cerebral microvascular acid phosphatase isoenzymes may contribute to the histamine-induced changes...

NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 24, NO. 3 125

CEREBRAL MICROVASCULAR ACID PHOSPHATASE ISOENZYMES MAY CONTRIBUTE

TO THE HISTAMINE-INDUCED CHANGES IN THE BLOOD-BRAIN BARRIER

PERMEABILITY

Laszlo Nemeth a*, Csilla A. Szabob, Maria A. Delib, Jozsef Kovacsa, Istvan A. Krizbaib, Csongor S. Abrahamb

aDepartment of Pediatrics, Albert Szent-Gydrgyi Medical University, P.O. Box 471; and bLaboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre, P.O. Box 52 1, H-6701

Szeged, Hungary *Corresponding author (present address: Children’s Research Centre; Our Lady’s Hospital for Sick Children, Crumlin, Dublin 12, Ireland; Fax:+353- l-4550201 ; E-mail: [email protected])

(Accepted March 13, 1999)

SUMMARY It was previously suggested that lysosomes and lysosomal acid phosphatase enzyme (AcP, ortho-

phosphoric monoester hydrolase, EC 3.1.3.2.) might have influence on the blood-brain barrier (BBB) permeability. In the present study, we examined the effect of intracarotid histamine administration on the activity of two AcP isoenzymes, lysosomal high molecular weight (HMW; mw > 100,000) and cytosolic low molecular weight (LMW; mw < 20,000) isoforms, and on the changes in BBB permeability for sodium fluorescein (mw: 376) and Evans blue-albumin (mw: 67,000) in newborn pigs. A marked, dose- dependent increase in the activity of LMW AcP and a moderate elevation in the activity of HMW AcP was found in isolated microvessels, but not in brain tissue, concomitantly with a dose-dependent BBB opening for both tracers. It is proposed that HMW and LMW AcP isoenzymes may have a role in the regulation of the para- and transcellular BBB permeability.

KEY WORDS: acid phosphatase, blood-brain barrier, histamine, paracellular and transcellular permeability

INTRODUCTION

The blood-brain barrier (BBB) is formed by cerebral microvascular endothelial cells (CMEC) having

specific morphological (presence of tight intercellular junctions, paucity of pinocytotic vesicles, lack of

0 1999 Wiley-Liss, Inc.

126 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 24, NO. 3

endothelial fenestrations) and functional (barrier properties, polarity, carrier functions) characteristics (1).

CMEC are functioning in co-operation with the neighbouring astrocytes, neurons, pericytes and microglial

cells and maintaining the homeostasis of the brain. A solute can permeate from blood to brain through the

BBB either transcellularly or paracellularly, both pathways being regulated by sophisticated machineries

(2,3). Transendothelial permeation of a macromolecule by adsorptive transcytosis including endocytosis,

transcellular passage, and exocytosis, is suggested to involve the Golgi complex, endosomes, and transport

vesicles (3). Paracellular permeability is thought to be regulated by the complex interaction of different

tight junction proteins (2). Endogenous compounds, pharmaceuticals, or diseases may initiate a series of

molecular events in cerebral endothelium, which later can have an effect on the regulation of the BBB

permeability.

Though CMEC have relatively few lysosomes, a role for these cell organelles in the regulation of

macromolecular transport through the BBB is proposed (4). Specific lysosomal enzymes, such as acid

phosphatase (AcP), trimethaphosphatase, phosphoprotein phosphatase, P-galactosidase and aryl sulphatase

have been identified in cerebral endothelium (4), and increased AcP activity was supposed to be involved

in the enhancement of transendothelial transport ($6). AcP enzyme (orthophosphoric monoesther

hydrolase, EC 3.1.3.2) have multiple molecular isoforms in the brain, such as high molecular weight

(HMW), low molecular weight (LMW) and Zn 2+-dependent isoenzymes which differ from each other in 7

their subcellular localisation, molecular weight, sensitivity for inhibitors, and substrate requirements (7,8).

HMW AcP (mw > lOO,OOO), present mainly in lysosomal fraction, can be blocked by tartrate, and it

nonspecifically hydrolyses phosphomonoesters, while LMW AcP (mw < 20,000), found predominantly in

cytosol, is tartrate-resistant, and it also has phosphotyrosine protein phosphatase activity (7). The effect of

AcP isoenzymes on the regulation of the BBB has not completely revealed yet. In vitro, histamine

treatment increased the AcP activity in an immortalised rat brain endothelial cell line, which effect could

be reduced by antagonists of HI-receptor in case of HMW, and Hx-receptor in case of LMW isoforms (9).

In vivo, histamine-induced activation of capillary AcP enzyme correlated with a vasogenic brain oedema

formation (10,ll).

Histamine released from different cerebral pools, such as histaminergic neurons, perivascular mast

cells, and CMEC, plays important roles in neuronal transmission, regulation of cerebral blood flow, and

brain oedema formation (12). Previous studies revealed that both Hz-receptor-dependent adenylate

cyclase-mediated and HI -receptor-dependent phosphoinositol-mediated mechanisms could contribute to


the histamine-induced changes in the BBB permeability (see for review: 1,12). We have recently found

that histamine increased the transcellular passage of albumin, but not the permeability for tight junction

markers sucrose and inuline, through monolayers of CMEC co-cultured with astrocytes (13), so we could

not exclude the possibility that histamine had a diverse effect on the regulation of para- and transcellular

permeability of the BBB. The aim of this study was to examine the possible role of HMW and LMW AcP

isoforms in histamine-induced BBB permeability changes. Histamine was given in doses corresponding to

literature data (12) and concentrations found in porcine brain during neonatal asphyxia (14).

MATERIALS AND METHODS Newborn pigs of either sex (1,160-l ,420 g) were anesthesized with pentobarbital(30 mg kg-l), then one

of the umbilical arteries was catheterised, and cardiovascular, blood gas, and acid-base parameters were monitored (15). The right internal carotid artery of the animals was exposed and catheterised through the external branch. Histamine diluted in 0.5 ml isotonic saline was given in slow intra-arterial injection in the following doses: 0 mol; 10-e mol; 5x10-6 mol; lo-5 mol; 5x10-5 mol; and 10-a mol (n=12 in each group). Then the catheter was removed and the external carotid artery was ligated. After 1 h, 6 piglets from each group were sacrificed, brain tissue samples were taken from parietal, frontal, and occipital cortex; hippocampus, and periventricular white matter; and cortical microvessels were also isolated using the method of Tontsch and Bauer (16). In the remaining animals, BBB permeability in the same brain regions were measured by sodium fluorescein (SF, mw: 376, Stokes radius: 0.55 nm) and Evans blue labelled albumin (EBA, mw: 67,000; Stokes radius: 3.5 m-n) tracers (both dyes from Sigma) 1 h after the histamine injection, as it was described (lo,1 5). Pigs were intravenously given a solution of both dyes in isotonic saline (2%, 5 ml kg-l) 30 min before the end of experiments. After sacrifice intravascular tracers were removed by a perfusion with 200 ml kg-l isotonic saline. Extravasation was expressed as brain tissue concentration divided by final serum concentration: mg dye x mg-1 brain tissue x (mg dye x ml-l serum)-l . The absorbency of EBA was measured at 620 nm, while the emission of SF at 525 nm after excitation at 440 nm by a fluorimeter. The experimental procedures were in accordance with the NIH Guidelines for the care and use of laboratory animals and were approved by the local Ethical Committee on Animal Investigation.

AcP enzyme activity was measured by the rate of hydrolysis of p-nitrophenylphosphate both in homogenized brain tissue and in isolated cortical microvessels (7,9). Samples in triplicates were incubated in 96-well microtiter plates in 160 ~1 solution containing 0.1 M acetate buffer (pH 5.5) and 2.5 mM p- nitrophenylphosphate at 37°C for 1 h with or without L-(+)tartrate (10 mM). After incubation, 45 ~1 of 1 M sodium hydroxide was added to stop the reaction and the absorbency was read at 405 nm by an ELISA reader (Labsystems Multiskan Biochromatic type 348). Total, and tar&ate-resistant LMW enzyme activities were determined from a calibration curve using increasing concentrations of purified AcP (AcP Lin-trol, Sigma), while tartrate-sensitive HMW activity was calculated. Each activity was expressed as mU mg- l protein.

All data presented are means * S.E.M, n=6 in each group. The values were compared between groups using the Kruskal-Wallis one way analysis of variance on ranks followed by the Dunn’s test. The differences between ipsi- and contralateral hemispheres were evaluated by Mann-Whitney rank sum test. Changes were considered statistically significant at P < 0.05.


RESULTS

Intracarotid histamine administration resulted in a dose-dependent increase in total AcP activity in

isolated microvessels, but not in brain tissue samples (data not shown). Each dose of histamine, but 10-e

mol, significantly (P < 0.05) increased the tartrate-resistant LMW AcP activity in ipsilateral cortical

microvessels of newborn pigs compared to that from control animals, and there was a similar tendency in

contralateral side, too (Fig.1 A.). However, tarn-ate-sensitive HMW AcP activity was only increased in

capillaries from both sides after the administration of the 2 highest doses (Fig. 1C.). Both LMW and HMW

AcP activities in brain homogenates were unchanged 1 h after histamine treatment in 5 regions examined,

representative data from parietal cortex are presented in Fig.lB. and 1D.

CORTICAL MICROVESSELS HOMOGENIZED CORTEX

300

200

2$ ‘4

; 100

ii 0

M a

\ 120

3

a 8o

40

0

tartrate-resistant LMW acid phosphatase Ii

U

0 166sx16616s5mis1~4 0 1ti65kP16%x1i551i5’ WI

tartrate-sensitive HMW aoid phosphatase a I I 121 \

C d

s, I3 I 3

T 14 a

m ipsilateral I::::::/ contralateral

Figure 1 The activity of acid phosphatase isoenzymes in isolated cortical microvessels (AC), and homogenized parietal cortical tissue (B,D) of newborn pigs 1 h after the intracarotid administration of histamine. Changes in the activity of tartrate-resistant low molecular weight (A,B) and tartrate-sensitive high molecular weight (C,D) isoforms were expressed as mU mg-1 protein. Symbols indicate significant differences (P < 0.05) compared to the following treatments: a: 0 mol; b: 10-e mol; c: 5x10-6 mol; d: lo-5 mol histamine* while * means significant difference between ipsi- and contralateral sides in the same 3 animal group.


Histamine increased the BBB permeability in each brain region (data from parietal cortex, hippocampus,

and periventricular white matter are shown in Fig.2.), but only the higher doses resulted in significant (P <

0.05) changes: 10-4 mol for SF, and 5x10-5 and 10-J mol for EBA transport. The highest dose caused a 3-

to 4-fold increase in SF, and a 5- to 6-fold increase in albumin extravasation compared to the control

permeability values in each brain region.

SODIUM FLUORESCEIN EVANS BLUE-ALBUMIN

parietal cortex

5 - R. 2

0 166sx166l6*sx16s164

ts a

hippocampus 2

-- 0 a

1665x1661655r165164

2 perivantricu

0 lbC5r16616ssx1651iF4

m ipsilateral

1d6sx166llPsx16s164 - bo

ar white nmttet

I?::::::] contralateral

Figure 2 Blood-brain barrier permeability changes in parietal cortex (A,B), hippocampus (C,D), and periventricular white matter (E,F) of newborn pigs 1 h after the intracarotid administration of histamine. The permeability markers were: sodium fluorescein (A,C,E) and Evans blue albumin (B,D,F). Extravasations were expressed as 10-Z mg dye x mg-1 brain tissue x (mg dye x ml-l serum)-1 for both dyes. Symbols indicate significant differences (P < 0.05) compared to the following treatments: a: 0 mol; b: 10-h mol; c: 5x1 O-6 mol histamine; while * means significant difference between ipsi- and contralateral sides in the same animal group.


DISCUSSION

As soon as 1 h after intracarotid histamine administration, a marked, dose-dependent increase in the

activity of LMW AcP with a moderate elevation in the activity of HMW AcP was found in isolated

microvessels, but not in homogenised brain tissue. A concomitant BBB opening was also seen. The change

in permeability for albumin, which is thought to cross the BBB by transcytosis (3,4), tended to be higher

than that for SF, which is suggested to pass the BBB mainly by the paracellular pathway (17).

Histamine has long been known to increase the BBB permeability by Hz-receptor-dependent ways

(1,12,1 S), most probably by the activation of adenylate cyclase enzyme (1). Histamine, similarly to cyclic

adenosine 3‘Vmonophosphate (CAMP), increased the formation of pinocytotic vesicles in CMEC in vivo

(1). However, in an in vitro reconstituted model of the BBB, CAMP treatment resulted in a rapid decrease

in paracellular permeability (19), while histamine administration did not alter significantly the

permeability of tight junction markers with a concomitant increase in albumin transport (13). Recently,

Mayhan published evidences that histamine could induce the BBB permeability by a nitric oxide-mediated

activation of guanylate cyclase enzyme (20). In peripheral endothelial cells, a similar mechanism involving

consequent phospholipase C activation, release of Ca 2+ from intracellular stores, induction of nitric oxide

synthase, stimulation of guanylate cyclase, and the formation of cyclic guanosine 3‘5‘monophosphate

(cGMP) is proved to be responsible for the histamine-induced increase in albumin permeability (21). In rat

CMEC, histamine elevated the intracellular [Ca2+] concentrations in vitro (21), while cGMP increased the

rate of pinocytosis in vivo (1). On the other hand, one can not exclude, that similarly to other mediators

(13), histamine could also increase the albumin permeability in CMEC by a rearrangement of endothelial

actin cytoskeleton, which mechanism contributed to the histamine-induced changes in peripheral

endothelium (24).

In order to understand the potential effect of AcP isoenzymes on the regulation of the BBB integrity,

we outline the possible connections between them. Lysosomal HMW AcP may have a role in the

regulation of transendothelial macromolecular transport. Broadman and Salcman (25) suggested that

lysosomal system of organelles with acid hydrolase activity in CMEC and in pericytes of cerebral

capillaries would function as a deterrent to the BBB transport of blood-borne substances. Endothelial

lysosomes fusing with AcP positive transcytotic structures were proposed to play a role in the increased

macromolecular transport in CMEC of stroke-prone rats (6), and brain-injured mice (5). We can only

speculate that cytosolic LMW AcP, an enzyme with known phosphotyrosine protein phosphatase activity,

may also have an effect on the BBB permeability. Tyrosine phosphorylation of proteins associated with


intercellular tight junctions, such as 20-1, 20-2, and p-catenin, could increase the paracellular

permeability (26). In vitro experiments performed either on CMEC (26,27) or epithelial cells (2,26), both

of which have tight junctions, revealed that phosphotyrosine protein phosphatase activity might tighten the

junctions and might decrease the paracellular permeability. Moreover, the endogenous substrate of LMW

AcP in the brain was an epidermal growth factor receptor (8), and transforming growth factors were

reported to have an in vitro influence on the tight junction barrier through this receptor (27,28). On the

other hand, protein tyrosine phosphatase activity might influence to the transendothelial albumin

permeability, too. Histamine treatment, similarly to protein tyrosine phosphatase inhibition, stimulated

tyrosine phosphorylation of two focal adhesion-associated proteins paxillin and p~l25~*~, and produced a

high albumin permeability through coronary endothelium, while inhibition of protein tyrosine

phosphorylation could block the hyperpermeability (29). In summary, it seems possible that an induction

of microvascular AcP isoenzymes by histamine may contribute to permeability changes: HMW form may

be involved in the increased transendothelial transport, while LMW form may tighten the interendothelial

junctions. It may serve as an explanation for the histamine-induced selective albumin permeation without

an increase in paracellular permeability in vitro (13).

This study is the first to suggest that HMW and LMW isoforms of cerebral microvascular AcP enzymes

could participate, by complex and sometimes controversial modes of action, in the regulation of the BBB

permeability. However, the validity of these pronosed mechanisms should be confirmed bv further

molecular and cell biological experiments.

Supported in part by the Hungarian Research Hungarian Ministry of Health (ETT TO4 232/96,

Fund (OTKA F-16682, F-25984, F-26504), and the TO7 154/96). The authors are grateful to Mrs. Ildiko r

Wellinger and Mrs. Ngo Thi Khue Dung for their skilful technical assistance.

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