Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin

C. H. Kang-Rotondo, S. Major, T. M. Chiang, Linda Myers, E. S. Kang

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [3H]L-citrulline using labeled L-arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykinin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2-48.3 mJ/cm2 but was inhibitory after 64.4 and 80.5 mJ/cm2. Thirty min after 10-6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M(r) 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.

Original languageEnglish (US)
Pages (from-to)57-65
Number of pages9
JournalPhotodermatology Photoimmunology and Photomedicine
Volume12
Issue number2
DOIs
StatePublished - Jan 1 1996

Fingerprint

Bradykinin
Keratinocytes
Nitric Oxide Synthase
Up-Regulation
Protein Isoforms
Nitric Oxide
Enzymes
Citrulline
Skin
NADP
Arginine
Monoclonal Antibodies
Antibodies
Brain

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology
  • Radiology Nuclear Medicine and imaging
  • Dermatology

Cite this

Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin. / Kang-Rotondo, C. H.; Major, S.; Chiang, T. M.; Myers, Linda; Kang, E. S.

In: Photodermatology Photoimmunology and Photomedicine, Vol. 12, No. 2, 01.01.1996, p. 57-65.

Research output: Contribution to journalArticle

@article{5bfe8fc70097421193b9df1a984237e0,
title = "Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin",
abstract = "Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [3H]L-citrulline using labeled L-arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykinin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2-48.3 mJ/cm2 but was inhibitory after 64.4 and 80.5 mJ/cm2. Thirty min after 10-6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M(r) 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.",
author = "Kang-Rotondo, {C. H.} and S. Major and Chiang, {T. M.} and Linda Myers and Kang, {E. S.}",
year = "1996",
month = "1",
day = "1",
doi = "10.1111/j.1600-0781.1996.tb00176.x",
language = "English (US)",
volume = "12",
pages = "57--65",
journal = "Photodermatology Photoimmunology and Photomedicine",
issn = "0905-4383",
publisher = "Blackwell Munksgaard",
number = "2",

}

TY - JOUR

T1 - Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin

AU - Kang-Rotondo, C. H.

AU - Major, S.

AU - Chiang, T. M.

AU - Myers, Linda

AU - Kang, E. S.

PY - 1996/1/1

Y1 - 1996/1/1

N2 - Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [3H]L-citrulline using labeled L-arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykinin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2-48.3 mJ/cm2 but was inhibitory after 64.4 and 80.5 mJ/cm2. Thirty min after 10-6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M(r) 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.

AB - Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [3H]L-citrulline using labeled L-arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykinin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2-48.3 mJ/cm2 but was inhibitory after 64.4 and 80.5 mJ/cm2. Thirty min after 10-6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M(r) 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.

UR - http://www.scopus.com/inward/record.url?scp=0029786850&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029786850&partnerID=8YFLogxK

U2 - 10.1111/j.1600-0781.1996.tb00176.x

DO - 10.1111/j.1600-0781.1996.tb00176.x

M3 - Article

VL - 12

SP - 57

EP - 65

JO - Photodermatology Photoimmunology and Photomedicine

JF - Photodermatology Photoimmunology and Photomedicine

SN - 0905-4383

IS - 2

ER -