Boron neutron capture therapy for clear cell sarcoma (CCS): biodistribution study of p-borono-L-phenylalanine in CCS-bearing animal models.

Clear cell sarcoma (CCS) is a rare melanocytic malignant tumor with a poor prognosis. Our previous study demonstrated that in vitro cultured CCS cells have the ability to highly uptake l-BPA and thus boron neutron capture therapy could be a new option for CCS treatment. This paper proved that a remarkably high accumulation of (10)B (45-74 ppm) in tumor was obtained even in a CCS-bearing animal with a well-controlled biodistribution followed by intravenous administration of L-BPA-fructose complex (500 mg BPA/kg).

Fiber-modified adenovirus vectors containing the TAT peptide derived from HIV-1 in the fiber knob have efficient gene transfer activity.

The interaction between viral capsid proteins and specific molecules exposed on the plasma membrane of the cells is involved in the viral tropism. A human adenovirus (Ad) belonging to subgroups A, C, D, E and F infects cells via the interaction between the fiber knob and the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Conventional human adenovirus type 5 (hAd5) vectors show efficient transduction in CAR-positive cells; in contrast, hAd5 vector application is limited by poor transduction into cells lacking CAR expression. In the present study, to broaden the tropism of hAd5 vectors, we generated hAd5 vectors containing the TAT peptide, which is a protein transduction domain derived from human immunodeficiency virus, in the HI loop of the fiber knob (Ad-TAT(HI)-L2) or the C-terminus of the fiber knob (Ad-TAT(C)-L2). In CAR-negative adherent cells, Ad-TAT(HI)-L2 and Ad-TAT(C)-L2 showed approximately 50- to 500-fold higher gene expression than the conventional hAd5 vector (Ad-L2). Ad-TAT(HI)-L2 was also more efficient than Ad-L2 in blood cell lines and in two types of primary cultured human vascular smooth muscle cells, which are almost refractory to Ad-L2. Furthermore, Ad-TAT(HI)-L2 was more efficient than other types of fiber-modified Ad vectors, which harbor an RGD (Arg-Gly-Asp) or a poly-lysine (KKKKKKK;K7) peptide in the HI loop or the C-terminus of the fiber knob, respectively. Ad-TAT(HI)-L2 efficiently transduced the organs in levels and patterns that were roughly similar to those of Ad-L2 after being systemically injected into mice. To the best of our knowledge, this study is the first report showing that hAd5 vectors containing the TAT peptide in the fiber knob could efficiently transduce cells independently of CAR. These Ad vectors should be useful for gene functional analysis and gene therapy.

Expressions of bradykinin B2-receptor, kallikrein and kininogen mRNAs in the heart are altered in pressure-overload cardiac hypertrophy in mice.

Angiotensin-converting enzyme inhibitors prevent cardiac hypertrophy in vivo, and a component of this ameliorative effect has been attributed to accumulation of kinins in cardiac tissues. However, little is known regarding the levels of kallikrein-kinin components in the heart during the development of cardiac hypertrophy. The objectives of the present study were to define the effects of pressure-overload cardiac hypertrophy on cardiac levels of kininogen, kallikrein and bradykinin B2 receptor mRNAs. The pressure-overload induced by aortic constriction produced cardiac hypertrophy in mice after 14 and 28d, assessed from the increased ratios of heart weight to body weight and elevation of brain natriuretic peptide mRNA in the heart. B2 receptor mRNA rapidly decreased in the heart within 7 d after the operation, subsequently returning to those of sham-operated animals. In contrast, levels of both low-molecular-weight kininogen and tissue kallikrein mRNAs were increased 7, 14 and 28 d after aortic constriction. These findings suggest that the mechanical load or stretch in cardiac tissue by pressue-overload rapidly produces the downregulation of B2 receptor expression during the initial stage which may allow the promotion of cardiac hypertrophy induced by a mediation of hypertophic factors such as angiotensin II, while upregulation of kininogen and kallikrein mRNAs during the chronic stage may lead to an enhancement of local kinin generation in the heart, from which further progression of cardiac hypertrophy during later stages may be regulated.

Tissue-Specific expression of rat kininogen mRNAs.

To characterize the local kallikrein-kinin system, tissue distribution of mRNAs for kininogens, precursor proteins of kinins, such as high-molecular-weight (H-), low-molecular-weight (L-) and T-kininogens, were studied in the rat by means of reverse-transcription polymerase chain reaction (RT-PCR) using a fluorophore Cy5-labeled 5'-primer. High levels of these three kininogen mRNAs were present in the liver. Relatively high levels of H-kininogen mRNA were also detected in the skin, lung, kidney, and testis in a descending order, whereas L-kininogen mRNA was detectable in the lung and brain, but not in the kidney, skin, testis, heart, adrenal gland, or skeletal muscle. T-Kininogen mRNA was present in these tissues, except for skeletal muscle. These findings suggest that the expression of each kininogen mRNA is regulated by the tissue-dependent mechanisms which is closely associated with functions of the kallikrein-kinin system in the tissue.

Bradykinin B(1) receptor mediates inhibition of neointima formation in rat artery after balloon angioplasty.

We evaluated the effects of the kallikrein-kinin system on the proliferation and migration of primary cultured vascular smooth muscle cells (VSMCs) in vitro and neointima formation in balloon-injured rat carotid arteries in vivo. In cultured rat VSMCs, tissue kallikrein inhibited cell proliferation, and this inhibitory effect was blocked by Sar-Tyr-Aca(epsilon)-Lys [D-betaNal(7), Ile(8)]-des-Arg(9)-bradykinin, a bradykinin B(1) receptor antagonist, and by icatibant, a bradykinin B(2) receptor antagonist. Platelet-derived growth factor significantly increased the expression of the B(1) receptor but not the B(2) receptor in VSMCs. Platelet-derived growth factor-induced cell migration was significantly attenuated by des-Arg(9)-bradykinin and to a lesser degree by bradykinin. Endogenous B(1) receptor mRNA increased in rat carotid arteries after balloon angioplasty. After local delivery of adenovirus carrying the human tissue kallikrein gene into the rat carotid artery, we observed a 54% reduction in the intima/media ratio at the injured site compared with the control ratio (n=7, P:<0.01). Administration of the B(1) receptor antagonist via minipumps blocked the protective effect of kallikrein and partially reversed the intima/media ratio toward the control ratio. Kallikrein gene delivery results in the regeneration of endothelium compared with the control groups, and the B(1) receptor antagonist abolished this effect. Nitrite/nitrate, cGMP, and cAMP levels in balloon-injured arteries significantly increased after kallikrein gene delivery, whereas the B(1) receptor antagonist abolished these increases (n=4 or 5, P:<0.05). These results indicate that the B(1) receptor contributes to the reduction of neointima formation via the promotion of reendothelialization and inhibition of VSMC proliferation and migration through NO-cGMP and cAMP signaling pathways. This study provides significant implications in treating restenosis after revascularization.

Expression of kininogen, kallikrein and kinin receptor genes by rat cardiomyocytes.

To ascertain the existence of the kallikrein-kinin system in the heart, we have studied in vivo and in vitro whether rat cardiac tissue expresses kininogen, kallikrein and kinin receptor mRNAs. The reverse transcription-polymerase chain reaction demonstrated that the ventricular myocardium of adult male rats expressed mRNAs for T- and low-molecular-weight (L-) kininogens, tissue kallikreins such as true kallikrein and T-kininogenase, and bradykinin B2 receptor, but not those for high-molecular-weight kininogen and B1 receptor. Lipopolysaccharide (LPS; 0.5 mg/kg, i.v.) increased the levels of mRNA for T-kininogen at 12 h and the bradykinin B1 receptor at 24 h without affecting that for other components. All of these mRNAs for the kallikrein-kinin system were also detected in cultured cardiomyocytes derived from neonatal rat ventricles; dibutyryl cyclic AMP, LPS or inflammatory cytokines such as interleukin-1 and tumor necrosis factor, up-regulated mRNA expression of T-kininogen, T-kininogenase, or B1 receptor in these cells in vitro. These results suggest that there are two kinin-generating systems in rat myocardium comprising T-kininogen/T-kininogenase and L-kininogen/true kallikrein respectively, and that the former may be relatively important in inflammatory diseases or conditions in which cAMP levels increase in cardiomyocytes.

Expression of kininogen genes by rat cardiomyocytes.

To determine the existence of the kallikrein-kinin system in the heart, we have studied in vitro and in vivo whether rat heart expresses kininogens (KGNs). The reverse transcription-polymerase chain reaction (RT-PCR) for KGN mRNAs demonstrated that the cardiac tissue of adult male rats expresses T-KGN mRNA but not high-molecular-weight (H-) KGN mRNA. An intravenous injection of lipopolysaccharide (LPS) resulted in a significant increase in T-KGN mRNA levels of rat heart within 12 h. Polyacrylamide gel electrophoresis of cDNA products generated by RT-PCR from heart mRNA using primers specific for either T- or low-molecular-weigh (L-) KGN revealed that rat heart expressed not only T-KGN gene but also L-KGN gene, and that LPS injection exclusively stimulated the expression of T-KGN but not of L-KGN gene. T-KGN mRNA was also detected in cultured myocytes derived from fetal rat heart, and the expression was markedly enhanced by an addition of LPS to cultures. These results demonstrated that rat cardiomyocytes are the source of T- and L-KGNs but not of H-KGN, and that their expression of T-KGN mRNA is stimulated by LPS, probably via LPS-receptor CD14.

Kallikrein gene delivery inhibits vascular smooth muscle cell growth and neointima formation in the rat artery after balloon angioplasty.

Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells (VSMCs). To explore potential roles of the kallikrein-kinin system in vascular biology, we evaluated the effects of adenovirus-mediated human kallikrein gene delivery on the growth of primary cultured VSMCs and in balloon-injured rat artery in vivo. Kallikrein gene transfer into cultured rat VSMCs resulted in time-dependent secretion of recombinant human tissue kallikrein and inhibition of cell proliferation. Balloon angioplasty reduced endogenous rat tissue kallikrein mRNA and protein levels at the injured site. In rats that received adenovirus-mediated human kallikrein gene delivery, we observed a 39% reduction in intima/media ratio at the injured vessel after delivery compared with that of rats that received control virus (n=8, P<0.01). Icatibant, a specific bradykinin B(2) receptor antagonist, blocked the protective effect and reversed the intima/media ratio to that of the control rats (n=5, P<0.01). After gene delivery, human kallikrein mRNA was identified at the injured vessel and a 3-fold increase occurred in kininogenase activity. cAMP and cGMP levels in balloon-injured aorta increased significantly at 4, 7, and 14 days after kallikrein gene delivery, but icatibant abolished the increase. These results provide new insights into the role of the vascular kallikrein-kinin system and have significant implications for gene therapy to treat restenosis or atherosclerosis.

Atrial natriuretic peptide gene delivery attenuates gentamycin-induced nephrotoxicity in rats.

BACKGROUND: Atrial natriuretic peptide (ANP) is a cardiac hormone which exerts potent natriuretic and vasorelaxant activities. The aim of this study is to investigate potential protective effects of ANP gene delivery in gentamycin-induced nephrotoxicity. METHODS: Adenovirus (Ad.RSV-ANP) carrying the human ANP gene or carrying the LacZ gene (Ad.RSV-LacZ) under the control of the Rous sarcoma virus promoter were delivered intravenously on the first day of gentamycin administration. Sprague Dawley rats were injected subcutaneously with gentamycin daily for 10 days. RESULTS: A single systemic injection of Ad.RSV-ANP at a dose of 1.2x10(10) pfu results in a significant increase in urine excretion, water intake, urinary sodium and potassium excretion. Adenovirus-mediated ANP gene delivery significantly increased renal blood flow, glomerular filtration rates and urine flow as well as attenuated the elevation of blood urea nitrogen levels. Histological evaluations revealed that ANP delivery attenuated gentamycin-induced renal tubular damage, cellular necrosis, and lumenal protein casts. The expression of human ANP mRNA was identified in rat kidney, heart, aorta and liver. Immunoreactive human ANP was detected in the heart and kidney of rats injected with Ad.RSV-ANP but not in rats injected with Ad.RSV-LacZ. Cyclic GMP levels in the kidney were significantly increased in rats receiving ANP gene delivery. CONCLUSIONS: This study shows that ANP gene delivery exhibits protection against gentamycin-induced nephrotoxicity and raises the potential to use ANP gene therapy for the treatment of drug-induced renal failure.

Kininogen expression by rat vascular smooth muscle cells: stimulation by lipopolysaccharide and angiotensin II.

To identify the presence of a local kallikrein-kinin system in vascular wall, we have studied whether rat vascular smooth muscle cells (VSMC) express kininogen in vitro and in vivo. Western blots using anti-T-kininogen antibody revealed the presence of T-kininogen in conditioned medium of cultured VSMC. T-Kininogen secretion by VSMC was markedly enhanced by the addition of lipopolysaccharide (LPS), angiotensin II (AII) and phorbol 12-myristate 13-acetate (PMA) to the culture. Experiments using specific inhibitors for protein kinases and on the PMA-induced down-regulation of protein kinase C suggested that a protein kinase C-dependent or unidentified pathway is involved in AII or LPS action, respectively. The intravenous injection of LPS (0.5 mg/kg) resulted in an increase in T-kininogen mRNA levels in the vascular smooth muscle of rat aorta, peaking at 16 h. Polyacrylamide gel electrophoresis of cDNA products generated by reverse transcription-polymerase chain reaction (RT-PCR) from aortic mRNA using primers specific for either T- or low-molecular-weight kininogen revealed that rat vascular smooth muscle expressed T-kininogen gene but not low-molecular-weight kininogen gene, and that LPS exclusively stimulated T-kininogen expression. The mRNA for high-molecular-weight kininogen was undetectable in either aortic smooth muscle or cultured VSMC by means of RT-PCR analysis. RT-PCR using specific primers for rat tissue kallikrein genes showed that aortic smooth muscle expressed KLK1 (true kallikrein) mRNA, but not KLK10 (T-kininogenase) mRNA. These results demonstrated that rat VSMC are a source of T-kininogen but not of low-molecular-weight- or high-molecular-weight kininogen, in contrast to the expression of true kallikrein but not of T-kininogenase by these cells.

Expression of low-molecular-weight kininogen in mouse vascular smooth muscle cells.

To determine the existence of the kallikrein-kinin system in vascular wall, the expression of low-molecular-weight (LMW) kininogen, a precursor protein of kinins, was studied in mouse aortic smooth muscle in vivo or in cultured vascular smooth muscle cells (VSMC) derived from mouse aorta in vitro. Although LMW-kininogen mRNA was undetectable in aortic smooth muscle of untreated mice using either Northern blotting or reverse transcription-polymerase chain reactions (RT-PCR) followed by Southern blotting, administration of lipopolysaccharide (LPS; 1 mg/kg, i.v.) induced the expression of LMW-kininogen mRNA at levels detectable by RT-PCR within 12 h. Cultured VSMC not only expressed LMW-kininogen mRNA at levels easily detectable by RT-PCR, but also secreted LMW-kininogen-like protein that was immunoreactive to anti-mouse LMW-kininogen antibody. These results demonstrate that VSMC are a source of LMW-kininogen in the mouse, and suggest the presence of a local kallikrein-kinin system in vascular tissue. LPS-induced up-regulation of LMW-kininogen expression suggests a role for vascular LMW-kininogen in tissue trauma or inflammation.

Kallikrein gene delivery attenuates hypertension and cardiac hypertrophy and enhances renal function in Goldblatt hypertensive rats.

To demonstrate potential therapeutic effects of kallikrein gene delivery, we delivered adenovirus (Ad.CMV-cHK) carrying the human tissue kallikrein gene into two-kidney, one-clip Goldblatt hypertensive rats. A single intravenous injection of the recombinant adenovirus caused a delay of blood pressure increase that began 1 day after injection and continued for 24 days. A maximal blood pressure reduction was observed in rats receiving kallikrein gene delivery compared with control rats receiving Ad.CMV-LacZ (160+/-5 versus 186+/-7 mm Hg, n=6, P<.01). The expression of human tissue kallikrein mRNA was identified in the kidney, heart, aorta, and liver of rats receiving kallikrein gene delivery. Immunoreactive human kallikrein levels were measured in rat serum and urine in a time-dependent manner. Adenovirus-mediated kallikrein gene delivery caused a significant reduction in the left ventricular mass and cardiomyocyte size, as well as an increase in renal blood flow, urine flow, glomerular filtration rates, electrolyte output, and urine excretion. Enhanced renal responses were accompanied by significant increases in urinary kinin, nitrite/nitrate, and cyclic GMP levels. These findings show that the expression of human tissue kallikrein via gene delivery has protective effects against renovascular hypertension and cardiovascular and renal dysfunction.

Human kallikrein gene delivery protects against gentamycin-induced nephrotoxicity in rats.

The tissue kallikrein-kinin system has been shown to play important roles in cardiovascular and renal function. The aim of this study was to investigate potential protective effects of kallikrein gene delivery in gentamycin-induced nephrotoxicity. Rats were injected subcutaneously with gentamycin daily for 10 to 14 days. Adenovirus, Ad.CMV-cHK carrying the human tissue kallikrein gene or Ad.CMV-LacZ carrying the beta-galactosidase gene under the control of the cytomegalovirus promoter, were delivered intravenously on the first day of gentamycin administration. The expression of human tissue kallikrein mRNA was identified in the kidney, aorta, heart and liver and immunoreactive human kallikrein levels were measured in the serum and urine of rats receiving kallikrein gene delivery. Adenovirus-mediated kallikrein gene delivery significantly increased the renal blood flow, glomerular filtration rates, and urine flow while it attenuated renal tubular damage, cellular necrosis, lumenal protein casts and reduced ventricular weight and cardiomyocyte size. Kallikrein gene delivery caused a decrease in blood urea nitrogen levels and increases in urinary kinin and nitrite/nitrate levels. This study shows that kallikrein gene delivery exhibits protection against gentamycin-induced nephrotoxicity, and raises the potential for kallikrein gene therapy to treat drug-induced renal diseases.

Molecular cloning of cDNAs for mouse low-molecular-weight and high-molecular-weight prekininogens.

We isolated cDNAs encoding low-molecular-weight (L-) and high-molecular-weight (H-) prekininogens from a mouse liver cDNA library using rat T-kininogen cDNA and rat H-kininogen cDNA respectively, as probes. The signal peptide, the heavy chain, and the bradykinin moiety, which are common between the two prekininogens, consist of 20, 359, and 9 amino acids, respectively, while the light chains of the L- and H-prekininogens are composed of 44 and 273 amino acids, respectively. All 19 cysteine residues present in both mouse prekininogens are located at the same positions relative to those of human origin. The light chain of H-prekininogen contains a characteristic 15-repeated His-Gly sequence and a conserved sequence for binding prekallikrein or factor XI. Northern blotting or reverse transcription-polymerase chain reaction followed by Southern blotting using mouse L- and H-kininogen cDNAs demonstrated that both L- and H-kininogens are predominantly expressed in the liver and kidney. L-Kininogen mRNA was also expressed in other tissues, such as the adrenal gland, brain, spinal cord, testis, lung, heart, and skin, while levels of H-kininogen mRNA in these tissues were too low to detect, suggesting that L-kininogen is synthesized in various tissues of mouse, while H-kininogen is exclusively synthesized in the liver and kidney. A genomic Southern blot using H-prekininogen cDNA revealed that the L- and H-prekininogen mRNAs in mouse are probably encoded by a single gene, as is the case in both human and bovine.

Effects of ethanol on the levels of brain 6R-L-erythro-5, 6, 7, 8-tetrahydrobiopterin in the inbred strains of mice. DBA/2J, C3H/HeJ and C57BL/6J with different alcohol preferences.

6R-L-erythro-5, 6, 7, 8-tetrahydrobiopterin (6R-BH4) is a coenzyme for tyrosine, tryptophan and phenylalanine hydroxylases, the former two of which are the initial and the rate-limiting enzymes in the biosynthesis of the catecholamines and serotonin, respectively. The present study was designed to determine the changes in concentrations of 6R-BH4 in striatum and midbrain of the inbred strains of mice, DBA/2J, C3H/HeJ and C57BL/6J, with different genetically determined alcohol preferences, following the injection of ethanol (EtOH). The intraperitoneal administration of EtOH (0, 1, 2 and 4 g/kg) significantly and dose-dependently reduced the levels of striatal and midbrain 6R-BH4 in DBA/2J mice with the lowest alcohol preference, and EtOH (4 g/kg, i.p.) reduced the level of striatal 6R-BH4 in C3H/HeJ with medium alcohol preference. Following the administration of EtOH (4 g/kg, i.p.), brain 6R-BH4 levels in C57BL/6J mice with high alcohol preference were lowered compared with the control group, but the difference did not reach statistic significance. EtOH has a tendency to reduce the brain 6R-BH4 levels in mice with lower alcohol preference or higher sensitivity to EtOH. Based on these findings, it was proposed that differences in alcohol drinking behavior in the inbred strains of mice was influenced by brain 6R-BH4.

Effects of ion channel blockers on rapid postmortem changes in extracellular dopamine and serotonin levels in the rat nucleus accumbens.

In the present study, we used in vivo brain microdialysis to examine the effects of ion channel blockers tetrodotoxin (TTX), EGTA-free Ca2+ and verapamil on rapid postmortem changes in extracellular levels of dopamine (DA), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in the ACC of freely moving rats. Extracellular ACC DA levels decreased following the perfusion of the three ion channel blockers in freely moving rats, and then, at death by cervical dislocation, maximum respective 220-, 60- and 90-fold increases were observed in the extracellular output of DA in animals treated with EGTA, verapamil and TTX, respectively. Also, ACC 5-HT decreased following perfusion with the three blockers in the freely moving rats, and then maximum increases of 80-, 30- and 45-fold in the extracellular output of 5-HT were observed at death in animals treated with EGTA, verapamil and TTX, respectively, compared to the baseline. Cervical dislocation-induced rapid postmortem changes were inhibited markedly by perfusion with CSF containing the CA2+ entry blocker verapamil. These observations suggested that rapid postmortem changes in ACC DA and 5-HT release were associated with the action of calcium ion channels and/or voltage gated channels in the CNS.

Possibility of 5-HT3 receptor involvement in alcohol dependence: a microdialysis study of nucleus accumbens dopamine and serotonin release in rats with chronic alcohol consumption.

The present study was performed to examine the involvement of serotonin-3 (5-HT3) receptors in the rat nucleus accumbens (ACC) in alcohol dependence. In alcohol-treated rats, perfusion of 40 mM K+ and 100 mM ethanol (EtOH) through the microdialysis probe increased the extracellular levels of ACC dopamine (DA), compared with controls. Perfusion of the serotonin (5-HT) uptake inhibitor sertlarine enhanced the extracellular levels of ACC 5-HT in both groups. Increased 5-HT availability in the synaptic clefts on the ACC further activated ACC DA release in the alcohol-treated rats, in comparison with controls. In the final experiments, perfusion of the 5.0 microM 5-HT3 receptor agonist 2-methyl-5-HT (2-Me-5-HT) through the microdialysis probe enhanced the extracellular levels of ACC DA. Magnitude of 2-Me-5-HT-induced DA release was significantly higher in alcohol-treated rats than in controls. On the other hand, 40 mM K(+)- and 100 mM EtOH-induced extracellular 5-HT release in alcohol-treated rats were markedly inhibited. These results show that (1) chronic alcohol intake increases the sensitivity of 5-HT3 receptors, (2) 5-HT3 receptors regulate DA release in the ACC, (3) the dopaminergic neuronal systems associated with 5-HT3 ionophore in the ACC were upregulated after chronic alcohol exposure, and (4) chronic alcohol intake desensitizes the serotonergic neuronal systems in rat ACC. These findings suggest that neurochemical functions of 5-HT3 receptors in regulating DA release in the ACC after alcohol exposure compensate for the dysfunction of serotonergic activity to restore the original properties in processing alcohol tolerance and that the development of alcohol dependence may be mediated by ACC 5-HT3 receptors.

Expression of low-molecular-weight kininogen mRNA in human fibroblast WI38 cells.

Expression of the low-molecular-weight kininogen (L-kininogen) mRNA in the human fibroblast cell line WI38 was examined by means of the reverse-transcription polymerase chain reaction and Southern blotting using human L-kininogen cDNA as a probe. The results demonstrated that WI38 fibroblasts expressed L-kininogen mRNA and that the expression was stimulated by 1 mM dibutyryl cAMP or 10 microM prostaglandin E2. Dexamethasone (1 microM) inhibited the stimulatory effect of prostaglandin E2. These results suggest that human fibroblasts supply L-kininogen, a protein precursor of the inflammatory mediator kinins, to connective tissues in response to inflammatory stimuli and that glucocorticoids may exert the antiinflammatory effect in part by inhibiting the local production of L-kininogen.

Fibroblasts synthesize kininogen in response to inflammatory mediators.

Mouse fibroblasts in vitro secret kininogen (KGN). Rat fibroblasts also synthesized and secreted T-KGN in vitro. KGN production by these fibroblasts is greatly stimulated by dibutyryl cAMP, prostaglandin E2 and tumor necrosis factor. Human fibroblast WI-38 cells also express the L-KGN gene, which was stimulated by dibutyryl cAMP and prostaglandin E2. These results demonstrate that fibroblasts express the KGN gene, and suggest that the expression is regulated by inflammatory mediators. RT-PCR, using specific primers for the T-KGN and L-KGN genes, reveals that the rat hind-paw express both T- and L-KGN mRNAs, and the expression of both KGN mRNAs was increased in the hind-paw 24 h after inducing inflammation by injecting Freund's complete adjuvant into the paw. Thus, it is suggested that local connective tissues express the KGN gene, and that the expression is enhanced under pathological conditions, such as inflammation.

Distribution of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin in regional brain areas of inbred strains of rats and mice with different alcohol preferences.

6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) is a coenzyme for tyrosine, tryptophan and phenylalanine hydroxylases, the former two of which are the initial and the rate-limiting enzymes in the biosynthesis of catecholamines and serotonin, respectively. The present study was conducted to determine the levels of 6R-BH4 in the inbred strains of rats BN and F344, and the inbred strains of mice C57BL/6J, C3H/HeJ and DBA/2J, with different genetically determined alcohol preferences. Previous studies have shown that BN and F344 rats exhibit a high and low alcohol preference, respectively; that C57BL/6J and DBA/2J mice show a high and low alcohol preference, respectively, and that C3H/HeJ mice exhibit a moderate alcohol preference. The levels of 6R-BH4 were measured in five regional brain areas of rats and in two regional brain areas of mice. There was about a 3-fold difference in 6R-BH4 levels across the rat brain areas assayed, ranging from a low level in the frontal cortex to a high level in the striatum and midbrain. Midbrain 6R-BH4 levels in F344 rats were higher than those of BN rats (p < 0.05). On the other hand, striatal 6R-BH4 levels in DBA/2J mice were higher than those of the other two strains of mice (p < 0.05). These results indicate that 6R-BH4 is distributed throughout the nigro- and mesostriatal dopaminergic nervous systems, and that brain 6R-BH4 levels may be involved in the genetic differences in alcohol-drinking behavior in animal models.