Purification of a Ca<sup>2+</sup>/calmodulin‐dependent nitric oxide synthase from porcine cerebellum

Mayer, Bernd; John, Mathias; Böhme, Eycke

doi:10.1016/0014-5793(90)80848-d

Cited by 426 publications

(205 citation statements)

References 25 publications

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“…In reality, basal [Ca 2ϩ ] i is still a prerequisite of this so-called "calcium-independent" activation of eNOS (49), since purified eNOS and broken endothelial cell preparations containing eNOS are unable to produce NO in the absence of Ca 2ϩ in the reaction mixtures (18,35 (25), it failed to fully block the increase in NO in response to ATP in P-UAE. This confirms that, in P-UAE at least, there is some component of cell signaling that can activate eNOS in a [Ca 2ϩ ] i -insensitive manner, as predicted by our studies in UAEC.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous imaging of [Ca²⁺]_iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Feng

Magness

Bird

2005

American Journal of Physiology-Regulatory, Integrative and Comp

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Yi, Fu-Xian, Ronald R. Magness, and Ian M. Bird. Simultaneous imaging of [Ca 2ϩ ] i and intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy. Am J Physiol Regul Integr Comp Physiol 288: R140 -R148, 2005. First published August 5, 2004; doi:10.1152/ajpregu. 00302.2004.-Pregnancy and the follicular phase of the ovarian cycle show elevation of uterine blood flow and associated increases in uterine artery endothelium (UAE) endothelial nitric oxide (NO) synthase (eNOS) expression. Nonetheless, a role for increased NO production during pregnancy and the follicular phase has only been inferred by indirect measures. The recent development of a uterine artery endothelial cell model further suggests that pregnancy is associated with reprogramming of cell signaling, such that eNOS may become more Ca 2ϩ sensitive and be subject to regulation by Ca 2ϩ -independent kinases. This study describes for the first time the direct and simultaneous monitoring of NO production and intracellular free Ca 2ϩ concentration ([Ca 2ϩ ]i) in freshly isolated UAE from pregnant, follicular, and luteal sheep. The pharmacological agonists ionomycin (calcium ionophore) and thapsigargin (TG; endoplasmic reticulum Ca 2ϩ pump inhibitor) were used to maximally elevate [Ca 2ϩ ]i and fully activate eNOS as a measure of eNOS expression. NO production stimulated by ionomycin (5 M) and TG (10 M) were 1.95-and 2.05-fold, respectively, in pregnant-UAE and 1.34-and 1.37-fold in follicular-UAE compared with luteal-UAE. In contrast, the physiological agonist ATP (100 M) stimulated a 3.43-fold increase in NO in pregnant-UAE and a 1.90-fold increase in follicular-UAE compared with luteal-UAE, suggesting that pregnancy and follicular phase enhance eNOS activation beyond changes in expression in vivo. 2-aminoethoxydiphenyl borate (APB; an inositol 1,4,5-trisphosphate receptor blocker) totally prevented the ATP-induced [Ca 2ϩ ]i response but only partially inhibited NO production. Thus pregnancy-enhanced eNOS activation in UAE is mediated through [Ca 2ϩ ]i-insensitive pathways as well as through a greater eNOS sensitivity to [Ca 2ϩ ]i. endothelium; nitric oxide; follicular; kinase; programming; intracellular free calcium concentration NITRIC OXIDE (NO) is a ubiquitous intracellular signaling molecule, synthesized from L-arginine by NO synthase in diverse cells and tissues. Endothelial NO synthase (eNOS), first identified in endothelial cells, plays a major role in the control of blood pressure and vascular homeostasis. In the vascular endothelium, production of NO results in vascular smooth muscle relaxation, which, in turn, reduces blood pressure. eNOS is highly dependent on intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) (1, 12) and is activated by [Ca 2ϩ ] i -mobilizing agonists of diverse G-protein-coupled receptors, such as acetylcholine, bradykinin, and extracellular ATP (40). A complication, however, is that the expression of eNOS in vascular endothelium does not automatically mean...

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Section: Discussionmentioning

confidence: 99%

Simultaneous imaging of [Ca²⁺]_iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Feng

Magness

Bird

2005

American Journal of Physiology-Regulatory, Integrative and Comp

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“…The particulate activity did not decline following repeated washes with 1 M KC1 indicating that it represents an integral membrane protein or is anchored in the membrane. As described for NO synthase from brain (Mayer et al, 1990;Schmidt et al, 1991) both soluble and particulate enzymes from rat anococcygeus required NADPH and BH4. The finding that the particulate enzyme had a greater requirement for BH4 than the soluble could be explained by higher levels of endogenous BH4 in the soluble fraction.…”

mentioning

confidence: 89%

Characterization of nitric oxide synthases in non‐adrenergic non‐cholinergic nerve containing tissue from the rat anococcygeus muscle

Mitchell

Sheng

Förstermann

et al. 1991

British J Pharmacology

103

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Tissue homogenates prepared from rat anococcygeus muscle converted L-arginine to L-citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in. crude and partially-purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8-tetrahydrobiopterin and calcium, and inhibited by NG-nitro-L-arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non-adrenergic non-cholinergic neurones, had no NO synthase activity.

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“…Characterization of the endothelial NOS promoter/␤-galactosidase reporter transgenic mice has been previously reported. 22 …”

Section: ␤-Galactosidase Stainingmentioning

confidence: 99%

“…23 Briefly, whole testes dissected from transgene-positive mice were fixed in Bouin's solution at 23°C for 16 hours and embedded in paraffin after dehydration in ascending alcohol concentrations. Six-m sections were mounted onto slides precoated with chrome-gelatin and immunolabeled with a rabbit anti-human nNOS polyclonal antibody (1:1000 dilution), 22 or a mouse anti-human nNOS monoclonal antibody (1:100 dilution) (Transduction Laboratories, Lexington, KY), or a mouse anti-rat cytochrome P 450 side-chain cleavage enzyme (P 450 scc), a Leydig cell marker, monoclonal antibody (1:200 dilution) (Chemicon International, Hofheim, Germany). To visualize the hybridization signals, a secondary biotinylated anti-rabbit IgG or antimouse IgG (Dakopatts, Glostrup, Denmark) was applied (1:250 dilution) followed by a tertiary rabbit (1:200 dilution) or mouse (1:100 dilution) peroxidase anti-peroxidase complex (Dakopatts) and finally an Elite avidinbiotin-peroxidase complex (ABC) (1:250 dilution; Vector Laboratories, Burlingame, CA).…”

Section: Immunohistochemistrymentioning

confidence: 99%

An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

Wang¹,

Newton²,

Miller³

et al. 2002

The American Journal of Pathology

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Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of ␤-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. ␤-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P 450 scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, ␤-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of ␤-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, ␤-galactosidase activity was expressed only in endothelial cells of large-and medium-sized arterial blood vessels. Transcriptional activity of the Of the three known human nitric oxide synthases (NOS) isoforms, the neuronal nitric oxide synthase (nNOS) has unique properties. The nNOS (NOS1) isoform is expressed from a very complex human genomic locus spanning 240 kb at 12q24.2.

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Purification of a Ca²⁺/calmodulin‐dependent nitric oxide synthase from porcine cerebellum

Cited by 426 publications

References 25 publications

Simultaneous imaging of [Ca²⁺]_iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Simultaneous imaging of [Ca²⁺]_iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Characterization of nitric oxide synthases in non‐adrenergic non‐cholinergic nerve containing tissue from the rat anococcygeus muscle

An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

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Purification of a Ca2+/calmodulin‐dependent nitric oxide synthase from porcine cerebellum

Cited by 426 publications

References 25 publications

Simultaneous imaging of [Ca2+]iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Simultaneous imaging of [Ca2+]iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Characterization of nitric oxide synthases in non‐adrenergic non‐cholinergic nerve containing tissue from the rat anococcygeus muscle

An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

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Purification of a Ca²⁺/calmodulin‐dependent nitric oxide synthase from porcine cerebellum

Simultaneous imaging of [Ca²⁺]_iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Simultaneous imaging of [Ca²⁺]_iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy