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Ca2+, calmodulin and phospholipids regulate nitricoxide synthase activity in the rabbit submandibular gland

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Abstract

Nitric oxide (NO) plays an important role as an intra- and intercellular signaling molecule in mammalian tissues. In the submandibular gland, NO has been suggested to be involved in the regulation of secretion and in blood flow. NO is produced by activation of NO synthase (NOS). Here, we have investigated the regulation of NOS activity in the rabbit submandibular gland. NOS activity was detected in both the cytosolic and membrane fractions. Characteristics of NOS in the cytosolic and partially purified membrane fractions, such as Km values for l-arginine and EC50 values for calmodulin and Ca2+, were similar. A protein band that cross-reacted with anti-nNOS antibody was detected in both the cytosolic and membrane fractions. The membrane-fraction NOS activity increased 1.82-fold with treatment of Triton X-100, but the cytosolic-fraction NOS activity did not. The NOS activity was inhibited by phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP2). The inhibitory effects of phospholipids on the NOS activity were relieved by an increase in Ca2+ concentrations. These results suggest that the Ca2+- and calmodulin-regulating enzyme nNOS occurs in cytosolic and membrane fractions, and PA and PIP2 regulate the NOS activity in the membrane site by regulating the effect of Ca2+ in the rabbit submandibular gland.

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Abbreviations

BSA :

bovine serum albumin

[Ca2+]i:

intracellular Ca2+ concentrations

DTT :

dithiothreitol

H 4 BP :

(6R)-5,6,7,8-tetrahydro-L-biopterin

L-NAME:

NG-nitro-l-arginine methyl ester

NO :

nitric oxide

NOLA :

NG-nitro-l-arginine

NOS :

NO synthase

PA :

phosphatidic acid

PC :

phosphatidylcholine

PE :

phosphatidylethanolamine

PIP 2 :

phosphatidylinositol 4,5-bisphosphate

PMSF :

phenylmethylsulfonyl fluoride

PNF :

post-nucleus fraction

PS :

phosphatidylserine

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Acknowledgements

We thank Dr. Yuka Mitsui for helping to determine NOS activities. This study was supported in part by a Nihon University Multidisciplinary Research Grant for 2003–2004, a Grant-in-Aid for 2003 Multidisciplinary Research Project from MEXT, and a Grant-in-Aid for Scientific Research from JSPS (#16390534).

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Authors and Affiliations

  1. Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, 271-8587, Chiba , Japan

    Y. Yamamoto

  2. Department of Physiology and Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Matsudo, 271-8587, Chiba , Japan

    O. Katsumata, S. Furuyama & H. Sugiya

Authors
  1. Y. Yamamoto
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  2. O. Katsumata
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  3. S. Furuyama
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  4. H. Sugiya
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Correspondence to H. Sugiya.

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Communicated by I.D. Hume

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Yamamoto, Y., Katsumata, O., Furuyama, S. et al. Ca2+, calmodulin and phospholipids regulate nitricoxide synthase activity in the rabbit submandibular gland. J Comp Physiol B 174, 593–599 (2004). https://doi.org/10.1007/s00360-004-0448-y

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  • DOI: https://doi.org/10.1007/s00360-004-0448-y

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