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(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

Studies were conducted to clarify the effects of nitric oxide donors NOR 3 ((±)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide, FK409), SIN-1 (3-morpholinosydnonimine) and SNAP (S-nitroso-N-acetylpenicillamine) on the accumulation of cGMP and cAMP and Ca 2+ mobilization as well as ketogenesis from oleate in isolated rat hepatocytes. NOR 3 caused inhibition of ketogenesis from oleate along with stimulation of cGMP accumulation in rat hepatocytes, whereas SIN-I and SNAP exerted no effect on ketogenesis despite their marked stimulation of cGMP accumulation. Although the nitric oxide trapping agent, carboxy-PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), antagonized the stimulation by NOR 3 of cGMP accumulation, it failed to modulate the anti-ketogenic action of NOR 3. Furthermore, neither 8-bromoguanosine-3',5'-cyclic monophosphate nor N 2 ,2'-O-dibutyrylguanosine-3',5'-cyclic monophosphate mimicked the anti-ketogenic action of NOR 3. It is concluded in the present study that NOR 3-induced inhibition of ketogenesis in rat hepatocytes is not mediated by cGMP. The present study revealed that the remaining structure of NOR 3 from which nitric oxide had been spontaneously released had no anti-ketogenic action. We first and clearly demonstrated that nitrite production was dramatically enhanced when NOR 3 was incubated in the presence of rat hepatocytes. The mechanism whereby NOR 3 inhibits ketogenesis in rat hepatocytes will be discussed.

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a mouse brain cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 994 base pairs. The coding region encoded a protein of 241 amino acid residues with a calculated molecular mass of 27,014 daltons. The deduced amino acid sequence of mouse GTP cyclohydrolase I was found to be highly homologous to rat (96%) and human type 1 (89%) enzymes.

The possible role of abnormal T cell-dependent B-cell activation in Graves' disease was investigated by comparing lymphocyte subset distribution and the production of soluble CD8 (sCD8), sCD23, IL-10 and IL-12 by peripheral blood cells (PBMC) and thyroid-infiltrating lymphocytes (TL) in vitro. In TL, the percentage of CD8(+) cells was slightly higher and the sCD8 concentration was significantly higher than in PBMC. The ratio CD23(+) cells to CD20(+) cells (activated B/pan B cells) was increased in TL compared to PBMC from Graves' or normal controls, although the percentage of CD20(+) cells was decreased. Compared to PBMC in Graves' disease, the relative ratio of IL-10 to IL-12 release (IL-10/IL-12) by unstimulated TL was increased, despite a lack of significant difference between PBMC and TL in mean values for either IL-10 or IL-12 secretion. Incubating PBMC with a combination of anti-CD40 monoclonal antibodies and interleukin-4 (IL-4) resulted in B cell activation, reflected in an increase in the sCD23 level in both controls and Graves' patients, but especially prominent in the latter. Stimulation with anti-CD40 antibody and IL-4 also decreased the percentage of CD8(+) cells in PBMC but not TL from both Graves' disease and normal controls, and the percentage of CD8(+) cells in TL was higher than PBMC after the stimulation. The sCD23 concentration in TL was decreased compared to PBMC both in patients with Graves' disease and normal controls. However, in contrast to the increased responses observed in Graves' PBMC or normal controls after stimulation, sCD23 levels remained the same in stimulated TL from Graves' patients. This combination of B cell stimulants increased production of IL-10 in PBMC but not in TL obtained from patients with Graves' disease, and the increased IL-10/IL-12 ratio declined to a value no different from that in PBMC group after stimulation. Thus, T cell-dependent B-cell activation via a CD40 pathway may cause a shift in the Th(1)/Th(2) balance to Th(2) dominance in Graves' disease, while increased CD8(+) cells in TL may suppress sCD23 production and IL-10-producing Th(2) cells.

We investigated for the first time the effect of lipopolysaccharide and the signal transduction pathway on the biosynthesis of tetrahydrobiopterin [(6R-L-erythro-1',2'-dihydroxypropyl) -2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine], the cofactor for the enzymatic hydroxylation of the aromatic amino acids, in the murine neuroblastoma cell line N1E-115, which synthesizes tetrahydrobiopterin constitutively. Activation of N1E-115 cells with 1 microgram/ml lipopolysaccharide resulted in statistically significant increases in both intracellular tetrahydrobiopterin contents and the activity (Vmax) of GTP cyclohydrolase I, a rate-limiting enzyme in tetrahydrobiopterin de novo biosynthesis. Following simultaneous addition of the inhibitors of protein tyrosine kinases and GTP-binding proteins into serum-free culture media with lipopolysaccharide, we analyzed the transduction pathway of lipopolysaccharide signal toward the tetrahydrobiopterin biosynthetic system in N1E-115 cells. Our data indicate the following conclusions: (a) Protein tyrosine kinase systems are involved in mediating lipopoly-saccharide signal to tetrahydrobiopterin production, and (b) there may be a cross-talk between GTP-binding protein and the protein tyrosine kinase system in mediating lipopolysaccharide signal. These observations suggest that a neuronal cell such as N1E-115, which barely expresses CD14 on its cell surface, responds to lipopolysaccharide like macrophages and monocytes in the absence of soluble CD14.

The human aromatic L-amino acid decarboxylase (AADC) gene is transcribed in a tissue-specific manner by an alternative promoter. In this study using human cultured cell lines, we analyzed the alternative promoter that regulates tissue-specific expression of AADC. Neither neuronalnor nonneuronal-type mRNA of AADC was detected in HeLa cells, nonneuronal-type mRNA of AADC was expressed in HepG2 cells, and the neuronal-type was expressed in the SK-N-SH cell line. We examined the promoter activities located in 5′- and 3′-flanking regions of exon N1 and exon L1 by transfection experiments. Plasmids containing 5′-flanking regions of exon L1, the shortest of which was 0.3kb, could promote specifically high expression of the reporter gene in HepG2 cells. On the other hand, plasmids containing 5′-flanking regions of exon N1 (3.6 kb to 0.5 kb) could promote the reporter gene expression not only in SK-N-SH cells but also in HeLa and HepG2. More enhanced expression were observed by transfection of plasmids containing parts of the first intron in these cell lines. Thus, these results suggest that the basal liver-specific promoter activity is located in the 5′-flanking region of exon L1 and that the first intron may also be needed for enhanced expression rather than determination of cell-specificity.

We examined the metabolic effects of glibenclamide, a potent second-generation sulfonylurea, in isolated rat hepatocytes incubated in the absence of extracellular Ca2+. We first demonstrated in the present study that glibenclamide caused a significant increase in basal glucose release and lactate production without any modification of intracellular Ca2+ concentration or cAMP levels in isolated rat hepatocytes. Furthermore, glibenclamide inhibited the noradrenaline-induced increase in cAMP accumulation, while activation of glycogenolysis by noradrenaline was not suppressed by this agent. Our data indicate that glibenclamide exerts its metabolic effects independent of intracellular Ca2+ mobilization and cAMP accumulation.

Studies were conducted to clarify the effects of noradrenaline on oleate metabolism in isolated hepatocytes from fed rats. Noradrenaline caused an inhibition of ketogenesis from oleate along with a stimulation of glucose release through α 1 -adrenergic receptors. Anti-ketogenic action of noradrenaline was confirmed by the suppression of the formation of radioactive acid-soluble products from [1- 14 C]oleate in response to this agent. Noradrenaline increased the conversion of [1- 14 C]oleate into 14 CO 2 but failed to affect [1- 14 C]oleate esterification. When hepatocytes were incubated in a medium containing 1 mM EGTA but no Ca 2+ , the effects of noradrenaline on oleate oxidation were negated. On the other hand, noradrenaline-induced increase in glucose release remained unchanged even in the absence of Ca 2+ in the incubation medium. Decrease in ketogenesis and increase in glucose release produced by vasopressin was completely abolished by calcium depletion. Noradrenaline caused a significant increase in cAMP levels in both the presence and absence of Ca 2+ , although the effect was more marked in the latter. Vasopressin did not affect it. The noradrenaline-induced increase in cAMP and glucose release in the absence of Ca 2+ was also mediated by α 1 -adrenergic receptors. These data are discussed and it is suggested that α 1 -adrenergic agonists may control hepatic ketogenesis and glycogenolysis through two separate signal transduction mechanisms, i.e., a calcium-mobilizing system which is common with vasopressin, and a cAMP generation system which vasopressin lacks.

Fasting in normal rats produced a fall in hepatic triglyceride lipase (H-TGL) activity as well as lipoprotein lipase (LPL) activities of adipose tissue and psoas minor muscle. On the other hand, LPL activities of heart and diaphragm were not decreased by fasting; the former, in fact, was increased significantly. Changes in tissue specific lipase activity caused by withdrawal of insulin from insulin-treated diabetic animals paralleled in direction the changes induced by starvation of normal rats. Furthermore, it was shown in the present paper that the tissue specific lipase activity of diabetic rats became stuck in the starve phase of the starve-feed cycle regardless of dietary intake. The changes of the tissue specific lipase activities, especially of liver, adipose tissue and heart, appeared to coincide with those of plasma insulin levels. These results strongly suggest that the tissue specific lipase system is under hormonal regulation by insulin. Streptozotocin diabetes produced hypertriglyceridemia. The possible mechanism of the hypertriglyceridemia in diabetic animals was discussed in connection with the role of the tissue specific lipase system in the serum triglyceride metabolism.

The interaction between alpha- and beta-adrenergic agonists was examined with respect to amylase release from rat parotid glands. The effect of hypothyroid status on this interaction was also compared with that in euthyroid rats. Both methoxamine and clonidine potentiated isoproterenol-induced amylase release from parotid glands of eu- and hypothyroid rats, but the alpha 2-adrenoceptor-mediated response disappeared in the hypothyroid rats. Dibutyryl cyclic AMP, a second messenger of beta-adrenergic agonists, also showed essentially the same results as those of isoproterenol, but potentiation of dibutyryl cyclic AMP-induced amylase release with alpha-adrenergic agonists was mediated through only alpha 1-adrenoceptors in both groups. Calcium ion plays an important role in the interaction between alpha- and beta-adrenergic agonists. These results suggest that the potentiating effect of alpha-adrenergic agonists may be mediated at least in part through an unknown mechanism at the step distal to cyclic AMP formation in both eu- and hypothyroid rats.

The dog heart-lung preparations were prepared. The "equilibrium point", which could be defined as the point at which the cardiac output (CO)-curve and the venous return (VR)-curve crossed, when the CO and VR were plotted against the right atrial pressure, was recorded directly by utilizing an X-Y recorder. The CO-curve was obtained, as a locus of the equilibrium point, by raising and lowering the level of blood in the venous reservoir (competence test). The meaning of the procedure was shown to increase or decrease the mean systemic pressure, and to cause the corresponding parallel shift in the VR-curve. The VR-curve was obtained by changing myocardial contractility. When heart failure was induced by pentobarbital or by chloroform, the equilibrium point shifted downwards to the right, depicting the VR-curve. During development of the failure, the slopes of CO-curves decreased gradually. Effects of cinobufagin and norepinephrine were also analyzed. Utilization of the X-Y recorder enabled us to settle the uniform experimental conditions more easily, and to follow the effects of drugs continuously on a diagram equating the CO- and VR-curves (Guyton's scheme).

Studies were conducted to compare the metabolic effects of vasopressin, 4β-phorbol-12-myristate-13-acetate (PMA) and A23187 on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats. Vasopressin inhibited the formation of acid-soluble products from [1-14C]oleate (0.25 mM, 0.5 mM and 1 mM), the inhibition being most marked at low (0.25 mM) concentration of oleate. Conversion of [1-14C]oleate into14CO2 and esterified products was stimulated by vasopressin. The stimulatory effect of this hormone on14CO2 production was most marked at high (1 mM) concentration of oleate, whereas that on [1-14C]-oleate esterification was most marked at low (0.25 mM) concentration of oleate. These vasopressin actions were abolished when hepatocytes were incubated in the absence of calcium in the medium. Our results strongly suggest that both increase in esterification and increase in oxidation to CO2 contribute to the anti-ketogenic action of vasopressin when oleate is added as substrate, although the relative extent of their contribution varies according to the oleate concentration. The anti-ketogenic action of vasopressin was mimicked by PMA but not by A23187. PMA also caused a stimulation of [1-14C]oleate esterification although the effect was diminished at 1 mM [1-14C]oleate. A23187 failed to affect [1-14C]oleate esterification. The metabolic effects of PMA were elicited in the absence of extracellular calcium, too. Conversion of [1-14C]oleate into14CO2 was only slightly increased by both PMA and A23187 when 1 mM [1-14C]oleate was added as substrate. The marked stimulatory effect of vasopressin on14CO2 production from [1-14C]oleate was not reproduced even by the combination of PMA and A23187. The possible involvement of protein kinase C and calcium mobilization in the regulation of oleate metabolism is discussed.

Studies were conducted to see whether exogenous phospholipase C from Clostridium perfringens, phospholipase A2 from Crotalus adamanteus venom, arachidonic acid and 1-oleoyl-2-acetyl-sn-glycerol (OAG) mimic the anti-ketogenic action of vasopressin in isolated rat hepatocytes. Exogenous phospholipase C inhibited ketogenesis in the presence of 0.5 mM oleate. Experiments employing [1-14C]oleate, however, indicated that the mechanism involved in the anti-ketogenic action of exogenous phospholipase C is distinct from that of vasopressin. The decreased rate of the production of acid-soluble products from [1-14C]oleate in response to vasopressin could be explained by the sum of the increased rates of 14CO2 formation and [1-14C]oleate esterification. By contrast, exogenous phospholipase C suppressed not only the formation of acid-soluble products but also 14CO2 production and [1-14C]oleate esterification. Indeed, phospholipase C greatly inhibited [1-14C]oleate uptake into hepatocytes. It is suggested that the alteration of the architecture of plasma membrane by exogenous phospholipase C may lead to the disturbance of oleate uptake and consequent general suppression of oleate metabolism. Exogenous phospholipase A2, arachidonic acid and OAG increased ketogenesis regardless of the presence of oleate. The ketogenic effects may be attributed to the supply of fatty acids by these agents to hepatocytes.

Searching for an experimental condition to produce pulmonary edema of uniform high grade severity in rabbits, five different doses of epinephrine (38,60, 100, 160 and 260 mug/ml) were infused at a rate of 0.29 ml/min for 20 min into the femoral vein of fed and fasted animals. Body temperature was maintained at 38 to 40 degrees C. The incidence of pulmonary edema was not dose-dependent, and was approx. 70% by the administration of 60 to 260 mug/ml of epinephrine solutions. In general, the edema was more severe in the fed group than in the fasted. The highest dose of epinephrine was often fatal in the fasted group. One hundred mug/ml (approx. 10 mug/kg/min), fed was regarded as a favourable condition for the experiment. The amounts of lung lipids increased in edema (+) cases of the fed groups. The plasma potassium level was elevated in proportion to the dose of epinephrine. The clotting time of blood was markedly prolonged in edema (+) rabbits. The significance of these observations was discussed in regard to the mechanism of epinephrine-induced pulmonary edema.

Studies were conducted to clarify the effects of vasopressin, angiotensin II and phenylephrine on hepatic ketogenesis and fatty acid synthesis. Hepatocytes from fed rats were incubated with oleate or octanoate. Vasopressin stimulated fatty acid synthesis as well as lactate and pyruvate accumulation in the presence of oleate. In accordance with this action, vasopressin caused a marked decrease in ketogenesis from oleate. When octanoate was added as a substrate, vasopressin failed to inhibit ketogenesis. Neither angiotensin II nor phenylephrine affected ketogenesis or fatty acid synthesis. The results in the present study show that there are vasopressin-mediated reciprocal changes in ketogenesis from oleate and fatty acid synthesis in isolated hepatocytes.

The change in responsiveness to a-adrenergic stimulation and to calcium ions in amylase release from parotid glands of hypothyroid rats was compared with that in euthyroid rats. Calcium depletion and addition of verapamil or W-7 in the medium caused a decrease in methoxamine-induced amylase release in both eu- and hypothyroid rats. Moreover, addition of calcium ions to Ca2+-free medium markedly increased the methoxamine-induced amylase release in proportion to its concentration in hypothyroid rats. These results suggest that calcium ions play an important role in methoxamine-induced amylase release from parotid glands of hypothyroid rats as well as those of the euthyroid ones.

The tumor-promoting agent 4 beta-phorbol-12-myristate-13-acetate (TPA) is shown to be a potent stimulator of fatty acid synthesis in isolated rat hepatocytes. The maximal effect of TPA is seen at 10(-6) M, and the concentration for half-maximal effect is ca. 10(-8) M. Stimulation of fatty acid synthesis by TPA is shown not to require the presence of extracellular Ca++. TPA produces a significant increase in lactate and pyruvate accumulation. The possible involvement of protein kinase C in short-term regulation of fatty acid synthesis in the liver is discussed.