PURPOSE: To determine whether alpha(1)-adrenergic receptors use the nitric oxide (NO)/cGMP pathway to stimulate protein secretion by rat lacrimal gland. METHODS: Identification and cellular location of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were determined by Western blot and immunofluorescence techniques, respectively. Rat lacrimal gland acini were isolated by collagenase digestion, and protein secretion stimulated by phenylephrine, an alpha(1)-adrenergic agonist, was measured with a fluorescence assay system. Acini were preincubated with inhibitors for 20 minutes before addition of phenylephrine (10(-4) M). NO and cGMP were measured in response to phenylephrine stimulation. Activation of p42/p44 MAPK was determined by Western blot analysis with an antibody against phosphorylated (active) p42/p44 MAPK. RESULTS: eNOS and nNOS were both present in lacrimal gland. eNOS appeared to be localized with caveolae, whereas nNOS was present in the nerves surrounding the acini. Inhibition of eNOS with N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-6) M) completely inhibited phenylephrine-stimulated protein secretion, whereas the inactive isomer d-NAME and inhibition of nNOS with S-methyl-l-thiocitrulline did not. Phenylephrine increased NO production in a time- and concentration-dependent manner, but the increase was abolished by the alpha(1D)-adrenergic receptor inhibitor BMY-7378. Inhibition of guanylate cyclase with oxadiazoloquinoxalin (ODQ) also inhibited phenylephrine-induced protein secretion, whereas phenylephrine caused a 2.2-fold increase in cGMP. In addition, preincubation with l-NAME and ODQ inhibited phenylephrine-stimulated p42/p44 MAPK activation. CONCLUSIONS: alpha(1D)-Adrenergic agonists stimulate eNOS to produce NO, leading to production of cGMP by guanylate cyclase, to transduce the extracellular signal through the cell and stimulate protein secretion in rat lacrimal gland.
PURPOSE: To determine whether alpha(1)-adrenergic receptors use the nitric oxide (NO)/cGMP pathway to stimulate protein secretion by rat lacrimal gland. METHODS: Identification and cellular location of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were determined by Western blot and immunofluorescence techniques, respectively. Rat lacrimal gland acini were isolated by collagenase digestion, and protein secretion stimulated by phenylephrine, an alpha(1)-adrenergic agonist, was measured with a fluorescence assay system. Acini were preincubated with inhibitors for 20 minutes before addition of phenylephrine (10(-4) M). NO and cGMP were measured in response to phenylephrine stimulation. Activation of p42/p44 MAPK was determined by Western blot analysis with an antibody against phosphorylated (active) p42/p44 MAPK. RESULTS: eNOS and nNOS were both present in lacrimal gland. eNOS appeared to be localized with caveolae, whereas nNOS was present in the nerves surrounding the acini. Inhibition of eNOS with N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-6) M) completely inhibited phenylephrine-stimulated protein secretion, whereas the inactive isomer d-NAME and inhibition of nNOS with S-methyl-l-thiocitrulline did not. Phenylephrine increased NO production in a time- and concentration-dependent manner, but the increase was abolished by the alpha(1D)-adrenergic receptor inhibitor BMY-7378. Inhibition of guanylate cyclase with oxadiazoloquinoxalin (ODQ) also inhibited phenylephrine-induced protein secretion, whereas phenylephrine caused a 2.2-fold increase in cGMP. In addition, preincubation with l-NAME and ODQ inhibited phenylephrine-stimulated p42/p44 MAPK activation. CONCLUSIONS: alpha(1D)-Adrenergic agonists stimulate eNOS to produce NO, leading to production of cGMP by guanylate cyclase, to transduce the extracellular signal through the cell and stimulate protein secretion in rat lacrimal gland.
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Authors: Larry P Solomonson; Brenda R Flam; Laura C Pendleton; Bonnie L Goodwin; Duane C Eichler Journal: J Exp Biol Date: 2003-06 Impact factor: 3.312
Authors: LiLi Chen; Robin R Hodges; Chika Funaki; Driss Zoukhri; Robert J Gaivin; Dianne M Perez; Darlene A Dartt Journal: Am J Physiol Cell Physiol Date: 2006-06-07 Impact factor: 4.249
Authors: Carlos Belmonte; Jason J Nichols; Stephanie M Cox; James A Brock; Carolyn G Begley; David A Bereiter; Darlene A Dartt; Anat Galor; Pedram Hamrah; Jason J Ivanusic; Deborah S Jacobs; Nancy A McNamara; Mark I Rosenblatt; Fiona Stapleton; James S Wolffsohn Journal: Ocul Surf Date: 2017-07-20 Impact factor: 5.033