How porphyrinogenic drugs modeling acute porphyria impair the hormonal status that regulates glucose metabolism. Their relevance in the onset of this disease.

This work deals with the study of how porphyrinogenic drugs modeling acute porphyrias interfere with the status of carbohydrate-regulating hormones in relation to key glucose enzymes and to porphyria, considering that glucose modulates the development of the disease. Female Wistar rats were treated with 2-allyl-2-isopropylacetamide (AIA) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) using different doses of AIA (100, 250 and 500mg/kg body weight) and a single dose of DDC (50mg DDC/kg body weight). Rats were sacrificed 16h after AIA/DDC administration. In the group treated with the highest dose of AIA (group H), hepatic 5-aminolevulinic acid synthase (ALA-S) increased more than 300%, phosphoenolpyruvate carboxykinase (PEPCK) and glycogen phosphorylase (GP) activities were 43% and 46% lower than the controls, respectively, plasmatic insulin levels exceeded normal values by 617%, and plasmatic glucocorticoids (GC) decreased 20%. GC results are related to a decrease in corticosterone (CORT) adrenal production (33%) and a significant reduction in its metabolization by UDP-glucuronosyltransferase (UGT) (62%). Adrenocorticotropic hormone (ACTH) stimulated adrenal production 3-fold and drugs did not alter this process. Thus, porphyria-inducing drugs AIA and DDC dramatically altered the status of hormones that regulate carbohydrate metabolism increasing insulin levels and reducing GC production, metabolization and plasmatic levels. In this acute porphyria model, gluconeogenic and glycogenolytic blockages caused by PEPCK and GP depressed activities, respectively, would be mainly a consequence of the negative regulatory action of insulin on these enzymes. GC could also contribute to PEPCK blockage both because they were depressed by the treatment and because they are positive effectors on PEPCK. These disturbances in carbohydrates and their regulation, through ALA-S de-repression, would enhance the porphyria state promoted by the drugs on heme synthesis and destruction. This might be the mechanism underlying the "glucose effect" observed in hepatic porphyrias. The statistical correlation study performed showed association between all the variables studied and reinforce these conclusions.

MDCK cells express serotonin-regulable 11beta-hydroxysteroid dehydrogenase type 2.

Prior to this work, we found that adrenal as well as extra-adrenal factors activate the response of renal 11beta-hydroxysteroid dehydrogenase 2 to stressful situations. These results -showing ways through which the organism hinders the pathological occupation of mineralocorticoid receptors by glucocorticoids leading to sodium retention and hypertension- prompted the present study on the nature of the above-mentioned extra-adrenal factors. Serotonin was chosen because of its properties as a widely distributed neurohormone, known to interact with glucocorticoids at many sites, also exhibiting increased levels and effects under stressful situations. We studied serotonin effects on 11beta-hydroxysteroid dehydrogenase 2 activity in a cell line derived from distal nephron polarized-epithelium, employing 3H-corticosterone as substrate. The end-product, 3H- 11 -dehydrocorticosterone was separated from the substrate by HPLC and quantified. Serotonin stimulated 1I beta-hydroxysteroid dehydrogenase 2 activity only at 2nM and 25pM, the magnitude of the response depending also on substrate concentration. The stimulation was blocked by the specific inhibitors methiothepin and ketanserin. We postulate that the organism partially prevents renal mineralocorticoid receptor occupancy by glucocorticoids, circulating at enhanced levels under stressful situations, through serotonin-mediated catabolic regulation of the 11beta-hydroxysteroid dehydrogenase 2 activity. Given many, mostly positive, interactions between both hormones, this might eventually pave the way to studies on a new regulatory axis.

Adrenal 11-beta hydroxysteroid dehydrogenase activity in response to stress.

This work studied the effect of stresses produced by simulated gavage or gavage with 200 mmol/L HCl two hours before adrenal extraction, on the activities of the 11beta-hydroxysteroid dehydrogenase 1 and 11beta-hydroxysteroid dehydrogenase 2 isoforms present in the rat adrenal gland. These activities were determined on immediately prepared adrenal microsomes following incubations with 3H-corticosterone and NAD+ or NADP+. 11-dehydrocorticosterone was measured as an end-product by TLC, and controls were adrenal microsomes from rats kept under basal (unstressed) conditions. 11beta-hydroxysteroid dehydrogenase 1 activity, but not 11beta-hydroxysteroid dehydrogenase 2 activity, was increased under both stress-conditions. Homeostatically, the stimulation of 11beta-hydroxysteroid dehydrogenase 1 activity would increase the supply of glucocorticoids. These, in turn, would activate the enzyme phenylethanolamine N-methyl transferase, thereby improving the synthesis of epinephrine as part of the stress-response.

Adrenal gland involvement in the regulation of renal 11beta-hydroxysteroid dehydrogenase 2.

Renal 11beta-hydroxysteroid dehydrogenase 2 (HSD2) catalyzes the conversion of active glucocorticoids to inert 11beta-keto compounds, thereby preventing the illicit binding of these hormones to mineralocorticoid receptors (MRs) and, thus, conferring aldosterone specificity. Absence or inhibition of HSD2 activity, originates a hypertensive syndrome with sodium retention and increased potassium elimination. Recent studies from our laboratory reported an increment of HSD2 activity in intact-stressed rats. To evaluate the adrenal involvement in this increase, we analyzed HSD2 activity and protein abundance in Intact, Sham-operated, and adrenalectomized rats under stress situations (gavage with an overload of 200 mM HCl (10 ml) and simulated gavage) or with corticosterone replacement. HSD2 activity was assessed in renal microsomal preparations obtained from different groups of animals. HSD2 protein abundance was measured by Western-blot. Circulating corticosterone was determined by radioimmunoassay. Sham-operated animals showed an increase in HSD2 activity and abundance compared to Intact and adrenalectomized rats suggesting the involvement of stress-related adrenal factors in HSD2 regulation. In the case of acidotic adrenalectomized animals, there was an increase in renal HSD2 activity when, along with the HCl overload, the rats were injected with corticosterone. This increment occurred without an increase in enzyme abundance. These results suggest the importance of circulating levels of glucocorticoids to respond to a metabolic acidosis, through regulation of HSD2 stimulation. The group subjected to a simulated gavage showed an increase in enzyme activity and protein abundance, thus demonstrating the need for both adrenal and extra-factors in the modulation of renal HSD2. The adrenalectomized animals injected with different doses of corticosterone, produced a progressive increase in enzyme activity and abundance, being significant for the dose of 68 microg corticosterone/100 g body weight. The highest dose (308 microg/100 g body weight) did not show any variation in activity and abundance compared to the control group. This biphasic effect of glucocorticoids could be explained taking into account their permissive and suppressive actions, depending on their blood levels. Knowing that stress induces multifactorial responses, it should not be surprising to observe a differential regulation in renal HSD2, confirming that different stressors act through different factors of both, adrenal and extra-adrenal origin.

Alteraciones en la enzimología de la producción de aldosterona / Alterations in the enzymology of aldosterone production

El último paso para la producción de aldosterona (11-desoxicorticosterona a aldosterona) en mitocondrias de zona glomerulosa de adrenal de rata es catalizado por la enzima CYP11B2. CYP11B1, en zona fasciculata, transforma 11-desoxicorticosterona en corticosterona o 18-hidroxi-11-desoxicorticosterona. CYO11B1 y CYP11B2 tienen alta homología y sus genes se hallan en tándem en el cromosoma 8q22. Mutaciones en el gen de CYP11B2 y recombinaciones genéticas entre éste y el gen de CYP11B1 serían las responsables de las alteraciones en la enzimología de la producción de aldosterona, dando una nueva denominación y explicación a las deficiencias anteriormente conocidas como de CMOI y CMOII

Alteraciones en la enzimología de la producción de aldosterona / Alterations in the enzymology of aldosterone production

El último paso para la producción de aldosterona (11-desoxicorticosterona a aldosterona) en mitocondrias de zona glomerulosa de adrenal de rata es catalizado por la enzima CYP11B2. CYP11B1, en zona fasciculata, transforma 11-desoxicorticosterona en corticosterona o 18-hidroxi-11-desoxicorticosterona. CYO11B1 y CYP11B2 tienen alta homología y sus genes se hallan en tándem en el cromosoma 8q22. Mutaciones en el gen de CYP11B2 y recombinaciones genéticas entre éste y el gen de CYP11B1 serían las responsables de las alteraciones en la enzimología de la producción de aldosterona, dando una nueva denominación y explicación a las deficiencias anteriormente conocidas como de CMOI y CMOII (AU)

Enzimología de la biosíntesis de aldosterona: una revisión / Enzymology of the biosynthesis of aldosterone: a review

La última etapa de la biosíntesis de aldosterona (ALDO) involucra la oxidación mitocondrial de 11-desoxicorticosterona (DOC), a través de varios caminos, que comienzan en sus metabolicos corticosterona (B) y 18-hidroxi-11-desoxicorticosterona (18OHDOC). Todas las reacciones de estos caminos son catalizadas por enzimas de la familia de los citocromos P450. El número y la identidad de cada una de ellas han sido objeto de investigaciones en los últimos treinta años. El modelo, actualmente en vigencia, postula que en la adrenal de la vaca todas las reacciones que llevan desde DOC a ALDO son catalizadas por un único citocromo P450-11ß/18 hidroxilasa/aldosintetasa, presente en toda la corteza adrenal. En cambio, en la adrenal de rata, ratón y humano, la catálisis total es llevada a cabo por el citocromo P450 CYP11B2 sólo presente en la zona glomerulosa, mientras que en la zona fasciculata existe el citocromo P450 CYP11B1, que sólo cataliza la transformación de DOC en B o en 18 OH-DOC. Este modelo, avalado por experimentos de bioquímica celular y molecular, no explica, sin embargo, algunos hechos experimentales

Enzimología de la biosíntesis de aldosterona: una revisión / Enzymology of the biosynthesis of aldosterone: a review

La última etapa de la biosíntesis de aldosterona (ALDO) involucra la oxidación mitocondrial de 11-desoxicorticosterona (DOC), a través de varios caminos, que comienzan en sus metabolicos corticosterona (B) y 18-hidroxi-11-desoxicorticosterona (18OHDOC). Todas las reacciones de estos caminos son catalizadas por enzimas de la familia de los citocromos P450. El número y la identidad de cada una de ellas han sido objeto de investigaciones en los últimos treinta años. El modelo, actualmente en vigencia, postula que en la adrenal de la vaca todas las reacciones que llevan desde DOC a ALDO son catalizadas por un único citocromo P450-11ß/18 hidroxilasa/aldosintetasa, presente en toda la corteza adrenal. En cambio, en la adrenal de rata, ratón y humano, la catálisis total es llevada a cabo por el citocromo P450 CYP11B2 sólo presente en la zona glomerulosa, mientras que en la zona fasciculata existe el citocromo P450 CYP11B1, que sólo cataliza la transformación de DOC en B o en 18 OH-DOC. Este modelo, avalado por experimentos de bioquímica celular y molecular, no explica, sin embargo, algunos hechos experimentales (AU)