate some processes involved in the functioning of the nervous system. The Qhysiological significance of this relationship remains to be elucidated. However, it is clear that these first results concerning the effects of insulin on alkaline phosphatase activity in brain microvessels represent first, an example of the neural target for insulin action and second, the alterations of a key enzyme of the blood-brain barrier by the hormone. pyridoxal
phosphate of enzymes transaminase,
ACKNOWLEDGMENTS: The authors are indebted to Mr. J. Palacin for his valuable assistance. We gratefully acknowledge support and encouragement given by Prof. F. Mayor. This work was supported in part by grants from the FIS and CAICYT.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Pernov, N. (1983) Pharmacol. Rev. 35, 85-141. Haskell, J.F., Meezan, E. and Pillion, D.J. (1985) Am. J. Physiol. 248, E 115-E 125. Havrankova, J., Brownstein, M. and Roth, J. (1981) Diabetologia 20, 268-273. Clarke, D.W., Mudd, L., Floyd, F.T. Jr., Fields, M. and Raizada, M.K. (1986) J. Neurochem. 47, 831-836. Roger, L.J. and Fellows, R.E. (1980) Endocrinology 106, 619-625. Yang, J.W., Raizada, M.K. and Fellows, R.E. (19811 J. Neurochem. 36. 1050-1057. Catalan, R.E., Martinez, A.M., Mata, F. and Aragones, M. D. (1981) Biochem. Biophys. Res. Commun. 101, 1216-1220. Vorbrodt, A.W., Lossinsky, A.S. and Wisniewski, H.M. (1986) Dev. Neurosci. 8, l-3. Goldstein, G.W., Wolinsky. N.S., Csejtey, 3. and Diamond, I. (1976) Brain Res. 110, 361-365. Palmer, G.C. (1983) Life Sci. 32, 365-374. Lowry, O-H., Roberts, N.R., Wu, M.L., Hixo, W.S. and Crawford, W.S. (1954) J. Biol. Chem. 207, 19-37. 589
Vol. 150, No. 2, 1988
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R. J. (1951) J. Biol. Chem. 1993, 265-275. Almira, E. and Reddy, W. (1979) Endocrinology 104, 205-211. Iiizumi. Y., Hirano, K., Sugiura, M., Iino, S., Suzuki, H. and Oda, T. (1986) Chem. Pharm. Bull. 31, 772-775. Register, T.C. and Wuthier, R.E. (1984) J. Biol. Chem. 259, 3511-3518. Shechter, Y. and Karlish, S.J.D. (1980) Nature 284, 556-558. Dawson. H. (1964) General Physiology. 3rd ed . Chuchill, London. pp. 492. Crone, C. (1965) J. Physiol. (London) 181, 103-113. Spatz, M., Mrsrulja, B.B., Micic, D., Mrsulja, B.J. and Klatzo. I. (1977) Brain Res. 120, 141-145. Diuricic. B.M. and Mrsulja, B.B. (1977) Brain Res. 138, 561-564. Moos, F. and Glazier, H.S. (1972) Camp. Biochem. Physiol. 42, 321-336. Wilson, I.B., Dayan, J. and Cry, K. (1964) J. Biol. Chem. 239, 4182-4188. Petilclerc, C. and Planta, G.E. (1981) Can. J. Physiol. 59, 34-39. Sorimachi, K. and Yasumura. Y. (1986) Biochim. Biophys. Acta 885, 272-281. Goldstein, D.J. and Harris, H. (1981) J. Neurochem. 36, 53-57. Dorai, D.T. and Bachhawat. B.K. (1977) J. Neurochem. 29, 503-511.
590
Vol. 150, No. 2, 1988 January 29, 1988
INSULIN
R.E.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 583-590
ACTION ON BRAIN MICROVESSELS; ALKALINE PHOSPHATASE Catalan'*. B.G.
A.M. Miguel'
EFFECT ON
Martfnez2. M.D. Aragones' and A. Robles 2
1 Departamento
de Bioquimica y Biologia Molecular Centro de Biologia Molecular (CSIC-UAM) Universidad Aut6noma de Madrid, E-28049, Madrid, Spain and 2Departamento de Bioquimica, Facultad de Ciencias Universidad Complutense, E-28040. Madrid, Spain Received December 4, 1987
SUMMARY: The effects of insulin on brain alkaline phosphatase activity have been examined. Insulin inhibited the activity of alkaline phosphatase on brain microvessels . . vitro experiments. The inhibition observed was of the ni:-cA:petitive type. These observations indicate that the hormone is able to induce neurochemical modifications revealed in this case as chanses in the phosphate transfer enzymes in brain microvessels. 0 1988
Academic
Press.
Inc.
Until recently, the central nervous system has not been generally considered to be a target tissue for insulin action and research on the effects of the hormone on nervous tissue has been a matter of controversy. The demonstration of the presence of insulin and specific insulin receptors in the brain have reevaluated the role played by insulin in the central nervous system (1). In this regard, the abundant receptors for insulin in brain microvessels that make up the blood-brain barrier have a capacity for binding insulin rivaling that of the liver plasma membrane (2); and, although the exact role of the microvessel insulin receptor remains unknown, it is quite logical to imagine a possible relationship between insulin and the physiology of the blood-brain barrier. Several studies have led to the suggestion that insulin may function as a neuromodulator and/or * To whom correspondence
should
be addressed 0006-291X/88
583
$1.50
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
neurotransmitter (3).Likewise a very recent study has demonstrated that neural cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions, a fact that strongly qualifies insulin as a neurotransmitter (4). Some reports have indicated the possibility of a direct action of insulin on ornithine decarboxylase (5,6) and acetylcholinesterase (7) from rat brain adding new data on the neuromodufator action of insulin. On the other hand, it has been reported that alkaline phosphatase activity is one of the main enzyme activities found in the brain microvessels (8). The function of the enzyme in the walls of blood vessels is unknown although the suggestion has been made that it may be involved with transendothelial transport and vascular permeability (8). Accordingly, the present studies were undertaken to determine if insulin is able to affect alkaline phosphatase in brain microvessels.
MATERIALS Preparation
AND METHODS
of microvesselq
Routinely, the bovine brains were obtained from a local slaughterhouse for each experiment and were used to prepare microvesssels, were used immediately. For optimum which preservation of metabolic activity the brains were transported to the laboratory in cold oxiqenated buffer (Krebs-Ringerminimize the effects of glucose (10 mM1 buffer, pH 7.2) to previously anoxia. The method was a slight modification of described methods (9.10). Pial vessels were carefully removed and discarded: the pieces of cerebral cortical qray matter were homogenized by hand-driven pestle (10 strokes 1 in 1 vol of ice-cold Krebs-Ringer-bicarbonate buffer, pH 7.2, containing 10 mM glucose, 1% bovine serum albumin and 15 mM HEPES. The homogenate was poured onto a 150 urn pore-size nylon sieve. The pellet consisted of two layers, the upper one containing finely divided material and the lower one with not entirely disrupted neuronic material. After disposal of supernatant fluid, the upper layer was dispersed in a small volume of the last buffer and removed with a Pasteur pipet. The lower layer was homogenized in 3 vol of fresh medium (10 strokes by hand) and centrifuqed as above: divided the upper layer of finely particles was removed and added to the remaining the first; granular bottom layer was dispersed in 1 vol medium by homogenization (10 This was added to the combined strokes). particle suspension from the two former centrifugations. The overall mixture was further disrupted by homogenization (10 strokes) and centrifuged at 1000 x g for 10 min. The resulting pellet was homogenized in 2 vol of sucrose 0.25 M. This suspension was centrifuged at 15000 x g for 10 min; the pellet was resuspended in 0.25 M sucrose and layered on a sucrose 584
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
density gradient consisting of 1 M and 1.5 M of sucrose (3 ml each) and centrifuged at 13500 x g for 10 min. The final pellet (capillary-enriched fraction) was homogenized (1:30) in a Krebs-Ringer-glucose buffer, pH 7.4 and the homogenate was utilized for determining the alkaline phosphatase activity.
Alkaline phosphatase using disodium activity was measured phenylphosphate as substrate. 0.4 ml of homogenate (180 ug protein) were incubated in a medium containing: 10 mM sodium bicarbonate buffer, pH 10, 5 mM substrate in the presence or the absence of insulin or vanadate in a total volume of 6.8 ml. The reaction was started by the addition of the substrate, disodium phenylphosphate. The mixture was incubated at 37OC for 30 min. Then, the reaction was stopped by addition of Folin reagent (11). After centrifugation at 3000 x g for 5 min, supernatants were treated with 20% Na CO and the phenol released was measured spectrophotometricJll? at 680 nm. Control lacking the enzyme as a blank was used. Protein concentration was measured by the method of Lowry et al. (12) with bovine serum albumin as the standard. A unit of alkaline phosphatase activity was defined as the formation of ug of phenol per min per ug protein.
Student's t-test was used to test the significance differences between means. Differences with a P value of than 0.05 were considered statisticallv siqnificant. RESULTS
of less
AND DISCUSSION
The availability of isolated brain microvessels provides the opportunity to study the structural and enzymatic properties of the blood-brain barrier at a biochemical level; in this regard it can be pointed out that the purity of brain microvesels used in these experiments was quite satisfactory for both morphological and biochemical criteria (Fig. 1). The effect of insulin on alkaline phosphatase activity is illustrated in Fig. 2 which shows a dose-response curve; it can be seen that a significant inhibition of the enzyme takes place at all the concentrations of insulin tested. In Fig. 3 the time-course is shown; the maximum inhibition was observed a 30 min incubation. This time is coincident with some data on the binding of insulin in bovine cerebral microvessels because in this case, binding is more rapid at 30X and reaches a maximum within 30 min (2). A slight decrease in the amount of specific binding was observed between 45 and 75 min at 3OOC. This phenomenon has also been reported for insulin binding to liver plasma membranes (13). This data could explain why our results indicate a lesser inhibition after 30 min incubation. 585
Vol. 150, No. 2, 1988
Fig.
BIOCHEMICAL
1. Photomicroqraph of isolated bovine microvessels consisting primarily Magnification x 400.
Taking
into
account
hat; been described addition, an in reported
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(IC;),
that
one of
the
brain cortical capillaries. of
main effects
(311the alkaline phosphatase (14,15) insulin-like effect of vanadate
a comparison
was carried
out.
The
of vanadate and that, has been comparative
-/:+y& i l “%,,,, * 1
,
-8
10
I -7
10
10
I
1o-5
10
“***+
-4
10
-3
(MI
Concentration
Fig.
,
-0
phosphatase 2. Dose dependent inhibition of alkaline Alkaline phoephatase activity is activity by insulin. shown as a function of insulin ( l ) or vanadate ( o ) indicated. deviations are concentration. Standard Control value: 3.6 + 0.1 units. 586
Vol.
150,
No.
2, 1988
BIOCHEMICAL
5
AND
15
30 Time
Fig.
BIOPHYSICAL
RESEARCH
45
COMMUNICATIONS
60
(min)
3. Time course of the action of insulin on alkaline phosphatase activity. Microvessels were incubated $ the absence or the presence of insulin 110 M concentration). Standard deviations are indicated.
results
are
takes
place in In order
shown
experiments these results designed alkaline
in
Fig.
2 and demonstrate
a similar fashion. to elucidate
were carried possible.
out Fig.
this
that
an
inhibition
inhibition
phenomenon, to make a better understanding of 4 shows the results of experiments
to test how insulin alters phosphatase. A non-competitive
the kinetic inhibition
parameters of was observed
A
-1
12
34
56
78910
12345678910
SUBSTRATE
Fig.
M-l
4. Comparison of the double-reciprocal plots of alkaline phosphatase activity from insulin-treated (A) or vanadate-treated (Bi brain microvessels. Alkaline phosphatase activity was assayed at different substrate concentrations and at different insulin or vanadate concentrations (Hf. Control = saline. 587
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the case of insulin (part A) while a competitive scheme of variation was observed in the case of vanadate (part B). In this regard, the effect of vanadate on alkaline phosphatase has already been described in some tissues. Likewise a competitive inhibition effect has been described in intestinal segments and matrix vesicle-enriched fractions from chicken ewphyseal cartilage (14,15) in agreement with our results herein described. For a long time the function of selective transport of substance from the blood to the brain has been attributed to cerebral microvessels (17-191. Since some metabolites appear to be transported across the blood-brain barrier, metabolic studies of microvessels isolated from brain lead to the may understanding of the factors that regulate permeability of the blood-brain barrier. Some authors (20) have demonstrated that brain microvessels have extremely high levels of butyrylcholinesterase, y-glutamyl transpeptidase and alkaline phosphatase compared with other enzymes, indicating this fact roles in the blood-brain that these enzymes may play important As has been indicated earlier, barrier functioning (20). alkaline phosphatase has been demonstrated to occur in cerebral capillaries but scarce data available (fundamentally the histochemical localization) do not provide enough support to for the enzyme in the blood-brain adscribe a specific function barrier: the suggestion has been made that it may be involved with transendothelial transport and vascular permeability. alkaline Moreover, we must emphasize that, in general, although phosphatase was described many years ago there is no clear Alkaline phosphatase is evidence for its physiological function. known to catalyze the hydrolysis of various phosphate compounds. phosphate transferase activity Hydrolysis of phosphoesters (21), (22) and transport of phosphate (23) have been suggested as The specific effects of functions of alkaline phosphatase. insulin on alkaline phosphatase activity have not been described (although not in detail and some reports in the literature in agreement with our closely related to our experiments) are slightly results because insulin decreased alkaline phosphatase in in vitro experiments in rat hepatoma cells (24). It is important to point out that alkaline phospbatase inactivation of enzymes be involved in the activation or in the metabolism dephosphorylation and may have a role 588
may by of
Vol. 150, No. 2, 1988
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Since Qyridoxal phosphate is a (25). as glutamate decarboxylase and cofactor such involved in the metabolism of glutamate enzymes is quite logical to imagine a possible neural tissue, it relation between alkaline Qhosphatase and neural enzymes that Pyridoxal can utilize pyridoxal phosphate as a cofactor. phosphate Qhosphatase activity has been demonstrated in purified sheeQ brain alkaline phosphatase (16). Likewise, in our case it has been detected that in brain capillaries pyridoxal phosphate is a good substrate for alkaline phosphatase, the affinity value of which is approximately five times higher than in the case of phenylphosphate utilized as a substrate (not shown). via In view of these facts we can speculate that insulin, alkaline Qhosphatase can modulate some processes involved in the functioning of the nervous system. The Qhysiological significance of this relationship remains to be elucidated. However, it is clear that these first results concerning the effects of insulin on alkaline phosphatase activity in brain microvessels represent first, an example of the neural target for insulin action and second, the alterations of a key enzyme of the blood-brain barrier by the hormone. pyridoxal
phosphate of enzymes transaminase,
ACKNOWLEDGMENTS: The authors are indebted to Mr. J. Palacin for his valuable assistance. We gratefully acknowledge support and encouragement given by Prof. F. Mayor. This work was supported in part by grants from the FIS and CAICYT.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Pernov, N. (1983) Pharmacol. Rev. 35, 85-141. Haskell, J.F., Meezan, E. and Pillion, D.J. (1985) Am. J. Physiol. 248, E 115-E 125. Havrankova, J., Brownstein, M. and Roth, J. (1981) Diabetologia 20, 268-273. Clarke, D.W., Mudd, L., Floyd, F.T. Jr., Fields, M. and Raizada, M.K. (1986) J. Neurochem. 47, 831-836. Roger, L.J. and Fellows, R.E. (1980) Endocrinology 106, 619-625. Yang, J.W., Raizada, M.K. and Fellows, R.E. (19811 J. Neurochem. 36. 1050-1057. Catalan, R.E., Martinez, A.M., Mata, F. and Aragones, M. D. (1981) Biochem. Biophys. Res. Commun. 101, 1216-1220. Vorbrodt, A.W., Lossinsky, A.S. and Wisniewski, H.M. (1986) Dev. Neurosci. 8, l-3. Goldstein, G.W., Wolinsky. N.S., Csejtey, 3. and Diamond, I. (1976) Brain Res. 110, 361-365. Palmer, G.C. (1983) Life Sci. 32, 365-374. Lowry, O-H., Roberts, N.R., Wu, M.L., Hixo, W.S. and Crawford, W.S. (1954) J. Biol. Chem. 207, 19-37. 589
Vol. 150, No. 2, 1988
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R. J. (1951) J. Biol. Chem. 1993, 265-275. Almira, E. and Reddy, W. (1979) Endocrinology 104, 205-211. Iiizumi. Y., Hirano, K., Sugiura, M., Iino, S., Suzuki, H. and Oda, T. (1986) Chem. Pharm. Bull. 31, 772-775. Register, T.C. and Wuthier, R.E. (1984) J. Biol. Chem. 259, 3511-3518. Shechter, Y. and Karlish, S.J.D. (1980) Nature 284, 556-558. Dawson. H. (1964) General Physiology. 3rd ed . Chuchill, London. pp. 492. Crone, C. (1965) J. Physiol. (London) 181, 103-113. Spatz, M., Mrsrulja, B.B., Micic, D., Mrsulja, B.J. and Klatzo. I. (1977) Brain Res. 120, 141-145. Diuricic. B.M. and Mrsulja, B.B. (1977) Brain Res. 138, 561-564. Moos, F. and Glazier, H.S. (1972) Camp. Biochem. Physiol. 42, 321-336. Wilson, I.B., Dayan, J. and Cry, K. (1964) J. Biol. Chem. 239, 4182-4188. Petilclerc, C. and Planta, G.E. (1981) Can. J. Physiol. 59, 34-39. Sorimachi, K. and Yasumura. Y. (1986) Biochim. Biophys. Acta 885, 272-281. Goldstein, D.J. and Harris, H. (1981) J. Neurochem. 36, 53-57. Dorai, D.T. and Bachhawat. B.K. (1977) J. Neurochem. 29, 503-511.
590