Spontaneous pulmonary emphysema in mice lacking all three nitric oxide synthase isoforms.

The roles of endogenous nitric oxide (NO) derived from the entire NO synthases (NOSs) system have yet to be fully elucidated. We addressed this issue in mice in which all three NOS isoforms were deleted. Under basal conditions, the triple n/i/eNOSs-/- mice displayed significantly longer mean alveolar linear intercept length, increased alveolar destructive index, reduced lung elastic fiber content, lower lung field computed tomographic value, and greater end-expiratory lung volume as compared with wild-type (WT) mice. None of single NOS-/- or double NOSs-/- genotypes showed such features. These findings were observed in the triple n/i/eNOSs-/- mice as early as 4 weeks after birth. Cyclopaedic and quantitative comparisons of mRNA expression levels between the lungs of WT and triple n/i/eNOSs-/- mice by cap analysis of gene expression (CAGE) revealed that mRNA expression levels of three Wnt ligands and ten Wnt/ß-catenin signaling components were significantly reduced in the lungs of triple n/i/eNOSs-/- mice. These results provide the first direct evidence that complete disruption of all three NOS genes results in spontaneous pulmonary emphysema in juvenile mice in vivo possibly through down-regulation of the Wnt/ß-catenin signaling pathway, demonstrating a novel preventive role of the endogenous NO/NOS system in the occurrence of pulmonary emphysema.

Mechanisms for establishment of GABA signaling in adrenal medullary chromaffin cells.

γ-Aminobutyric acid (GABA) is thought to play a paracrine role in adrenal medullary chromaffin (AMC) cells. Comparative physiological and immunocytochemical approaches were used to address the issue of how the paracrine function of GABA in AMC cells is established. GABAA receptor Cl- channel activities in AMC cells of rats and mice, where corticosterone is the major glucocorticoid, were much smaller than those in AMC cells of guinea-pigs and cattle, where cortisol is the major. The extent of enhancement of GABAA receptor α3 subunit expression in rat pheochromocytoma (PC12) cells by cortisol was larger than that by corticosterone in parallel with their glucocorticoid activities. Thus, the species difference in GABAA receptor expression may be ascribed to a difference in glucocorticoid activity between corticosterone and cortisol. GABAA receptor Cl- channel activity in mouse AMC cells was enhanced by allopregnanolone, as noted with that in guinea-pig AMC cells, and the enzymes involved in allopregnanolone production were immunohistochemically detected in the zona fasciculata in both mice and guinea pigs. The expression of glutamic acid decarboxylase 67 (GAD67), one of the GABA synthesizing enzymes, increased after birth, whereas GABAA receptors already developed at birth. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, but not nicotinic or muscarinic receptors, in PC12 cells, resulted in an increase in GAD67 expression in a protein-kinase A-dependent manner. The results indicate that glucocorticoid and PACAP are mainly responsible for the expressions of GABAA receptors and GAD67 involved in GABA signaling in AMC cells, respectively.

Angiotensin II promotes pulmonary metastasis of melanoma through the activation of adhesion molecules in vascular endothelial cells.

Hypertension is considered as one of the cancer progressive factors, and often found comorbidity in cancer patients. Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and angiotensin II (Ang II) is well known pressor peptide associated with RAS. Ang II has been reported to accelerate progression and metastasis of cancer cells. However, its precise mechanisms have not been fully understood. In this study, we sought to elucidate the mechanisms by which Ang II exacerbates hematogenous metastasis in mouse melanoma cells, focusing the adhesion pathway in vascular endothelial cells. For this purpose, B16/F10 mouse melanoma cells, which do not express the Ang II type 1 receptor (AT1R), were intravenously injected into C57BL/6 mice. Two weeks after cell injection, the number of lung metastatic colonies was significantly higher in the Ang II-treated group (1 µg/kg/min) than in the vehicle-treated group. The AT1R blocker valsartan (40 mg/kg/day), but not the calcium channel blocker amlodipine (5 or 10 mg/kg/day), significantly suppressed the effect of Ang II. In endothelium-specific Agtr1a knockout mice, Ang II-mediated acceleration of lung metastases of melanoma cells was significantly diminished. Ang II treatment significantly increased E-selectin mRNA expression in vascular endothelial cells collected from lung tissues, and thus promoted adherence of melanoma cells to the vascular endothelium. Ang II-accelerated lung metastases of melanoma cells were also suppressed by treatment with anti-E-selectin antibody (20 mg/kg). Taken together, Ang II-treatment exacerbates hematogenous cancer metastasis by promoting E-selectin-mediated adhesion of cancer cells to vascular endothelial cells.

Protective Role of Myelocytic Nitric Oxide Synthases against Hypoxic Pulmonary Hypertension in Mice.

RATIONALE: Nitric oxide (NO), synthesized by NOSs (NO synthases), plays a role in the development of pulmonary hypertension (PH). However, the role of NO/NOSs in bone marrow (BM) cells in PH remains elusive. OBJECTIVES: To determine the role of NOSs in BM cells in PH. METHODS: Experiments were performed on 36 patients with idiopathic pulmonary fibrosis and on wild-type (WT), nNOS (neuronal NOS)-/-, iNOS (inducible NOS)-/-, eNOS (endothelial NOS)-/-, and n/i/eNOSs-/- mice. MEASUREMENTS AND MAIN RESULTS: In the patients, there was a significant correlation between higher pulmonary artery systolic pressure and lower nitrite plus nitrate levels in the BAL fluid. In the mice, hypoxia-induced PH deteriorated significantly in the n/i/eNOSs-/- genotype and, to a lesser extent, in the eNOS-/- genotype as compared with the WT genotype. In the n/i/eNOSs-/- genotype exposed to hypoxia, the number of circulating BM-derived vascular smooth muscle progenitor cells was significantly larger, and transplantation of green fluorescent protein-transgenic BM cells revealed the contribution of BM cells to pulmonary vascular remodeling. Importantly, n/i/eNOSs-/--BM transplantation significantly aggravated hypoxia-induced PH in the WT genotype, and WT-BM transplantation significantly ameliorated hypoxia-induced PH in the n/i/eNOSs-/- genotype. A total of 69 and 49 mRNAs related to immunity and inflammation, respectively, were significantly upregulated in the lungs of WT genotype mice transplanted with n/i/eNOSs-/--BM compared with those with WT-BM, suggesting the involvement of immune and inflammatory mechanisms in the exacerbation of hypoxia-induced PH caused by n/i/eNOSs-/--BM transplantation. CONCLUSIONS: These results demonstrate that myelocytic n/i/eNOSs play an important protective role in the pathogenesis of PH.

Inhibitory effects of pine nodule extract and its component, SJ-2, on acetylcholine-induced catecholamine secretion and synthesis in bovine adrenal medullary cells.

Extract of pine nodules (matsufushi) formed by bark proliferation on the surface of trees of Pinus tabulaeformis or Pinus massoniana has been used as an analgesic for joint pain, rheumatism, neuralgia, dysmenorrhea and other complaints in Chinese traditional medicine. Here we report the effects of matsufushi extract and its components on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that matsufushi extract (0.0003-0.005%) and its component, SJ-2 (5-hydroxy-3-methoxy-trans-stilbene) (0.3-100 µM), but not the other three, concentration-dependently inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue. Matsufushi extract (0.0003-0.005%) and SJ-2 (0.3-100 µM) also inhibited 45Ca2+ influx induced by acetylcholine in a concentration-dependent manner, similar to its effect on catecholamine secretion. They also suppressed 14C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine. In Xenopus oocytes expressing α3ß4 nicotinic acetylcholine receptors, matsufushi extract (0.00003-0.001%) and SJ-2 (1-100 µM) directly inhibited the current evoked by acetylcholine. The present findings suggest that SJ-2, as well as matsufushi extract, inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

Decreased Bronchial Eosinophilic Inflammation and Mucus Hypersecretion in Asthmatic Mice Lacking All Nitric Oxide Synthase Isoforms.

BACKGROUND: Asthma is characterized by airflow limitation with chronic airway inflammation, hyperresponsiveness and mucus hypersecretion. NO is generated by three nitric oxide synthase (i/n/eNOSs) isoforms, but conflicting results have been reported using asthmatic mice treated with NOSs inhibitors and NOS-knockout mice. To elucidate the authentic role of NO/NOSs in asthma, we used asthmatic mice lacking all NOSs (n/i/eNOS(-/-)). METHODS: Wild-type and n/i/eNOS(-/-) mice were sensitized and challenged with ovalbumin. Pathological findings and expressions of interferon (IFN)-γ, interleukin (IL)-4, -5, -10, -13 and chemokines in the lung were evaluated. RESULTS: Decreased eosinophilic inflammation, bronchial thickening and mucus secretion, IL-4, -5 and -13, monocyte chemoattractant protein-1, eotaxin-1 and thymus and activation-regulated chemokine expressions were observed in n/i/eNOS(-/-) mice compared to wild-type, but expressions of IFN-γ and IL-10 were similar. CONCLUSION: Using asthmatic n/i/eNOS(-/-) mice, NO plays important roles in accelerating bronchial eosinophilic inflammation and mucus hypersecretion in the pathophysiology of asthma.

Ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppressing nicotinic acetylcholine receptor-ion channels in cultured bovine adrenal medullary cells.

Ikarisoside A is a natural flavonol glycoside derived from plants of the genus Epimedium, which have been used in Traditional Chinese Medicine as tonics, antirheumatics, and aphrodisiacs. Here, we report the effects of ikarisoside A and three other flavonol glycosides on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that ikarisoside A (1-100 µM), but not icariin, epimedin C, or epimedoside A, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. Ikarisoside A had little effect on catecholamine secretion induced by veratridine and 56 mM K(+). Ikarisoside A (1-100 µM) also inhibited (22)Na(+) influx and (45)Ca(2+) influx induced by acetylcholine in a concentration-dependent manner similar to that of catecholamine secretion. In Xenopus oocytes expressing α3ß4 nicotinic acetylcholine receptors, ikarisoside A (0.1-100 µM) directly inhibited the current evoked by acetylcholine. It also suppressed (14)C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine at 1-100 µM and 10-100 µM, respectively. The present findings suggest that ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

Effects of various pharmacological agents on the function of norepinephrine transporter.

The norepinephrine transporter is selectively expressed in noradrenergic nerve terminals, where it can exert spatial and temporal control over the action of norepinephrine. The norepinephrine transporter mediates the termination of neurotransmission via the reuptake of norepinephrine released into the extracellular milieu. In the present brief review, we report our recent studies about the effects of various pharmacological agents such as fasudil, nicotine, pentazocine, ketamine and genistein on norepinephrine transporter function.

Development of an experimentally useful model of acute myocardial infarction: 2/3 nephrectomized triple nitric oxide synthases-deficient mouse.

We investigated the effect of subtotal nephrectomy on the incidence of acute myocardial infarction (AMI) in mice deficient in all three nitric oxide synthases (NOSs). Two-thirds nephrectomy (NX) was performed on male triple NOSs(-/-) mice. The 2/3NX caused sudden cardiac death due to AMI in the triple NOSs(-/-) mice as early as 4months after the surgery. The 2/3NX triple NOSs(-/-) mice exhibited electrocardiographic ST-segment elevation, reduced heart rate variability, echocardiographic regional wall motion abnormality, and accelerated coronary arteriosclerotic lesion formation. Cardiovascular risk factors (hypertension, hypercholesterolemia, and hyperglycemia), an increased number of circulating bone marrow-derived vascular smooth muscle cell (VSMC) progenitor cells (a pro-arteriosclerotic factor), and cardiac up-regulation of stromal cell-derived factor (SDF)-1α (a chemotactic factor of the progenitor cells) were noted in the 2/3NX triple NOSs(-/-) mice and were associated with significant increases in plasma angiotensin II levels (a marker of renin-angiotensin system activation) and urinary 8-isoprostane levels (a marker of oxidative stress). Importantly, combined treatment with a clinical dosage of an angiotensin II type 1 receptor blocker, irbesartan, and a calcium channel antagonist, amlodipine, markedly prevented coronary arteriosclerotic lesion formation and the incidence of AMI and improved the prognosis of those mice, along with ameliorating all those pro-arteriosclerotic parameters. The 2/3NX triple NOSs(-/-) mouse is a new experimentally useful model of AMI. Renin-angiotensin system activation, oxidative stress, cardiovascular risk factors, and SDF-1α-induced recruitment of bone marrow-derived VSMC progenitor cells appear to be involved in the pathogenesis of AMI in this model.

Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms. METHODS: Wild-type, single NOS knockout and triple NOS knockout (n/i/eNOS-/-) mice were administered bleomycin (BLM) intraperitoneally at a dose of 8.0 mg/kg/day for 10 consecutive days. Two weeks after the end of the procedure, the fibrotic and inflammatory changes of the lung were evaluated. In addition, we evaluated the effects of long-term treatment with isosorbide dinitrate, a NO donor, on the n/i/eNOS-/- mice with BLM-induced pulmonary fibrosis. RESULTS: The histopathological findings, collagen content and the total cell number in bronchoalveolar lavage fluid were the most severe/highest in the n/i/eNOS-/- mice. Long-term treatment with the supplemental NO donor in n/i/eNOS-/- mice significantly prevented the progression of the histopathological findings and the increase of the collagen content in the lungs. CONCLUSIONS: These results provide the first direct evidence that a lack of all three NOS isoforms led to a deterioration of pulmonary fibrosis in a BLM-treated murine model. We speculate that the entire endogenous NO and NOS system plays an important protective role in the pathogenesis of pulmonary fibrosis.

New insights into the pharmacological potential of plant flavonoids in the catecholamine system.

Flavonoids are biologically active polyphenolic compounds widely distributed in plants. Recent research has focused on high dietary intake of flavonoids because of their potential to reduce the risks of diseases such as cardiovascular diseases, diabetes, and cancers. We report here the effects of plant flavonoids on catecholamine signaling in cultured bovine adrenal medullary cells used as a model of central and peripheral sympathetic neurons. Daidzein (0.01 - 1.0 µM), a soy isoflavone, stimulated (14)C-catecholamine synthesis through plasma membrane estrogen receptors. Nobiletin (1.0 - 100 µM), a citrus polymethoxy flavone, enhanced (14)C-catecholamine synthesis through the phosphorylation of Ser19 and Ser40 of tyrosine hydroxylase, which was associated with (45)Ca(2+) influx and catecholamine secretion. Treatment with genistein (0.01 - 10 µM), another isoflavone, but not daidzein, enhanced [(3)H]noradrenaline uptake by SK-N-SH cells, a human noradrenergic neuroblastoma cell line. Daidzein as well as nobiletin (≥ 1.0 µM) inhibited catecholamine synthesis and secretion induced by acetylcholine, a physiological secretagogue. The present review shows that plant flavonoids have various pharmacological potentials on the catecholamine system in adrenal medullary cells, and probably also in sympathetic neurons.

Effects of selective estrogen receptor modulators on plasma membrane estrogen receptors and catecholamine synthesis and secretion in cultured bovine adrenal medullary cells.

We previously reported the occurrence and function of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of raloxifene and tamoxifen, selective estrogen receptor modulators, on plasma membrane estrogen receptors and catecholamine synthesis and secretion in these cells. Raloxifene caused dual effects on the specific binding of [(3)H]17ß-estradiol to the plasma membranes isolated from bovine adrenal medulla; that is, it had a stimulatory effect at 1.0 - 10 nM but an inhibitory effect at 1.0 - 10 µM, whereas tamoxifen (1.0 nM - 10 µM) increased binding at all concentrations (except for 100 nM). Tamoxifen at 100 nM caused a significant increase in basal (14)C-catecholamine synthesis from [(14)C]tyrosine, whereas tamoxifen and raloxifene at higher concentrations attenuated basal and acetylcholine-induced (14)C-catecholamine synthesis. Raloxifene (0.3, 1.0, and 3 - 100 µM) and tamoxifen (10 - 100 µM) also suppressed catecholamine secretion and (45)Ca(2+) and (22)Na(+) influx, respectively, induced by acetylcholine. Raloxifene (1.0 µM) inhibited Na(+) current evoked by acetylcholine in Xenopus oocytes expressing α4ß2 neuronal nicotinic acetylcholine receptors. The present findings suggest that raloxifene and tamoxifen at low concentrations allosterically modulate plasma membrane estrogen receptors and at high concentrations inhibit acetylcholine-induced catecholamine synthesis and secretion by inhibiting Na(+) and Ca(2+) influx in bovine adrenal medulla.

Effects of food deprivation on the hypothalamic feeding-regulating peptides gene expressions in serotonin depleted rats.

We examined the effects of serotonin (5-HT) depletion induced by peripheral injection of 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA) on the expression of feeding-regulating peptides expressions by using in situ hybridization histochemistry in adult male Wistar rats. PCPA pretreatment had no significant effect on basal levels of oxytocin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), neuropeptide-Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) or orexin in the hypothalamus. Food deprivation for 48 h caused a significant decrease in CRH, TRH, POMC, and CART, and a significant increase in NPY, AgRP and MCH. After PCPA treatment, POMC and CART did not decrease despite food deprivation. NPY was significantly increased by food deprivation with PCPA, but was attenuated compared to food deprivation without PCPA. These results suggest that the serotonergic system in the hypothalamus may be involved in the gene expression of POMC, CART, and NPY related to feeding behavior.

Stimulatory effect of nobiletin, a citrus polymethoxy flavone, on catecholamine synthesis through Ser19 and Ser40 phosphorylation of tyrosine hydroxylase in cultured bovine adrenal medullary cells.

We previously reported the dual effects of nobiletin, a compound of polymethoxy flavones found in citrus fruits, on catecholamine secretion in cultured bovine adrenal medullary cells. Here, we report the effects of nobiletin on catecholamine synthesis in the cells. Nobiletin increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine in a time (20-30 min)- and concentration (1.0-100 µM)-dependent manner. Nobiletin (10-100 µM) also activated tyrosine hydroxylase activity. The stimulatory effect of nobiletin on (14)C-catecholamine synthesis was not observed when extracellular Ca(2+) was not present in the incubation medium. Protein kinase inhibitors including H-89, an inhibitor of cyclic AMP-dependent protein kinase, and KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, suppressed the stimulatory effects of nobiletin on catecholamine synthesis as well as tyrosine hydroxylase activity. Nobiletin also induced the phosphorylation of tyrosine hydroxylase at Ser(19) and Ser(40). Nobiletin (1.0-100 µM) inhibited (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that nobiletin, by itself, stimulates catecholamine synthesis through tyrosine hydroxylase phosphorylation at Ser(19) and Ser(40), whereas it inhibits catecholamine synthesis induced by acetylcholine in bovine adrenal medulla.

Pentazocine inhibits norepinephrine transporter function by reducing its surface expression in bovine adrenal medullary cells.

(±)-Pentazocine (PTZ), a non-narcotic analgesic, is used for the clinical management of moderate to severe pain. To study the effect of PTZ on the descending noradrenergic inhibitory system, in the present study we examined the effect of [(3)H]norepinephrine (NE) uptake by cultured bovine adrenal medullary cells and human neuroblastoma SK-N-SH cells. (-)-PTZ and (+)-PTZ inhibited [(3)H]NE uptake by adrenal medullary cells in a concentration-dependent (3-100 µM) manner. Eadie-Hofstee analysis of [(3)H]NE uptake showed that both PTZs caused a significant decrease in the V(max) with little change in the apparent K(m), suggesting non-competitive inhibition. Nor-Binaltorphimine and BD-1047, κ-opioid and σ-receptor antagonists, respectively, did not affect the inhibition of [(3)H]NE uptake induced by (-)-PTZ and (+)-PTZ, respectively. PTZs suppressed specific [(3)H]nisoxetine binding to intact SK-N-SH cells, but not directly to the plasma membranes isolated from the bovine adrenal medulla. Scatchard analysis of [(3)H]nisoxetine binding to SK-N-SH cells revealed that PTZs reduced the B(max) without changing the apparent K(d). Western blot analysis showed a decrease in biotinylated cell-surface NE transporter (NET) expression after the treatment with (-)-PTZ. These findings suggest that PTZ inhibits the NET function by reducing the amount of NET in the cell surface membranes through an opioid and σ-receptor-independent pathway.

Stimulation of norepinephrine transporter function by fasudil, a Rho kinase inhibitor, in cultured bovine adrenal medullary cells.

Norepinephrine transporter (NET) regulates noradrenergic synaptic transmission by controlling extracellular levels of norepinephrine (NE). The small GTPase, RhoA, and its downstream effector Rho kinase (ROCK) are involved in the regulation of actin cytoskeleton and focal adhesion/stress fiber formation, which may play an important role in various functions of the sympathetic nervous system. We report here the effect of fasudil, a ROCK inhibitor, on the functions of NET in cultured bovine adrenal medullary cells as a model of sympathetic neurons. Treatment of bovine adrenal medullary cells with fasudil caused an increase in [(3)H]NE uptake in time (8-120 h) and concentration (10-100 µM)-dependent manner. Another ROCK inhibitor, Y-27632 (10-100 µM, 1 day), also increased [(3)H]NE uptake by the cells. Kinetics analysis of the effect of fasudil on NE transport showed a significant increase in the V (max) of NE transport with little change in K (m). When both extracellular and intracellular Ca(2+) were removed by the deprivation of extracellular Ca(2+) and BAPTA-AM, a cell-permeable Ca(2+) chelator, [(3)H]NE uptake induced by fasudil was completely abolished. Nocodazole, an inhibitor of microtubule polymerization, but not cytochalasin D, an inhibitor of actin polymerization, suppressed the stimulatory effect of fasudil on [(3)H]NE uptake. The present findings suggest that the ROCK inhibitor fasudil up-regulates NET function in a Ca(2+)-dependent and/or nocodazole-sensitive pathway in adrenal medullary cells.

Crucial vasculoprotective role of the whole nitric oxide synthase system in vascular lesion formation in mice: Involvement of bone marrow-derived cells.

Although all three nitric oxide (NO) synthases (nNOS, iNOS, and eNOS) are expressed in injured arteries, it remains to be elucidated the role of the NOSs in their entirety in the vascular lesion formation. We addressed this issue in mice deficient in all NOS genes. Vascular injury was induced by permanent ligation of a unilateral carotid artery in wild-type (WT), singly, and triply NOS(-/-) mice. Two weeks after the procedure, constrictive vascular remodeling and neointimal formation were recognized in the ligated arteries. While constrictive remodeling was noted in the nNOS(-/-) and iNOS(-/-) genotypes, it was most accelerated in the n/i/eNOS(-/-) genotype. While neointimal formation was evident in the eNOS(-/-) and nNOS(-/-) genotypes, it was also most aggravated in the n/i/eNOS(-/-) genotype. Those lesions were reversed by long-term treatment with isosorbide dinitrate, a NO donor. Finally, we examined the involvement of bone marrow-derived cells in the vascular lesion formation. Bone marrow from the WT, singly, or triply NOS(-/-) mice was transplanted into the WT mice, and then the carotid ligation was performed. Intriguingly, constrictive remodeling and neointimal formation were both similarly most exacerbated in the case of the n/i/eNOS(-/-) bone marrow transplantation. These results indicate that the complete disruption of all the NOS genes causes markedly accelerated vascular lesion formation caused by blood flow disruption in mice in vivo, demonstrating the crucial vasculoprotective role of the whole endogenous NOS system. Our findings also suggest that the NOS system in bone marrow-derived cells may be involved in this vasculoprotective mechanism.

Stimulation of IFN-γ production by garlic lectin in mouse spleen cells: involvement of IL-12 via activation of p38 MAPK and ERK in macrophages.

Several lectins, present in beans and edible plant products, have immuno-potentiating and anti-tumor activities. We here report the effects of garlic lectin purified from garlic bulbs on the production of cytokines such as interleukin-12 (IL-12) and interferon-γ (IFN-γ) in the mouse. Garlic lectin induced IFN-γ production in spleen cells in a bell-shaped time (24-60 h)- and concentration (0.25-2.0 mg/ml)-dependent manner. The maximal enhancement was observed at 36 h with 0.5 mg/ml of garlic lectin. The stimulatory effect of garlic lectin on IFN-γ production was completely inhibited by both actinomycin D and cycloheximide, an inhibitor of ribosomal protein synthesis and DNA-dependent RNA polymerase, respectively, and was associated with an increase in IFN-γ mRNA level. Garlic lectin also induced IL-12 production in mouse peritoneal macrophages in a concentration (0.25-1.0 mg/ml)- and bell-shaped time (3-24 h)-dependent manner. The lectin increased the phosphorylation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) in macrophages. Furthermore, specific pharmacological inhibitors of ERK kinase (U0126) and p38 MAPK (SB203580) also suppressed the production of IL-12 induced by garlic lectin. The present findings suggest that garlic lectin induces IL-12 production via activation of p38 MAPK and ERK in mouse macrophages, which, in turn, stimulates IFN-γ production through an increase in IFN-γ mRNA in the spleen cells.

Complete disruption of all nitric oxide synthase genes causes markedly accelerated renal lesion formation following unilateral ureteral obstruction in mice in vivo.

The role of nitric oxide (NO) derived from all three NO synthases (NOSs) in renal lesion formation remains to be fully elucidated. We addressed this point in mice lacking all NOSs. Renal injury was induced by unilateral ureteral obstruction (UUO). UUO caused significant renal lesion formation (tubular apoptosis, interstitial fibrosis, and glomerulosclerosis) in wild-type, singly, and triply NOS(-/-) mice. However, the extents of renal lesion formation were markedly and most accelerated in the triply NOS(-/-) genotype. UUO also elicited the infiltration of inflammatory macrophages, up-regulation of transforming growth factor (TGF)-ß1, and induction of epithelial mesenchymal transition (EMT) in all of the genotypes; however, the extents were again largest by far in the triply NOS(-/-) genotype. Importantly, long-term treatment with the angiotensin II type 1 (AT(1))-receptor blocker olmesartan significantly prevented the exacerbation of those renal structural changes after UUO in the triply NOS(-/-) genotype, along with amelioration of the macrophage infiltration, TGF-ß1 levels, and EMT. These results provide the first evidence that the complete disruption of all NOS genes results in markedly accelerated renal lesion formation in response to UUO in mice in vivo through the AT(1)-receptor pathway, demonstrating the critical renoprotective role of all NOSs-derived NO against pathological renal remodeling.

Upregulation of norepinephrine transporter function by prolonged exposure to nicotine in cultured bovine adrenal medullary cells.

Nicotine acts on nicotinic acetylcholine receptors in the adrenal medulla and brain, thereby stimulating the release of monoamines such as norepinephrine (NE). In the present study, we examined the effects of prolonged exposure to nicotine on NE transporter (NET) activity in cultured bovine adrenal medullary cells. Treatment of adrenal medullary cells with nicotine increased [(3)H]NE uptake in both a time- (1-5 days) and concentration-dependent (0.1-10 muM) manner. Kinetic analysis showed that nicotine induced an increase in the V (max) of [(3)H]NE uptake with little change in K (m). This increase in NET activity was blocked by cycloheximide, an inhibitor of ribosomal protein synthesis, but not by actinomycin D, a DNA-dependent RNA polymerase inhibitor. [(3)H]NE uptake induced by nicotine was strongly inhibited by hexamethonium and mecamylamine but not by alpha-bungarotoxin, and was abolished by elimination of Ca(2+) from the culture medium. KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, attenuated not only nicotine-induced [(3)H]NE uptake but also (45)Ca(2+) influx in the cells. The present findings suggest that long-term exposure to nicotine increases NET activity through a Ca(2+)-dependent post-transcriptional process in the adrenal medulla.