L-Histidine decarboxylase protein and activity in rat brain microvascular endothelial cells.

OBJECTIVE AND DESIGN: L-Histidine decarboxylase (HDC) is the primary enzyme regulating histamine biosynthesis. This study was carried out to examine whether the cultured rat brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier (BBB), have the ability to form histamine, and whether HDC mRNA is expressed in rat BMEC. MATERIAL: Male, 3-week-old Wistar rats were used. For in vitro studies, rat BMEC were isolated from rat brains, and subculture cells were grown on collagen-coated culture flask and slide. METHODS: HDC assay, immunofluorescence analysis and expression of HDC mRNA by RT-PCR were performed in rat BMEC. RESULTS: The HDC activity of the BMEC was estimated to be 0.14 +/- 0.05 p mol/min/mg protein. This activity was completely inhibited by (S)-alpha-fluoromethylhistidine, a specific inhibitor of HDC. Using a polyclonal anti HDC antibody and immunofluorescence microscopy, we confirmed the presence of HDC protein in rat BMEC. RT-PCR also showed the expression of HDC mRNA in rat BMEC. CONCLUSIONS: L-Histidine uptaken by rat BMEC was shown to be converted to histamine, suggesting that HDC plays an important role in BBB.

Stereoselective blood-brain barrier transport of histidine in rats.

The transport characteristics of l- and d-histidine through the blood-brain barrier (BBB) were studied using cultured rat brain microvascular endothelial cells (BMEC). l-Histidine uptake was a saturable process. A decrease in incubation temperature from 37 to 0 degreesC or the addition of metabolic inhibitors (DNP and rotenone) reduced the uptake rate of l-histidine. Ouabain, an inhibitor of (Na+, K+)-ATPase, also reduced uptake of l-histidine. Moreover, the substitution of Na+ with choline chloride and choline bicarbonate in the incubation buffer decreased the initial l- and d-histidine uptake rates. These results suggested that l-histidine is actively uptaken by a carrier-mediated mechanism into the BMEC, with energy supplied by Na+. However, l-histidine uptake at 0 degreesC was not completely inhibited, and it was reduced in the presence of an Na+-independent System-L substrate, BCH, suggesting facilitated diffusion (the Na+-independent process) by a carrier-mediated mechanism into the BMEC. l-histidine uptake in rat BMEC also appeared to be System-N mediated since uptake was inhibited by glutamine, aspargine and l-glutamic acid gamma-monohydroxamate. System-N mediated transport was not pH sensitive. d-histidine transport was also studied in rat BMEC. d-histidine transport by rat BMEC has similar characteristics to l-histidine. However, System-N transport did not play a role in d-histidine uptake. The uptake of l-histidine was also greater than that of the d-isomer, indicating the stereoselective uptake of histidine in rat BMEC.

In-vivo microdialysis measurement of 5-hydroxytryptamine and its metabolites, 5-hydroxyindoleacetic acid and N-acetyl 5-hydroxytryptamine, in rat blood: effects of histamine-receptor antagonists.

The blood concentrations of 5-hydroxytryptamine (5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and N-acetyl 5-HT were assayed by in-vivo microdialysis and a highly sensitive HPLC procedure that was originally developed to analyse CNS mediators. We investigated the effects of histamine-receptor antagonists on 5-HT metabolism and its release into the blood of rats. The mean basal levels of 5-HT, 5-HIAA and N-acetyl 5-HT in the blood measured by in-vivo microdialysis were 77.2 +/- 9.4, 20.3 +/- 1.5 and 1.89 +/- 0.15 pmol mL-1, respectively. These levels were not significantly affected by an intraperitoneal injection of saline, and remained at constant levels for at least 8 h after administration of saline. After an intraperitoneal injection of 5-HT hydrochloride (0.5 mg kg-1), 5-HT was soon detected in the blood of the jugular vein. 5-HIAA also quickly appeared in the blood and declined monoexponentially from 60 min after injection. In contrast, N-acetyl 5-HT slowly appeared in the blood and it reached a maximal level at 270 min. The 5-HT and N-acetyl 5-HT levels in dialysates from rat jugular vein were significantly increased by intraperitoneal pyrilamine (2.0 mg kg-1), (+)-chlorpheniramine (2.0 mg kg-1) and cimetidine (20.0 mg kg-1). However, there was no increase in the 5-HIAA concentration after an intraperitoneal injection of these histamine-receptor antagonists, demonstrating that the 5-HT released from various cells containing 5-HT was predominantly metabolized to N-acetyl 5-HT by N-acetyltransferase. Moreover, thioperamide did not affect the basal levels of 5-HT, 5-HIAA or N-acetyl 5-HT. Because the recovered 5-HT, 5-HIAA and N-acetyl 5-HT in the dialysate is directly proportional to the free fraction in the blood, in-vivo microdialysis is a reliable method of examining 5-HT metabolism and its release into the blood.