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.

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.

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.

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.

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.

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.

Stimulatory effects of the soy phytoestrogen genistein on noradrenaline transporter and serotonin transporter activity.

We examined the effects of genistein, one of the major soy phytoestrogens, on the activity of noradrenaline transporter (NAT) and serotonin transporter. Treatment with genistein (10 nM-10 microM) for 20 min stimulated [(3)H]noradrenaline (NA) uptake by SK-N-SH cells. Genistein also stimulated [(3)H]NA uptake and [(3)H]serotonin uptake by NAT and serotonin transporter transiently transfected COS-7 cells, respectively. Kinetics analysis of the effect of genistein on NAT activity in NAT-transfected COS-7 cells revealed that genistein significantly increased the maximal velocity of NA transport with little or no change in the affinity. Scatchard analysis of [(3)H]nisoxetine binding to NAT-transfected COS-7 cells showed that genistein increased the maximal binding without altering the dissociation constant. Although genistein is also known to be an inhibitor of tyrosine kinases, daidzein, another soy phytoestrogen and an inactive genistein analogue against tyrosine kinases, had little effect on [(3)H]NA uptake by SK-N-SH cells. The stimulatory effects on NAT activity were observed by treatment of tyrphostin 25, an inhibitor of epidermal growth factor receptor tyrosine kinase, whereas orthovanadate, a protein tyrosine phosphatase inhibitor, suppressed [(3)H]NA uptake by NAT-transfected COS-7 cells. These findings suggest that genistein up-regulates the activity of neuronal monoamine transporters probably through processes involving protein tyrosine phosphorylation.

Insights into the pharmacological potential of estrogens and phytoestrogens on catecholamine signaling.

We report here the effects of estrogens and phytoestrogens on catecholamine signaling in cultured bovine adrenal medullary cells used as a model of catecholaminergic neurons in the brain. Treatment of the cells for 20 min with 17beta-estradiol (E(2)) (0.3-100 nM) or phytoestrogens such as daidzein (0.01-1.0 microM), a soy isoflavone, and resveratrol (0.1-1.0 microM), a grape polyphenol, stimulated (14)C-catecholamine synthesis from [(14)C]tyrosine, which was associated with the activation of tyrosine hydroxylase. The stimulatory effect of E(2) and phytoestrogens was not inhibited by ICI182,780, a nuclear estrogen receptor inhibitor, but abolished by U0126, an inhibitor of extracellular signal-regulated kinase1/2 (ERK1/2) kinase. E(2) enhanced the phosphorylation of ERK1/2. The plasma membrane isolated from the adrenal medulla showed two classes of specific binding sites of [(3)H]E(2). Resveratrol and daidzein at high concentrations (> or =1.0 microM) inhibited catecholamine secretion induced by various secretagogues. The present findings suggest that estrogens and phytoestrogens most likely stimulate catecholamine synthesis via estrogen receptors in the plasma membrane, but in high concentrations phytoestrogens inhibit catecholamine secretion induced by secretagogues in adrenal medullary cells, and probably in brain neurons.

Copper, zinc-superoxide dismutase enhances the mutagenicity in Salmonella typhimurium induced by 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole.

The mutagenicities of various carcinogens induced by liver microsomes are increased in the presence of liver cytosol in rodents. It still remains, however, to be clarified which factor or factors in the cytosol enhance(s) the microsome-mediated mutagenicities. In the present study, we sought to identify the enhancing factor in liver cytosol prepared from rats using the microsome-mediated Salmonella mutagenicity induced by 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole (Glu-P-1). By a series of chromatographic steps, we purified a 16-kDa protein on SDS-PAGE from the cytosol of rat livers. Partial amino acid sequences of this protein revealed that the 16-kDa protein was copper, zinc-superoxide dismutase (CuZn-SOD). The purified CuZn-SOD enhanced the microsome-mediated mutagenicities of several heterocyclic amines and aromatic amines. Furthermore, bovine and human CuZn-SOD also enhanced the microsome-mediated mutagenicity of Glu-P-1. The CuZn-SOD caused an increase in the mutagenicity of N-hydroxylated Glu-P-1 formed from Glu-P-1 by the microsomes, although CuZn-SOD did not affect either the formation or the stability of the N-hydroxylated derivative. These findings suggest that the enhancing cytosol factor for the mutagenicity of Glu-P-1 is CuZn-SOD, which stimulates the mutagenicity of N-hydroxylated Glu-P-1 without changing its metabolism.

Effects of resveratrol, a grape polyphenol, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells.

We report the effects of resveratrol, a polyphenol found in the skins of red grapes, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Resveratrol suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner (IC(50)=20.4, 11.0, and 62.8 microM, respectively). Resveratrol also inhibited catecholamine secretion induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels, at concentrations similar to those for (45)Ca(2+) influx. Resveratrol directly inhibited the current evoked by acetylcholine in Xenopus oocytes expressing alpha3beta4 neuronal nicotinic acetylcholine receptors (IC(50)=25.9 microM). Furthermore, resveratrol (IC(50)=5.32 microM) attenuated (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that resveratrol inhibits acetylcholine-induced catecholamine secretion and synthesis through suppressing ion influx in cultured bovine adrenal medullary cells.

Dual effects of daidzein, a soy isoflavone, on catecholamine synthesis and secretion in cultured bovine adrenal medullary cells.

We recently demonstrated the occurrence and functional roles of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of daidzein, a phytoestrogen of soybeans, on catecholamine synthesis and secretion in the cells. Incubation of cells with daidzein for 20 min increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine but not [(14)C]dihydroxyphenylalanine, in a concentration-dependent manner (10-1000 nm). The stimulatory effect of daidzein on (14)C-catecholamine synthesis was not inhibited by ICI182,780, a classical estrogen receptor inhibitor. Acetylcholine, a physiological secretagogue, stimulated the synthesis of (14)C-catecholamines, which was suppressed by daidzein at 1 mum. Daidzein at high concentrations (1-100 microm) suppressed catecholamine secretion induced by acetylcholine. Furthermore, daidzein (10-1000 nm) inhibited the specific binding of [(3)H]17beta-estradiol to plasma membranes isolated from bovine adrenal medulla. The present findings suggest that daidzein at low concentrations stimulates catecholamine synthesis through plasma membrane estrogen receptors but at high concentrations inhibits catecholamine synthesis and secretion induced by acetylcholine in bovine adrenal medulla. The latter effect of daidzein may be a beneficial action on the cardiovascular system.

Stimulation of catecholamine synthesis through unique estrogen receptors in the bovine adrenomedullary plasma membrane by 17beta-estradiol.

Incubation of cultured bovine adrenal medullary cells with 17beta-estradiol (E(2)) (0.3-100nM) or membrane-impermeable E(2)-bovine serum albumin (100nM) acutely increased (14)C-catecholamine synthesis from [(14)C]tyrosine. The stimulatory effect of E(2) was not inhibited by ICI182,780, a nuclear estrogen receptor inhibitor. E(2) also increased tyrosine hydroxylase activity and p44/42MAPK phosphorylation, the former of which was attenuated by U0126, an inhibitor of p44/42MAPK kinase. The plasma membrane isolated from the gland showed two classes of specific binding sites of [(3)H]E(2) with apparent K(d)s of 3.2 and 106nM, and B(max)s of 0.44 and 8.5pmol/mg protein, respectively. The high-affinity binding of [(3)H]E(2) was most strongly inhibited by E(2) and phytoestrogens, and to lesser extents by other steroid hormones, while it was enhanced by ICI182,780 and environmental estrogenic pollutants. These findings suggest that E(2) acutely stimulates catecholamine synthesis via activation of p44/42MAPK through unique estrogen receptors in the plasma membrane of bovine adrenal medulla.

Stimulation of catecholamine synthesis by environmental estrogenic pollutants.

Environmental estrogenic pollutants are compounds that have been shown to have estrogenic effects on fetal development and reproductive systems. Less attention, however, has been paid to their influence on neuronal functions. We report here the effects of estrogenic pollutants on catecholamine synthesis in bovine adrenal medullary cells used as a model system of noradrenergic neurons. Treatment of cultured bovine adrenal medullary cells with p-nonylphenol and bisphenol A at 10 nM for 3 d stimulated [14C]catecholamine synthesis from [14C]tyrosine and tyrosine hydroxylase activity, an effect that was not inhibited by ICI 182,780, an antagonist of estrogen receptors. Significant effects of p-nonylphenol on [14C]catecholamine synthesis were observed at 0.1 nM, which is 45 times lower than that of the international regulatory standard (4.5 nM), and the maximum effects were around 10-100 nM. The concentrations (0.1-10 nM) used in the present study are similar to the range observed in rivers in the United States or Europe. On the other hand, short-term treatment of cells with 10 nM p-nonylphenol for 10 min also activated tyrosine hydroxylase, which was suppressed by U0126, an inhibitor of MAPK kinase. Furthermore, treatment of cells with p-nonylphenol for 5 min increased the phospho-p44/42MAPK in a concentration-dependent (1-1000 nM) manner, whereas p-nonylphenol (100 nm, 2 d) enhanced both levels of non-phospho- and phospho-p44/42MAPK. These findings suggest that short-term and long-term treatment of cells with estrogenic pollutants at environmental concentrations stimulates catecholamine synthesis and MAPK through an estrogen receptor-independent pathway.