Journal of Neurochemistry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry
Rapid Communication
Endothelial Cells from Human Fetal Brain Microvessels May Be Cholinoceptive, but Do Not Synthesize Acetylcholine P. Kasa, M. Pakaski, *F. Job and ?A. Lajtha Central Research Laboratory, Albert Szent-Gyorgyi Medical University; *Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Szeged, Hungary; and tNathan Kline Institute for Psychiatric Research, Orangeburg, New York, U S A .
in the cytoplasm of cerebral endothelial cells (ECs), but it could not be excluded that the presence of its synthesizing enzyme, choline acetyltransferase (ChAT), in the isolated microvessel fraction was due to contamination by cholinergic nerve endings. To decide whether ECs contain the enzymes for ACh synthesis and/or breakdown, our aim in this investigation was to perform a comparative study of the presence of some of the elements of the cholinergic system [acetylcholinesterase (AChE), ChAT, and butyrylcholinesterase (BuChE)] in brain homogenate, in the CNS microvessel fraction, and in ECs cultured in vitro. In addition, the conditions were established for an in vitro human blood-brain bamer model system with a possibility of investigating the permeability and the transport function of the ECs.
Abstract: Brain homogenate, cerebral microvessels, and endothelial cells (ECs) were prepared from 15- 18-week-old human fetuses and analyzed biochemically for the presence of elements of the cholinergic system [acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and butyrylcholinesterase]. The ECs were cultured, and their purity was checked by light microscopic immunohistochemistry with the application of anti-human factor VIII and glial fibrillary acidic protein. The highest activity of ChAT was found in the brain homogenate and the lowest in the microvesselfraction. No ChAT activity could be detected in the cultured ECs, despite the presence of high AChE activity. It is suggested that human brain ECs may be under the control of acetylcholine released from cholinergic nerve terminals but that the cells do not produce the transmitter itself. In coculture experiments, when ECs were plated on the upper surface of a polycarbonate filter and glial cells were seeded on the lower surface, the electric resistance was measured. During the culture period, the resistance first increased up to 5 days in vitro (297 ? 17 ohm * cm2) but later gradually declined. These results demonstrate that human ECs cocultured with glial cells provide a useful model for study of the function of the blood-brain barrier in vitro. Key Words: Human brain-Microvessels-Endothelial cells-Coculture-Cholinergic system. Kasa P. et al. Endothelial cells from human fetal brain microvessels may be cholinmptive, but do not synthesize acetylcholine. J. Neurochem. 56,2143-2146 (1991).
MATERIALS AND METHODS Culture of human ECs From fetuses obtained by therapeutic abortion performed at 15-18 weeks, brain cortical samples were dissected and placed in cold Hanks’ balanced salt solution. The microvessels from the cortex were prepared according to the method of Diglio et al. (1982). The cells were cultured and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 1.25% glutamine, and antibiotics at 37°C in a humidified atmosphere of 5% C 0 2 and 95% air. After 8-10 days in vitro (DIV), the ECs developed a monolayer, and primary culture subcultures were produced from this by trypsinization. Between the third and seventh subcultures, the EC fractions were used for the different experiments.
Several lines of evidence suggest that a dysfunction of the blood-brain barrier may play a role in the pathogenesis of Alzheimer’s disease. The loss of cholinergic nerve cells from the basal forebrain (Whitehouse et al., 1981) and other transmitter-containing neurons from different parts of the brain (Bondareff et al., 1982) may lead to alterations in the neurogenic control of the microvessels, modulating cerebral blood flow (Arneric et al. 1988) and permeability in the CNS. Because of the profound structural and functional changes in the CNS microvasculature, it has been suggested that Alzheimer’s disease may be a capillary dementia (Scheibel et al., 1989). Previous studies on isolated cerebral microvessels have shown that acetylcholine (ACh) may be synthesized locally
The glial cell cultures were obtained from 15- 18-week-old human fetal brain. Small pieces of embryonic brain samples were triturated, and after centrifugation (1,000 g, 10 min), the cells from the supernatant were plated either onto a collagen-coated petri dish or onto glass coverslips. The subcul-
Received March 1, 1991; accepted March 4, 1991. Address correspondence and reprint requests to Dr. P. Kasa at Central Research Laboratory, A. Szent-Gyorgyi Medical University, Somogyi B. ut 4, 6720 Szeged, Hungary.
Abbreviations used: ACh, acetylcholine; AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; ChAT, choline acetyltransferase; DIV, days in vitro; EC, endothelial cell; GFAP, glial fibrillary acid protein.
Preparation of human astroglial cell cultures
2143
2144
P. KASA ET AL.
tures of these cells were used for the experiments, in which the glial cells were cocultured with ECs.
whereas ChAT activity was measured by the micromethod of Fonnum (1975).
Cocultures of glial cells with ECs
Measurements of electric resistance
In coculture experiments, the Millicell culture plate insert (Millipore, PICM 030 50) was turned upside down, and the human glial cells were seeded on the bottom of the transparent membrane filter. The cells were allowed to grow for several days, and ECs were then plated on the upper surface of the filter.
The electric resistance across human EC monolayers cultured on the polycarbonate filter was measured with a WPI EVOM apparatus. The ECs on the filter separated the chamber into two compartments: (a) a lower one, which is the petri dish, and (b) an upper one, the Millicell chamber. The electrodes were immersed to precisely the same distance from the base of the petri dish and from the surface of the filter in the Millicell chamber. For each experiment, the resistance of the polycarbonate filter with the glial cells on it was measured, and the resistance of cocultures (ECs cocultured with glial cells) was determined. The resistance of the filter with the glial cells was subtracted from the resistance of the cocultures to yield the resistance due to the EC monolayers themselves.
Characterization of ECs and glial cells by immunohistochemistry After different periods, the cultures (ECs and/or glial cells) on the glass coverslips were tested immunohistochemically for the presence of anti-human factor VIII and glial fibrillary acidic protein (GFAP) by means of a peroxidase-antiperoxidase technique (Sternberger, 1979).
Light and electron microscopy The morphological appearance of the EC cultures and the presence of tight junctions between the cells on the polycarbonate filter were studied at the light and electron microscopic levels. The samples were fixed in a solution containing 2% formaldehyde and 4% glutaraldehyde in 0.1 A4 sodium cacodylate buffer (pH 7.4). After dehydration, the samples were embedded in Durcupan (ACM; Fluka), sectioned with glass knives, and investigated in a JEOL model 100C electron microscope.
Biochemical determination of AChE, BuChE, and ChAT activities AChE and BuChE activities were determined according to the spectrophotometric method of Ellman et al. (196 l),
RESULTS AND DISCUSSION The results presented here show that ECs from human fetuses can be cultured equally well on a plastic surface, on a polycarbonate filter, and on a glass surface. On these surfaces, the cells were first round (Fig. 1A) but later became elongated. Immunocytochemically, it was demonstrated that the ECs retained an EC-specific marker, factor VIII-related antigen (Fig. 1B and C), and only a very few GFAP-positive cells were found among the ECs (Fig. ID). In the coculture experiments, on the upper surface of the filter the ECs developed as a monolayer, whereas the glial cells on the bottom surface were present in multiple layers (Fig. IE). Ultrastruc-
FIG. 1. A: Light microscopic, unstained appearance of ECs, DIV 4. Bar = 180 pm. B Low-power view of ECs culture stained for the presence of anti-human factor Vlll immunoreactivity. Bar = 45 pm. C: High-magnification view of factor Vlll positivity. Note the perikaryal localization of immunoreactivity and the absence in the nuclei, DIV 2. Bar = 180 pm. D: Demonstrationof GFAP-positive astrocytes (arrows) in the EC culture. Bar = 65 pm. E: Coculture of endothelial cells (DIV 5;*) and astrocytes (DIV 17;+)on the surface of a Millicell polycarbonate filter (F). Bar = 100 pm.
J. Neuroehem.. Vol. 56, No. 6 . 1991
ABSENCE OF C U T ACTIVITY FROM HUMAN BRAIN ECS turally, tight junctions could be observed among the ECs (data not shown). In the biochemical measurements, the capillary and/or EC fraction exhibited a higher AChE activity than the brain homogenate. The BuChE activity did not reveal an enrichment in the capillary samples compared with that of the brain homogenate, whereas the activity was much higher in the cultured ECs (Table l). AChE in the brain capillaries is thought to be of neuronal origin (Kreutzberg et al., 1979), because no positivity was found with cytochemical methods in the rough endoplasmic reticulum of the ECs or other nonneuronal cells in the CNS. In contrast, in vitro culture of human brain ECs demonstrated that the cells contain a high AChE activity. This may indicate that the cells are under the control of ACh. ChAT activity was measured in the homogenate and capillary fraction and in ECs isolated from the human fetal cortex. Differences were found among the fractions of the brain homogenate, microvessels, and ECs cultured in vitro. The highest activity was found in the brain homogenate, and no activity could be detected in the EC fraction. Earlier results demonstrated that the EC fraction isolated with collagenase (Estrada et al., 1983) contained significantly less ChAT activity than the intact capillaries, a finding suggesting that this enzyme was located in periendothelial structures removed by collagenase treatment. It seems likely that most of the ChAT activity in the microvessel fraction is localized in the nerve terminals associated with the basement membrane of the capillaries, rather than in the ECs (Estrada et al., 1983; but see Gonzhlez and Santos-Benito, 1987). Thus, the different levels of ChAT activity measured in vessel fractions from different brain areas (Estrada et al., 1983) may reflect regional variations in the density of cholinergic innervation of the vessels. This suggestion is supported by the fact that synaptic structures attached to the basement membrane of the isolated capillaries were demonstrated in morphological experiments (Suddith, 1980). The ChAT activity level in the capillary fraction from different brain areas was postulated to be an index for the presence of penvascular cholinergic nerves (Estrada et al., 1988). The presence of cholinergic receptor binding sites in the cerebral microvessels (Albroch, 1981;Hariketal., 1981;Estradaand Krause, 1982;Grammas et al., 1983; Spatz et al., 1989) strongly suggests that the ACh receptors are located in the membranes of capillary ECs. This result lends further support to the cholinergic regulation of ECs in different parts of the brain. When the transendothelial passage of electric current was investigated in ECs daily from DIV 2 to DIV 9 in cocultures, a transient increase in electric resistance was found (Table 2). The resistance of the cocultures first increased (DIV 5 TABLE 1. Cholinergic markers in homogenate, capillary fraction, and EC cultures from microvessels offetal human cortex Activity (nmol/mg of protein/min)
BuChE
ChAT
2.65 t- 0.76 2.39 k 0.93 6.84 f 0.93
0.04 f 0.01 0.003 k 0.001
AChE
Homogenate Capillaries ECs
*
8.72 2.34 28.87 f 5.55 27.02 2.68
+
Data are mean f SD values (n tectable.
=
ND
5 experiments). ND, not de-
2145
TABLE 2. Changes in electrical resistance of cocultures in the course of culturina ECs (DIV) plus glia (DIV) coculture (n)
Resistance (ohm -cm2)
DIV 2 plus DIV 14 (4) DIV 3 plus DIV 15 (4) DIV 4 plus DIV 16 (4) DIV 5 plus DIV 17 (4) DIV 6 plus DIV 18 (4) DIV 7 plus DIV 19 (4) DIV 8 plus DIV 20 (4) DIV 9 plus DIV 2 1 (4)
55 f 8 61 + 8 l l O + 12 297 f 17 2 7 0 k 17 194 12 78 f 8 61 2 8
+
Resistance data are mean f SEM values of the indicated total number of individual cultures tested (n). ECs plus DIV 17 glial cells) up to 438 ohm-cm2 (minus filter, 135 _+ 15 ohm-cm2; minus glial cells, 67 k 12 ohm cm2)but after 6 days started to decline. The results of these measurements demonstrate the development and maturation of tight junctions among the ECs but suggest that the maintenance of these interendothelial connections may be under the control of other, still unknown factors. In conclusion, it has been demonstrated that human brain capillary ECs probably do not synthesize ACh but that they may respond to the pericapillary cholinergic nerves. The primary cultures of human brain microvessel ECs cocultured with glial cells (Kasa et al., 1990) may provide a useful model for studying the properties of the blood-brain barrier. As far as we are aware, this study is the first to report on the elements of the cholinergic system and measurements of the electric resistance of cocultures of human CNS ECs with human glial cells. Further physiological studies are warranted to prove the cholinoceptive nature of cerebral ECs.
-
Acknowledgment: The authors thank V. Sapirstein and H. Sershen (Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, U.S.A.) for their help and continuous interest. Thanks are also due to the Department of Obstetrics and Gynecology (Director Prof. Dr. L. Kovacs) for providing the brain samples and to the Hungarian Ministry of Health (grant 526) and to the Hungarian Academy of Sciences (grant 518141) for support.
REFERENCES Albroch E. (1 98 1) Cholinergic receptors in the cerebral arteries of the goat, in Cerebral Microcirculation and Metabolism (CervcisNavarro J. and Fritschka E., eds), pp. 285-291. Raven Press, New York. Arneric S. P., Honig M. A., Milner T. A., Greco S., Iadecola C., and Reis D. J. (1988) Neuronal and endothelial sites of acetylcholine synthesis and release associated with microvessels in rat cerebral cortex: ultrastructural and neurochemical studies. Brain Rex 454, I 1-30.
Bondareff W., Mountjoy C. Q., and Roth M. (1982) Loss of neurons of origin of the adrenergic projection to cerebral cortex in senile dementia. Neurology 32, 164-168. Diglio C. A., Grammas P., Giacomelli F., and Wiener J. (1982) Primary culture of rat cerebral microvascular endothelial cells. Isolation, growth, and characterization. Lab. Invest. 46, 554-563. Ellman G. L., Courtney K. D., Andres V., and Featherstone R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Estrada C. and Krause D. N. (1982) Muscarinic cholinergic receptor J. Narrochem.. Vol. 56,No. 6. 1991
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P. U S A ET AL.
sites in cerebral blood vessels. J . Pharmacol. Exp. Ther. 221, 85-90. Estrada C., Hamel E., and Krause D. N. (1983) Biochemical evidence for cholinergic innervation of intracerebral blood vessels. Brain Res. 266, 261-270. Estrada C., Triguero D., Munoz J., and Sureda A. (1988) Acetylcholinesteraxantaining fibers and choline acetyltransferaseactivity in isolated cerebral microvessels from goats. Bruin Res. 453, 275-280. Fonnum F. (1975) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24,407-409. Gonzaez J. L. and Santos-Benito F. F. (1987) Synthesis of acetylcholine by endothelial cells isolated from rat brain cortex capillaries. Brain Res. 412, 148- 150. Grammas P., Diglio C. A,, Marks B. H., Giacomelli F., and Wiener J. (1983) Identification of muxarinic receptors in rat cerebral cortical microvessels. J. Neurochem. 40,645-65 1 . Hank S. I., Sharma V. K., Wetherbee J. R., Warren R. H., and Banerjee S. P. (1981) Adrenergk and cholinergic receptors of cerebral microvessels. J. Cereb. Blood F/ow Metab. 1, 329-338. Kasa P., Pakaski M., Joo F., Sershen H., and Lajtha A. (1990) Human in vitro blood-brain barrier model system for studying the effects
J. Neurochem., Vol. S6, No. 6, 1991
of drugs, in Abstracts of the 8th Meeting of the European Society for Neurochemistry, p. 163. Kreutzberg G. W., Kaiya H., and T6th L. (1979) Distribution and origin of acetylcholinesterase activity in the capillaries of the brain. Histochemistry 61, 1 11-122. Scheibel A. B., Doung T., and Jacobs R. (1989) Alzheimer’s disease as a capillary dementia. Ann. Med. 21, 103-107. Spatz M., Back F., McCarron R. M., Merkel N., Uematsu S., Long D. M.. and Bembry J. (1989) Cholinergic and histaminergic receptors in cultured endothelium derived from human cerebral microvessels, in Neurotransmission and CerebrovascularFunction I (Seylaz J. and MacKenzie E. T., eds), pp. 105-108. Elsevier, New York. Sternberger L. A. (1979) Immunohistochemistry, 2nd ed. Wiley, New York. Suddith R. L., Savage K. E., and Eisenberg H. M. (1980) Ultrastructural and histochemical studies of cerebral capillary synapses. Adv. Exp. Med. Biol 131, 139-145. Whitehouse P. J.. Price D. L., Clark A. W., Coyle J. T., and DeLong M. R. (1981) Alzheimer’s disease: evidence for selective loss cholinergic neurons in the nucleus basalis. Ann. Neurol. 10, 122126.
Rapid Communication
Endothelial Cells from Human Fetal Brain Microvessels May Be Cholinoceptive, but Do Not Synthesize Acetylcholine P. Kasa, M. Pakaski, *F. Job and ?A. Lajtha Central Research Laboratory, Albert Szent-Gyorgyi Medical University; *Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Szeged, Hungary; and tNathan Kline Institute for Psychiatric Research, Orangeburg, New York, U S A .
in the cytoplasm of cerebral endothelial cells (ECs), but it could not be excluded that the presence of its synthesizing enzyme, choline acetyltransferase (ChAT), in the isolated microvessel fraction was due to contamination by cholinergic nerve endings. To decide whether ECs contain the enzymes for ACh synthesis and/or breakdown, our aim in this investigation was to perform a comparative study of the presence of some of the elements of the cholinergic system [acetylcholinesterase (AChE), ChAT, and butyrylcholinesterase (BuChE)] in brain homogenate, in the CNS microvessel fraction, and in ECs cultured in vitro. In addition, the conditions were established for an in vitro human blood-brain bamer model system with a possibility of investigating the permeability and the transport function of the ECs.
Abstract: Brain homogenate, cerebral microvessels, and endothelial cells (ECs) were prepared from 15- 18-week-old human fetuses and analyzed biochemically for the presence of elements of the cholinergic system [acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and butyrylcholinesterase]. The ECs were cultured, and their purity was checked by light microscopic immunohistochemistry with the application of anti-human factor VIII and glial fibrillary acidic protein. The highest activity of ChAT was found in the brain homogenate and the lowest in the microvesselfraction. No ChAT activity could be detected in the cultured ECs, despite the presence of high AChE activity. It is suggested that human brain ECs may be under the control of acetylcholine released from cholinergic nerve terminals but that the cells do not produce the transmitter itself. In coculture experiments, when ECs were plated on the upper surface of a polycarbonate filter and glial cells were seeded on the lower surface, the electric resistance was measured. During the culture period, the resistance first increased up to 5 days in vitro (297 ? 17 ohm * cm2) but later gradually declined. These results demonstrate that human ECs cocultured with glial cells provide a useful model for study of the function of the blood-brain barrier in vitro. Key Words: Human brain-Microvessels-Endothelial cells-Coculture-Cholinergic system. Kasa P. et al. Endothelial cells from human fetal brain microvessels may be cholinmptive, but do not synthesize acetylcholine. J. Neurochem. 56,2143-2146 (1991).
MATERIALS AND METHODS Culture of human ECs From fetuses obtained by therapeutic abortion performed at 15-18 weeks, brain cortical samples were dissected and placed in cold Hanks’ balanced salt solution. The microvessels from the cortex were prepared according to the method of Diglio et al. (1982). The cells were cultured and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 1.25% glutamine, and antibiotics at 37°C in a humidified atmosphere of 5% C 0 2 and 95% air. After 8-10 days in vitro (DIV), the ECs developed a monolayer, and primary culture subcultures were produced from this by trypsinization. Between the third and seventh subcultures, the EC fractions were used for the different experiments.
Several lines of evidence suggest that a dysfunction of the blood-brain barrier may play a role in the pathogenesis of Alzheimer’s disease. The loss of cholinergic nerve cells from the basal forebrain (Whitehouse et al., 1981) and other transmitter-containing neurons from different parts of the brain (Bondareff et al., 1982) may lead to alterations in the neurogenic control of the microvessels, modulating cerebral blood flow (Arneric et al. 1988) and permeability in the CNS. Because of the profound structural and functional changes in the CNS microvasculature, it has been suggested that Alzheimer’s disease may be a capillary dementia (Scheibel et al., 1989). Previous studies on isolated cerebral microvessels have shown that acetylcholine (ACh) may be synthesized locally
The glial cell cultures were obtained from 15- 18-week-old human fetal brain. Small pieces of embryonic brain samples were triturated, and after centrifugation (1,000 g, 10 min), the cells from the supernatant were plated either onto a collagen-coated petri dish or onto glass coverslips. The subcul-
Received March 1, 1991; accepted March 4, 1991. Address correspondence and reprint requests to Dr. P. Kasa at Central Research Laboratory, A. Szent-Gyorgyi Medical University, Somogyi B. ut 4, 6720 Szeged, Hungary.
Abbreviations used: ACh, acetylcholine; AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; ChAT, choline acetyltransferase; DIV, days in vitro; EC, endothelial cell; GFAP, glial fibrillary acid protein.
Preparation of human astroglial cell cultures
2143
2144
P. KASA ET AL.
tures of these cells were used for the experiments, in which the glial cells were cocultured with ECs.
whereas ChAT activity was measured by the micromethod of Fonnum (1975).
Cocultures of glial cells with ECs
Measurements of electric resistance
In coculture experiments, the Millicell culture plate insert (Millipore, PICM 030 50) was turned upside down, and the human glial cells were seeded on the bottom of the transparent membrane filter. The cells were allowed to grow for several days, and ECs were then plated on the upper surface of the filter.
The electric resistance across human EC monolayers cultured on the polycarbonate filter was measured with a WPI EVOM apparatus. The ECs on the filter separated the chamber into two compartments: (a) a lower one, which is the petri dish, and (b) an upper one, the Millicell chamber. The electrodes were immersed to precisely the same distance from the base of the petri dish and from the surface of the filter in the Millicell chamber. For each experiment, the resistance of the polycarbonate filter with the glial cells on it was measured, and the resistance of cocultures (ECs cocultured with glial cells) was determined. The resistance of the filter with the glial cells was subtracted from the resistance of the cocultures to yield the resistance due to the EC monolayers themselves.
Characterization of ECs and glial cells by immunohistochemistry After different periods, the cultures (ECs and/or glial cells) on the glass coverslips were tested immunohistochemically for the presence of anti-human factor VIII and glial fibrillary acidic protein (GFAP) by means of a peroxidase-antiperoxidase technique (Sternberger, 1979).
Light and electron microscopy The morphological appearance of the EC cultures and the presence of tight junctions between the cells on the polycarbonate filter were studied at the light and electron microscopic levels. The samples were fixed in a solution containing 2% formaldehyde and 4% glutaraldehyde in 0.1 A4 sodium cacodylate buffer (pH 7.4). After dehydration, the samples were embedded in Durcupan (ACM; Fluka), sectioned with glass knives, and investigated in a JEOL model 100C electron microscope.
Biochemical determination of AChE, BuChE, and ChAT activities AChE and BuChE activities were determined according to the spectrophotometric method of Ellman et al. (196 l),
RESULTS AND DISCUSSION The results presented here show that ECs from human fetuses can be cultured equally well on a plastic surface, on a polycarbonate filter, and on a glass surface. On these surfaces, the cells were first round (Fig. 1A) but later became elongated. Immunocytochemically, it was demonstrated that the ECs retained an EC-specific marker, factor VIII-related antigen (Fig. 1B and C), and only a very few GFAP-positive cells were found among the ECs (Fig. ID). In the coculture experiments, on the upper surface of the filter the ECs developed as a monolayer, whereas the glial cells on the bottom surface were present in multiple layers (Fig. IE). Ultrastruc-
FIG. 1. A: Light microscopic, unstained appearance of ECs, DIV 4. Bar = 180 pm. B Low-power view of ECs culture stained for the presence of anti-human factor Vlll immunoreactivity. Bar = 45 pm. C: High-magnification view of factor Vlll positivity. Note the perikaryal localization of immunoreactivity and the absence in the nuclei, DIV 2. Bar = 180 pm. D: Demonstrationof GFAP-positive astrocytes (arrows) in the EC culture. Bar = 65 pm. E: Coculture of endothelial cells (DIV 5;*) and astrocytes (DIV 17;+)on the surface of a Millicell polycarbonate filter (F). Bar = 100 pm.
J. Neuroehem.. Vol. 56, No. 6 . 1991
ABSENCE OF C U T ACTIVITY FROM HUMAN BRAIN ECS turally, tight junctions could be observed among the ECs (data not shown). In the biochemical measurements, the capillary and/or EC fraction exhibited a higher AChE activity than the brain homogenate. The BuChE activity did not reveal an enrichment in the capillary samples compared with that of the brain homogenate, whereas the activity was much higher in the cultured ECs (Table l). AChE in the brain capillaries is thought to be of neuronal origin (Kreutzberg et al., 1979), because no positivity was found with cytochemical methods in the rough endoplasmic reticulum of the ECs or other nonneuronal cells in the CNS. In contrast, in vitro culture of human brain ECs demonstrated that the cells contain a high AChE activity. This may indicate that the cells are under the control of ACh. ChAT activity was measured in the homogenate and capillary fraction and in ECs isolated from the human fetal cortex. Differences were found among the fractions of the brain homogenate, microvessels, and ECs cultured in vitro. The highest activity was found in the brain homogenate, and no activity could be detected in the EC fraction. Earlier results demonstrated that the EC fraction isolated with collagenase (Estrada et al., 1983) contained significantly less ChAT activity than the intact capillaries, a finding suggesting that this enzyme was located in periendothelial structures removed by collagenase treatment. It seems likely that most of the ChAT activity in the microvessel fraction is localized in the nerve terminals associated with the basement membrane of the capillaries, rather than in the ECs (Estrada et al., 1983; but see Gonzhlez and Santos-Benito, 1987). Thus, the different levels of ChAT activity measured in vessel fractions from different brain areas (Estrada et al., 1983) may reflect regional variations in the density of cholinergic innervation of the vessels. This suggestion is supported by the fact that synaptic structures attached to the basement membrane of the isolated capillaries were demonstrated in morphological experiments (Suddith, 1980). The ChAT activity level in the capillary fraction from different brain areas was postulated to be an index for the presence of penvascular cholinergic nerves (Estrada et al., 1988). The presence of cholinergic receptor binding sites in the cerebral microvessels (Albroch, 1981;Hariketal., 1981;Estradaand Krause, 1982;Grammas et al., 1983; Spatz et al., 1989) strongly suggests that the ACh receptors are located in the membranes of capillary ECs. This result lends further support to the cholinergic regulation of ECs in different parts of the brain. When the transendothelial passage of electric current was investigated in ECs daily from DIV 2 to DIV 9 in cocultures, a transient increase in electric resistance was found (Table 2). The resistance of the cocultures first increased (DIV 5 TABLE 1. Cholinergic markers in homogenate, capillary fraction, and EC cultures from microvessels offetal human cortex Activity (nmol/mg of protein/min)
BuChE
ChAT
2.65 t- 0.76 2.39 k 0.93 6.84 f 0.93
0.04 f 0.01 0.003 k 0.001
AChE
Homogenate Capillaries ECs
*
8.72 2.34 28.87 f 5.55 27.02 2.68
+
Data are mean f SD values (n tectable.
=
ND
5 experiments). ND, not de-
2145
TABLE 2. Changes in electrical resistance of cocultures in the course of culturina ECs (DIV) plus glia (DIV) coculture (n)
Resistance (ohm -cm2)
DIV 2 plus DIV 14 (4) DIV 3 plus DIV 15 (4) DIV 4 plus DIV 16 (4) DIV 5 plus DIV 17 (4) DIV 6 plus DIV 18 (4) DIV 7 plus DIV 19 (4) DIV 8 plus DIV 20 (4) DIV 9 plus DIV 2 1 (4)
55 f 8 61 + 8 l l O + 12 297 f 17 2 7 0 k 17 194 12 78 f 8 61 2 8
+
Resistance data are mean f SEM values of the indicated total number of individual cultures tested (n). ECs plus DIV 17 glial cells) up to 438 ohm-cm2 (minus filter, 135 _+ 15 ohm-cm2; minus glial cells, 67 k 12 ohm cm2)but after 6 days started to decline. The results of these measurements demonstrate the development and maturation of tight junctions among the ECs but suggest that the maintenance of these interendothelial connections may be under the control of other, still unknown factors. In conclusion, it has been demonstrated that human brain capillary ECs probably do not synthesize ACh but that they may respond to the pericapillary cholinergic nerves. The primary cultures of human brain microvessel ECs cocultured with glial cells (Kasa et al., 1990) may provide a useful model for studying the properties of the blood-brain barrier. As far as we are aware, this study is the first to report on the elements of the cholinergic system and measurements of the electric resistance of cocultures of human CNS ECs with human glial cells. Further physiological studies are warranted to prove the cholinoceptive nature of cerebral ECs.
-
Acknowledgment: The authors thank V. Sapirstein and H. Sershen (Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, U.S.A.) for their help and continuous interest. Thanks are also due to the Department of Obstetrics and Gynecology (Director Prof. Dr. L. Kovacs) for providing the brain samples and to the Hungarian Ministry of Health (grant 526) and to the Hungarian Academy of Sciences (grant 518141) for support.
REFERENCES Albroch E. (1 98 1) Cholinergic receptors in the cerebral arteries of the goat, in Cerebral Microcirculation and Metabolism (CervcisNavarro J. and Fritschka E., eds), pp. 285-291. Raven Press, New York. Arneric S. P., Honig M. A., Milner T. A., Greco S., Iadecola C., and Reis D. J. (1988) Neuronal and endothelial sites of acetylcholine synthesis and release associated with microvessels in rat cerebral cortex: ultrastructural and neurochemical studies. Brain Rex 454, I 1-30.
Bondareff W., Mountjoy C. Q., and Roth M. (1982) Loss of neurons of origin of the adrenergic projection to cerebral cortex in senile dementia. Neurology 32, 164-168. Diglio C. A., Grammas P., Giacomelli F., and Wiener J. (1982) Primary culture of rat cerebral microvascular endothelial cells. Isolation, growth, and characterization. Lab. Invest. 46, 554-563. Ellman G. L., Courtney K. D., Andres V., and Featherstone R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Estrada C. and Krause D. N. (1982) Muscarinic cholinergic receptor J. Narrochem.. Vol. 56,No. 6. 1991
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