Inhibitory Effects of Different Retinoids (Vitamin A Analogues) on the Stimulated Rat Liver Guanylate Cyclase Activity

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The effect of retinylacetate (RA) was investigated on rat liver guanylate cyclase stimulated by nitroprusside (NP). The stimulated enzyme seemed to adhere to Michaëlis‐Menten kinetics with an apparent Km for MnGTP of 0.10 mM and a Vmax, of 410 pmol cGMP/min. × mg prot. RA (0.1–1mM) dose‐dependently inhibited the enzyme stimulated by NP (0.1–10 mM). In the presence of 1 mM RA the activity was only 10% of the control activity. The inhibitory action of RA seemed to be non‐competitive since it depressed Vmax without affecting the Km for MnGTP. In contrast, however, RA was instead found to stimulate the enzyme when low concentrations of NP (0.01–0.1 mM) were used. The concentration‐activity curve for NP was bell‐shaped showing an optimum at 0.5 mM. The inhibition induced by RA could not be surmounted by increasing the NP concentration indicating that RA did not compete with NP. A bell‐shaped activity curve was also seen when the enzyme activity was measured in the presence of increasing Mn2+ concentrations and during these conditions RA also caused inhibition. In the presence of the sulphydryl reductant dithiothreitol (DTT), the NP concentration needed for optimal enzyme activation was about 100‐fold less than in the absence of DTT. The maximal enzyme activity was also slightly increased. In the presence of DTT, RA was much less effective to induce inhibition of the stimulated enzyme, than when DTT was absent. (In the presence of DTT 1 mM RA caused only 30% inhibition compared to 90% inhibition in its absence). These results suggest that sulphydryl groups are of crucial importance in regulating guanylate cyclase activity and that the inhibitory effect of RA on the enzyme might be dependent on the interaction with these sulphydryl groups.

Section: Discussionmentioning

confidence: 99%

“…In some recent papers (Craven & DeRubertis 1977;Rydell et al 1982), a series of vitamin A analogues (retinoids) were observed to be potent inhibitors of the cyclic GMP (cGMP) forming enzyme guanylate cyclase (GTP pyrophosphatelyase (cyclizing), EC 4.6.1.2). Cyclic GMP has been proposed as a regulator of normal and malignant cell growth (Goldberg ef al.…”

mentioning

confidence: 99%

Effects of Retinylacetate on the Kinetics of Rat Liver Guanylate Cyclase: Possible Interaction with Sulfhydryl Groups

Rydell

Axelsson

Wikberg

1985

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“…The supernatant was recovered and stored in the dark under nitrogen at -80" until used in the guanylate cyclase assay. This was performed as described earlier (Rydell et al 1982).…”

Section: Methodsmentioning

confidence: 99%

The Relaxant Effect of Glyceryltrinitrate on Isolated Human Peripheral Vein and its Relation to Cyclic GMP Metabolism

Ahlner¹,

Andersson²,

Axelsson³

et al. 1986

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The relaxant effect of glyceryltrinitrate (GTN) on human vena saphena magna was studied in vitro. Vessels contracted by serotonin (0.25 μM) and phenylephrine (0.1 mM) were relaxed to the same extent (EC50 = 10 μM) by GTN, whereas in 100 mM K+‐depolarized vessels the relaxation was significantly lower. The relaxant effect produced by GTN was preceeded by an elevation of cyclic guanosine‐3′,5′‐monophosphate (cGMP). For 0.1 mM GTN there was a 3‐fold increase in cGMP after 3 min. A correlation between relaxation and increase in cGMP was established. When GTN was combined with dipyridamole (5 μM) the relaxant effect of GTN was significantly greater (EC50 = 0.1 μM). Phosphodiesterase inhibition, as a possible mechanism behind the observed better relaxation for the combination (GTN + dipyridamole), is briefly discussed. In conclusion, the relaxant effect of GTN on isolated human vena saphena magna seems to be i) dependent on the contractile stimuli used, ii) increased by the addition of DIP and iii) to be mediated via cGMP.

“…The supernatant was recovered and stored under nitrogen at -80" until use in the guanylate cyckdse assay. This was performed as described earlier (Rydell et a/. 1982) with varying concentrations of GTP and with 2 mM Mg2+ added in excess of substrate.…”

Section: Methodsmentioning

confidence: 99%

Nitroglycerin Tolerance in Vitro: Effect on cGMP Turnover in Vascular Smooth Muscle

Axelsson

Karlsson

1984

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Bovine mesenteric arteries (BMA) were made tolerant to nitroglycerin (GTN) by incubation with high concentrations of GTN at elevated pH. This treatment has previously been shown to reduce the relaxant and cGMP‐elevating action of a challenging dose of GTN. The stimulatory action of nitroprusside (NP) or GTN/cysteine on guanylate cyclase (GC) was reduced by 50–60% in GTN‐tolerant vessels as compared to control vessels. The stimulatory action of GTN and NP on GC has been suggested to occur through formation of S‐nitrosothiols, probably with a previous denitration step required for GTN. However, tolerance induction to GTN was not found to change the rate of nitrite formation from GTN, and exogenous addition of thiols in the GC assay, in order to increase S‐nitrosothiol formation, did not restore the GC activity in tolerant vessels back to control level. This is suggested to indicate a direct effect of GTN tolerance on GC. Since the cGMP‐phosphodiesterase activity was not affected in GTN‐tolerant vessels, the reduced GC activity may be of a crucial importance for the reduced cGMP response in GTN‐tolerant BMA as found earlier (Axelsson et al. 1982).