Csilla Andrea Szabó

Expression of glutamate receptors on cultured cerebral endothelial cells

Activation of glutamate receptors has been shown to mediate a large number of neuronal processes such as long‐term potentiation and ischemic damage. In addition to neurons and glia, glutamate receptors may occur on cerebral endothelial cells (CECs). The aim of the present study was to determine which glutamate receptors are expressed in CECs and to demonstrate the functional presence of such channels. By using reverse transcriptase‐polymerase chain reaction, we showed that primary cultures of rat CECs express N‐methyl‐D‐aspartate (NMDA) receptors (NR1 subunit, which is necessary for the formation of functional NMDA receptors, and NR2A–C subunits), 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐4‐isoxazolyl‐propionate (AMPA) receptors (GLUR1–4 subunits), and metabotropic receptors (mGLUR). Exposure of the cultures to 2 mM glutamate, a well‐established mediator of ischemic damage, for 30 min increased significantly the phosphorylation of calcium/calmodulin‐dependent protein kinase II even after 10‐ and 60‐min recovery times. This effect could be prevented by the NMDA blocker MK‐801. The presence of multiple glutamate receptor types may confer a finely tuned responsiveness of the cerebral endothelium to glutamate in physiological and pathological conditions. J. Neurosci. Res. 54:814–819, 1998.

Vasoactive substances produced by cultured rat brain endothelial cells

The vasoactive substances synthesized by primary cultures of rat brain endothelial cells were investigated and compared to those from two, immortalized cell lines, RBE4 and GP8. The vasoactivity of endothelium-derived substances was measured on isolated canine coronary artery. Vascular tone was significantly decreased by both primary and GP8, but not by RBE4 cells. Indomethacin pretreatment of primary and GP8 cells turned vasorelaxation into contraction while N(omega)-nitro-L-arginine pretreatment decreased the vasorelaxation induced by primary, but not by GP8 cells. Eicosanoid production was determined after incubation with [14C]arachidonic acid. The predominant vasoactive eicosanoid was prostaglandin E2 in both primary and GP8 cells. RBE4 cells synthetized mainly prostaglandin E2 and thromboxane B2 and significantly less prostaglandin E2 than did either primary or GP8 cells. The capacity of cerebral endothelium to regulate vascular tone by production of dilator and constrictor substances can be preserved under certain circumstances in immortalized cell lines.

Expression of G-protein subtypes in cultured cerebral endothelial cells

This paper describes Western-blotting evidence for the presence of various guanine nucleotide binding proteins, G-proteins in cultured rat cerebral endothelial cells (CECs) and two immortalized cerebral endothelial cell lines, RBE4 and GP8. By using specific antibodies raised against known sequences of appropriate G-protein types that were previously characterized, we demonstrated the presence of Gsalpha, Gi2alpha, Gi3alpha, Gq/11alpha, Goalpha and Gbeta in cell lysates of primary cultures of CECs, and plasma membranes of RBE4 and GP8 cells. The appearance of Goalpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previous studies. Isoproterenol and bradykinin displayed significant, dose-dependent stimulation of [35S]GTPgammaS binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on Galpha-subunits by receptor agonists, suggested that there were functional, G-protein coupled beta-adrenergic and bradykinin receptors in these systems. No significant stimulation of [35S]GTP7gammaS binding was noted with serotonin under our experimental conditions. Since stimulation of [35S]GTPgammaS binding by isoproterenol and bradykinin was additive, it was concluded that different Galpha proteins were activated by these two ligands. In analogy to other systems, activation of Gs is most likely by isoproterenol, while Gi and/or Gq/11 proteins might be activated by bradykinin receptors. The possible significance of the receptors and G-proteins detected is being discussed in the functioning of cerebral endothelium, and thus the blood-brain barrier.

Pole reversals and hypothalamic self-stimulation: Ascending spread of rewarding excitation

Abstract Direction of the spread of rewarding impulses has been studied by analysis the changes in the efficiency of monophasic square wave pulses established by reversing the position of cathode (which is the active pole for self-stimulation) and anode (under which blockade of neural conduction may occur) for the same two wires of such bipolar electrodes whose two poles were aligned sagittally in lateral hypothalamus of rats. Lower bipolar self-stimulation thresholds were observed to belong to the anterior cathodal and posterior anodal pole positions and the same current level was found to maintain higher rate of bar pressing in this case than when stimulations with reversed cathodal-anodal pole positions were applied. This finding suggests that rewarding excitation starts to spread in anterior direction from the active pole of lateral hypothalamic self-stimulation electrodes.

Intracarotid histamine administration results in dose-dependent vasogenic brain oedema formation in new-born pigs.

The existence of three pools of brain histamine (in neurons, perivascular mast cells, cerebral endothelium) suggests involvement of histamine released after physiological and pathological stimuli in neuronal transmission, regulation of cerebral blood flow and brain oedema formation [1]. Cerebral endothelial cells have been shown to take up histamine from either side but release it mainly luminally [2, 3]. Indirect evidence implicates histamine in the pathogenesis of neonatal brain oedema since histamine accumulated in brain compartments in asphyxia [3]. Studies with antihistamines suggested that H 2 receptors play a major role in the development of brain oedema in asphyxiated new-born pigs, but H1 receptors also contribute [4]. The direct effect of histamine in neonates has not yet been reported. Histamine regulates the activity of cerebral endothelial acid phosphatase in vitro, and might influence blood-brain barrier (BBB) permeability by increasing endocytic transport [5]. The present study investigated the effects of intra-carotid histamine administration on brain oedema formation and endothelial acid phosphatase activity in new-born pigs.

Histamine-Induced Vasogenic Brain Oedema Formation in Newborn Pigs

It is well-known that some specific morphological characteristics of cerebral endo-thelium, such as the presence of tight intercellular junctions, paucity of pinocytotic vesicles and the absence of fenestrations, contribute to the maintaining of the blood-brain barrier properties. Cerebral endothelial cells have relatively few lysosomes and the number of these compartments seems to parallel the presence of intracellular vesicles in different endothelia (1). On the other side, there is a growing evidence that endothelial lysosomes play a role in regulating entry of internalized macromolecules into the central nervous system despite limited expression and the significance of lysosomes is increased during pathological conditions (1,2). Specific lysosomal enzyme systems have been identified in cerebral endothelium, such as acid phosphatase, trimethaphosphatase, phospho-protein phosphatase, β-galactosidase and aryl sulphatase (1). These enzymes may participate in the alteration of the blood-brain barrier permeability and the pathogenesis of brain oedema during different diseases. Particularly, increased acid phosphatase activity was shown to be involved in the enhancement of transendothelial transport of macromolecules through the blood-brain barrier in stroke-prone spontaneously hypertensive rats (2). We have recently published that histamine regulated the activity of acid phosphatase enzyme in cultured cerebral endothelial cells and suggested that increased activity might have effect on blood-brain barrier permeability (3).

Effects of Histamine on the Acid Phosphatase Activity of Cultured Cerebral Endothelial Cells

Acid phosphatase (orthophosphoric monoester hydrolase, EC 3.1.3.2) is widely distributed in nature and has been studied thoroughly in both plants and animals (Hollander, 1971). The enzyme is activated in acidic environment, which is provided by the presence of H+-ATPase, in different subcellular organelles of the exo- and endocytotic pathway (Mellman, Fuchs and Helenius, 1986). Consequently, the low pH provides favourable conditions for enzymatic hydrolyses in lysosomes; the proton gradient is used as an energy source for the coupled transport of biogenic amines. whereas the difference in pH between the endosome and the extracellular environment is used by the cell in receptor mediated endocytosis to provide symmetry to the recycling circuit between the two compartments (Mellman, Fuchs and Helenius, 1986).

Receptor-mediated regulation by histamine of the acid phosphatase activity in cultured cerebral endothelial cells

Acid phosphatase (APase; othophosphoric monoester hydrolase, EC 3.1.3.2) is widely distributed in nature and has been studied in both plants and animals. Biochemical studies have revealed the presence of multiple molecular forms of acid phosphatase which differ in their molecular size, subcellular localization, sensitivity to inhibitors and substrate requirements [1-2]. The high molecular weight form ofthe enzyme (HMW; m.w. > 100,000 Da) can be completely inhibited by tartrate or fluoride and is present mainly in the lysosomal fraction nonspecifically hydrolysing various phosphomonoesters. A low molecular weight form (LMW; m.w. < 20,000 Da; tartrate-resistant form) is present predominantly in the cytosol and a third form of APase activity with a molecular weight of about 62,000 Da can be detected in the presence of Zn2+ ions [3]. Acid hydroloase activity was first demonstrated in primary cultures of cerebral endothelial cells (CEC) by Baranczyk-Kuzma et al. [4]. However, it remained to be seen if the enzyme activity could be modified in, or released from, the CEC by vasoactive substances. The present study was designed to check the possible effects of histamine on the activity of different molecular forms of acid phosphatase.