The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity

Basilia Zingarelli, Garry J. Southan, Elijah Gilad, Michael O'Connor, Andrew L. Salzman, Csaba Szabo

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

1. It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes. Aminoguanidine, N(G)-methyl-L-arginine (L-NMA) and N(G)-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes. 2. Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 μg ml-1) and interferon γ (IFNγ, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 μM) for 6h. This treatment induced a significant production of 6-keto-prostaglandin F(1α), (6-keto-PGF(1α), the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF(1α), with an IC50 of 20 μM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF(1α) in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 μM) for 30 min; this also induced a significant production of 6-keto-PGF(1α) and MEG (1 μM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF(1α). Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF(1α) production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 μM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins. 5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF(1α), production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF(1α), production in the HUVECs. 6. The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33 μM and 36 μM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3 mM) decreased COX-1 activity only, while L-NMA (up to 3 mM) had no effect on the activity of either enzyme. 7. These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.

Original languageEnglish (US)
Pages (from-to)357-366
Number of pages10
JournalBritish Journal of Pharmacology
Volume120
Issue number3
DOIs
StatePublished - 1997
Externally publishedYes

Fingerprint

Prostaglandin-Endoperoxide Synthases
Prostaglandins F
Arachidonic Acid
NG-Nitroarginine Methyl Ester
Human Umbilical Vein Endothelial Cells
Macrophages
Prostaglandins
Nitrites
Inhibitory Concentration 50
Protein Isoforms
Cyclooxygenase Inhibitors
Nitric Oxide Synthase
Indomethacin
Immunization

Keywords

  • Endotoxin
  • Inflammation
  • Mercaptoalkylguanidines
  • Prostaglandins nitric oxide
  • Shock

ASJC Scopus subject areas

  • Pharmacology

Cite this

The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity. / Zingarelli, Basilia; Southan, Garry J.; Gilad, Elijah; O'Connor, Michael; Salzman, Andrew L.; Szabo, Csaba.

In: British Journal of Pharmacology, Vol. 120, No. 3, 1997, p. 357-366.

Research output: Contribution to journalArticle

Zingarelli, B, Southan, GJ, Gilad, E, O'Connor, M, Salzman, AL & Szabo, C 1997, 'The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity', British Journal of Pharmacology, vol. 120, no. 3, pp. 357-366. https://doi.org/10.1038/sj.bjp.0700892
Zingarelli, Basilia ; Southan, Garry J. ; Gilad, Elijah ; O'Connor, Michael ; Salzman, Andrew L. ; Szabo, Csaba. / The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity. In: British Journal of Pharmacology. 1997 ; Vol. 120, No. 3. pp. 357-366.
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Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 μg ml-1) and interferon γ (IFNγ, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 μM) for 6h. This treatment induced a significant production of 6-keto-prostaglandin F(1α), (6-keto-PGF(1α), the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF(1α), with an IC50 of 20 μM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF(1α) in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 μM) for 30 min; this also induced a significant production of 6-keto-PGF(1α) and MEG (1 μM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF(1α). Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF(1α) production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 μM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins. 5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF(1α), production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF(1α), production in the HUVECs. 6. The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33 μM and 36 μM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3 mM) decreased COX-1 activity only, while L-NMA (up to 3 mM) had no effect on the activity of either enzyme. 7. These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.",
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T1 - The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity

AU - Zingarelli, Basilia

AU - Southan, Garry J.

AU - Gilad, Elijah

AU - O'Connor, Michael

AU - Salzman, Andrew L.

AU - Szabo, Csaba

PY - 1997

Y1 - 1997

N2 - 1. It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes. Aminoguanidine, N(G)-methyl-L-arginine (L-NMA) and N(G)-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes. 2. Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 μg ml-1) and interferon γ (IFNγ, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 μM) for 6h. This treatment induced a significant production of 6-keto-prostaglandin F(1α), (6-keto-PGF(1α), the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF(1α), with an IC50 of 20 μM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF(1α) in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 μM) for 30 min; this also induced a significant production of 6-keto-PGF(1α) and MEG (1 μM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF(1α). Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF(1α) production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 μM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins. 5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF(1α), production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF(1α), production in the HUVECs. 6. The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33 μM and 36 μM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3 mM) decreased COX-1 activity only, while L-NMA (up to 3 mM) had no effect on the activity of either enzyme. 7. These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.

AB - 1. It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes. Aminoguanidine, N(G)-methyl-L-arginine (L-NMA) and N(G)-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes. 2. Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 μg ml-1) and interferon γ (IFNγ, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 μM) for 6h. This treatment induced a significant production of 6-keto-prostaglandin F(1α), (6-keto-PGF(1α), the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF(1α), with an IC50 of 20 μM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF(1α) in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 μM) for 30 min; this also induced a significant production of 6-keto-PGF(1α) and MEG (1 μM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF(1α). Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 μM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF(1α) production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 μM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins. 5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF(1α), production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF(1α), production in the HUVECs. 6. The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33 μM and 36 μM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3 mM) decreased COX-1 activity only, while L-NMA (up to 3 mM) had no effect on the activity of either enzyme. 7. These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.

KW - Endotoxin

KW - Inflammation

KW - Mercaptoalkylguanidines

KW - Prostaglandins nitric oxide

KW - Shock

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