ACTH Modulates PTP-PEST Activity and Promotes Its Interaction With Paxillin.

Adrenocorticotropic hormone (ACTH) treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in-gel PTP assays have shown the activation of a 115-kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP-PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP-PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT-PCR studies revealed PTP-PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP-PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non-ACTH-treated rats increased KM and VM . Finally, in-gel PTP assays of immunoprecipitated paxillin from control and ACTH-treated rats suggested a hormone-mediated increase in paxillin-PTP115 interaction, while PTP-PEST and paxillin co-localize in Y1 cells. Taken together, these data demonstrate PTP-PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH-induced PTP-PEST-paxillin interaction. J. Cell. Biochem. 117: 2170-2181, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Association between Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in subgingival plaque and clinical parameters, in Argentine patients with aggressive periodontitis.

BACKGROUND: Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) have been associated with aggressive (AgP) and chronic periodontitis. OBJECTIVE: The aim of this study was to evaluate the levels of Aa and Pg in gingival crevicular fluid (GCF) of patients with AgP and its relation with clinical parameters. DESIGN: Sixteen females and fourteen males with clinical diagnosis of AgP aged 17-23 years and their match's controls, were included in this study. Clinical recording concerning probing pocket depth, clinical attachment level, plaque index and gingival bleeding index were performed at baseline, 30 and 60 days after baseline. After clinical examination GCF samples were analyzed for Aa and Pg with a real-time polymerase chain reaction technique. Patients group was treated with a combined of mechanical and oral antibiotic therapy (doxycycline 100 mg/day, during 21 days). A multivariate analysis was used to determine the relationship between Aa and Pg counts with clinical parameters. RESULTS: GCF from all subjects was positive for Aa and PG. In controls Pg concentration was higher than Aa (Pg: 42,420 ± 3,034 copies/ml; Aa: 66.6 ± 5.4 copies/ml p < 0.001) while in patients both microbes showed the same concentration (Aa: 559,878 ± 39,698 Pg: 572,321 ± 58,752). A significant and positive correlation was observed between counts of Aa and Pg (R square: 0.7965, p < 0.0001). Female showed more counts/ml. Aa might be closely associated with clinical parameters while Pg did not. At 30 and 60 days Aa counts in patients were similar to controls while Pg counts were equal to baseline. However, in spite of Pg presence a clinical improvement was observed in all patients. CONCLUSIONS: In our population the presence of Aa may be associated with AgP while Pg may be in GCF as an opportunistic pathogen which might caused disease when the ecological balance was favorable.

Oxidative stress status during the acute phase of haemolytic uraemic syndrome.

BACKGROUND: Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopaenia and acute renal failure. The aim of this study was to investigate the levels of oxidative stress (OS) during the acute phase of HUS. METHODS: This prospective study included 18 patients diagnosed with D + HUS, 6 age-matched healthy controls and 29 children with end-stage renal disease (ESRD) not caused by HUS under regular haemodialysis. Plasma lipid peroxidation and non-enzymatic antioxidant defences were measured as thiobarbituric acid-reactive substances (TBARs) and total reactive antioxidant potential (TRAP), respectively, during hospitalization and in control individuals. RESULTS: TBARs were significantly higher in both oliguric and non-oliguric patients at admission (1.8 ± 0.1; 1.7 ± 0.2 µM) and discharge (1.5 ± 0.1; 1.0 ± 0.1 µM) vs controls (0.5 ± 0.1 µM, P < 0.01) following disease progression. Maximal TBARs values differed significantly between oliguric and non-oliguric groups (4.5 ± 0.9 vs 2.4 ± 0.3 µM, P < 0.01) and were significantly higher (P < 0.05) than those found in ESRD patients (1.63 ± 0.1). TRAP values were significantly higher at admission and when the disease was fully established (measured here as highest TBARs record) vs controls (675 ± 51, 657 ± 60 and 317 ± 30 µM Trolox, P < 0.01), and were similar to control values at discharge (325 ± 33 µM Trolox). CONCLUSIONS: We demonstrate here increased levels of OS during the acute phase of HUS, with peak plasma lipid peroxidation values well above those registered in ESRD individuals, and suggest a connection between OS and the clinical course of HUS.

Angiotensin II-upregulated MAP kinase phosphatase-3 modulates FOXO1 and p21 in adrenocortical H295R cells.

MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processes including tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study of human adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggering ERK1/2 phosphorylation in a transient fashion. MKP-3 dephosphorylates ERK1/2 and, as recently reported, forkhead box protein 1 (FOXO1). Here, we analyzed MKP-3 expression in H295R cells and its putative regulation by Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulation of both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulation and declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1 activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effect of Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also the induction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation and p21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity and also triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the current findings reveal the participation of MKP-3 not only in turn-off but also in turn-on signals which control important cellular processes.

Self-medication in patients seeking care in a dental emergency service.

The aims of this work were: To determine what percentage of firsttime patients to the Dental Emergency Department at the School of Dentistry of Buenos Aires University had taken medications to relieve or treat their condition. To determine what percentage of these had used selfmedication, and which were the most frequently taken medicines. To determine whether there is an association between selfmedication and educational level, and between selfmedication and whether the patient has health coverage. This was an observational, crosssectional study which reviewed 567 clinical histories of patients who visited the Dental Emergency Department from March 2015 to September 2016. The following parameters were assessed: sex, age, reason for consultation, medication, dose, interval, duration and indication. Patients' educational level and whether they had health coverage were ascertained. Confidence intervals of 95% were calculated for percentages using the Wilson score method. Inferential analyses were performed using the Chisquare test (ᵪ2). Significance level was set at 5%. Eighty five percent (85%,.n=481) of the patients had taken at least one medication; 77% (n=372) had used selfmedication. The most frequently used medicines were nonsteroid antiinflammatory drugs (61%), antibiotics (34%) and glucocorticoids (2%). No association was found between selfmedication and patients' having health coverage (ᵪ2=13; p=0.08). No significant association was found between educational level and selfmedication (ᵪ2=10; p=0.22). Nevertheless, the lowest percentages of selfmedication were found in subjects with complete university studies (77%; CI95: 60% to 89%), while the highest percentages were found in subjects with incomplete primary education (89%; CI95: 69% to 97%), complete primary education (92%; CI95: 82% to 96%) and incomplete secondary educations (90%; CI95: 84% to 94%).High levels of selfmedication were found in the study population. Although no association was found between educational level and selfmedication behavior, the percentage of selfmedication was higher among patients with lower educational levels. The high level of selfmedication highlights the importance of conducting campaigns to raise awareness about the adequate use of medicines.

Los objetivos del presente trabajo fueron: Determinar qué porcentaje de pacientes que concurrió por primera vez al Servicio de Urgencias de la Facultad de Odontología de la Universidad de Buenos Aires consumió medicamentos para aliviar o tratar su dolencia. Determinar qué porcentaje de pacientes fueron automedicados, y cuáles fueron los medica mentos más utilizados. Determinar si existe relación entre la automedicación y el nivel de estudio y entre la automedicación y la presencia de cobertura médica. Se realizó un estudio observacional y transversal. Se relevaron 567 historias clínicas de pacientes que concurrieron entre marzo 2015 y septiembre 2016 y se valoraron los siguientes parámetros: sexo, edad, origen de la consulta, medicación, dosis, intervalo, duración, e indicación. Se indagó el nivel educacional alcanzado y la existencia de cobertura médica. Se calcularon intervalos de confianza al 95% para porcentajes mediante el método score de Wilson. Se realizaron análisis inferenciales mediante la prueba Chicuadrado (ᵪ2). Se fijó un nivel de significación del 5%. El 85% (n=481) de los pacientes había consumido al menos un medicamento. El 77% (n=372) de los pacientes estaba autome dicado. Los medicamentos más utilizados fueron antiinflamatorios no esteoroideos (61%), antibióticos (34%) y glucocorticoides (2%). No se encontró asociación entre la automedicación y la presencia de cobertura médica (ᵪ2=13; p=0,08). No se encontró asociación significativa entre el nivel de estudios y la automedicación (ᵪ2=10; p=0,22). Sin embargo, los sujetos con estudio universitario completo presentaron el menor porcentaje de automedicación (77%; IC95: 60% a 89%), mientras que los mayores porcentajes se encontraron en sujetos con primario incompleto (89%; IC95: 69% a 97%), primario completo (92%; IC95: 82% a 96%) y secundario incompleto (90%; IC95: 84% a 94%). Se encontraron niveles elevados de automedicación en la población estudiada. Si bien no se observó asociación entre nivel educativo y la conducta de automedicación, fue mayor el porcentaje de automedicación en pacientes con menor nivel educativo. La alta presencia de automedicación refuerza la importancia de realizar campañas de concientización sobre el consumo adecuado de medicamentos.

Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis.

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.

Reactive oxygen species mediate dopamine-induced signaling in renal proximal tubule cells.

Intrarenally-produced dopamine (DA) induces a large increase in urinary sodium excretion mainly due to the inhibition of tubular sodium reabsorption. We aimed to study the participation of reactive oxygen species (ROS) in DA signaling pathway in proximal tubule cells. Our results show that DA increased ROS production in OK cells and indicate the mitochondria as the main source of ROS. DA also increased ERK1/2, superoxide dismutase (SOD) and transcription factor κB (NF-κB) activity. These findings suggest that DA generates mitochondria-derived ROS that activate ERK1/2 and subsequently NF-κB and SOD activity at concentrations that exert a physiological regulation of renal function.

Modulation of albumin-induced endoplasmic reticulum stress in renal proximal tubule cells by upregulation of mapk phosphatase-1.

High amounts of albumin in urine cause tubulointerstitial damage that leads to a rapid deterioration of the renal function. Albumin exerts its injurious effects on renal cells through a process named endoplasmic reticulum (ER) stress due to the accumulation of unfolded proteins in the ER lumen. In addition, albumin promotes phosphorylation and consequent activation of MAPKs such as ERK1/2. Since ERK1/2 activation promoted by albumin is a transient event, the aims of the present work were to identify the phosphatase involved in their dephosphorylation in albumin-exposed cells and to analyze the putative regulation of this phosphatase by albumin. We also sought to determine the role played by the phospho/dephosphorylation of ERK1/2 in the cellular response to albumin-induced ER stress. MAP kinase phosphatase-1, MKP-1, is a nuclear enzyme involved in rapid MAPK dephosphorylation. Here we present evidence supporting the notion that this phosphatase is responsible for ERK1/2 dephosphorylation after albumin exposure in OK cells. Moreover, we demonstrate that exposure of OK cells to albumin transiently increases MKP-1 protein levels. The increase was evident after 15 min of exposure, peaked at 1 h (6-fold) and declined thereafter. In cells overexpressing flag-MKP-1, albumin caused the accumulation of this chimera, promoting MKP-1 stabilization by a posttranslational mechanism. Albumin also promoted a transient increase in MKP-1 mRNA levels (3-fold at 1 h) through the activation of gene transcription. In addition, we also show that albumin increased mRNA levels of GRP78, a key marker of ER stress, through an ERK-dependent pathway. In line with this finding, our studies demonstrate that flag-MKP-1 overexpression blunted albumin-induced GRP78 upregulation. Thus, our work demonstrates that albumin overload not only triggers MAPK activation but also tightly upregulates MKP-1 expression, which might modulate ER stress response to albumin overload.

MAP kinase phosphatase-3 (MKP-3) is transcriptionally and post-translationally up-regulated by hCG and modulates cAMP-induced p21 expression in MA-10 Leydig cells.

Luteinizing hormone (LH) activates ERK1/2, MAP kinases (MAPKs) necessary for its action on steroidogenesis and cell proliferation, and also induces MAPK phosphatase-1 (MKP-1), which rapidly dephosphorylates nuclear ERK1/2. MKP-3 is a cytoplasmic ERK-phosphatase up-regulated by proliferative stimuli. MKP-3 also dephosphorylates transcription factor FOXO1, promoting its transport to the nucleus. Here we analyzed MKP-3 expression in MA-10 Leydig cells and demonstrated that LH receptor (LHR) activation with human gonadotropin hormone (hCG) and an analog of its second messenger, 8Br-cAMP, up-regulates MKP-3 by transcriptional and post-translational mechanisms. It is known that FOXO1 drives the expression of the cell cycle inhibitor p21. Since the activation of this transcription factor by MKP-3 has been reported, we assessed the effect of shRNA against MKP-3 on p21mRNA levels. 8Br-cAMP increased these levels (2-fold at 2h) and MKP-3 down-regulation reduced this effect. Our work demonstrates that LH/hCG tightly up-regulates MKP-3 which in turn, dephosphorylates ERK1/2 and drives p21 expression. These events could contribute to counteract hormonal action on cell proliferation.

MAPK phosphatase-2 (MKP-2) is induced by hCG and plays a role in the regulation of CYP11A1 expression in MA-10 Leydig cells.

MAPKs such as ERK1/2 are dephosphorylated, and consequently inactivated, by dual specificity phosphatases (MKPs). In Leydig cells, LH triggers ERK1/2 phosphorylation through the action of protein kinase A. We demonstrate that, in MA-10 Leydig cells, LH receptor activation by human chorionic gonadotropin (hCG) up-regulates MKP-2, a phosphatase that dephosphorylates ERK1/2, among other MAPKs. After 2 hours, hCG and 8-bromo-cAMP (8Br-cAMP) significantly increased MKP-2 mRNA levels (3-fold), which declined to basal levels after 6 hours. MKP-2 protein accumulation exhibited a similar kinetic profile. In cells transiently expressing flag-MKP-2 protein, hCG/8Br-cAMP stimulation promoted the accumulation of the chimera (2.5-fold after 3 h of stimulation). Pharmacologic and biochemical approaches showed that the accumulation of flag-MKP-2 involves a posttranslational modification that increases MKP-2 half-life. MKP-2 down-regulation by a short hairpin RNA (MKP-2 shRNA) raised the levels of phosphorylated ERK1/2 reached by 8Br-cAMP stimulation. This effect was evident after 180 min of stimulation, which suggests that MKP-2 down-regulates the late phase of cAMP-induced ERK1/2 activity. Also, MKP-2 down-regulation by MKP-2 shRNA increased the stimulatory effect of 8Br-cAMP on both promoter activity and messenger levels of CYP11A1, which encodes for the steroidogenic enzyme P450scc and is induced by LH/hCG through protein kinase A and ERK1/2 activities. Our findings demonstrate, for the first time, that LH/hCG tightly regulates MKP-2 expression, which modulates the induction of CYP11A1 by 8Br-cAMP. MKP-2 up-regulation might control ERK1/2 activity in a specific temporal frame to modulate the expression of a finite repertory of ERK-dependent genes.

Self-medication in patients seeking care in a dental emergency service

The aims of this work were: To determine what percentage of firsttime patients to the Dental Emergency Department at the School of Dentistry of Buenos Aires University had taken medications to relieve or treat their condition. To determine what percentage of these had used selfmedication, and which were the most frequently taken medicines. To determine whether there is an association between selfmedication and educational level, and between selfmedication and whether the patient has health coverage. This was an observational, crosssectional study which reviewed 567 clinical histories of patients who visited the Dental Emergency Department from March 2015 to September 2016. The following parameters were assessed: sex, age, reason for consultation, medication, dose, interval, duration and indication. Patients' educational level and whether they had health coverage were ascertained. Confidence intervals of 95% were calculated for percentages using the Wilson score method. Inferential analyses were performed using the Chisquare test (ᵪ2). Significance level was set at 5%. Eighty five percent (85%,.n=481) of the patients had taken at least one medication; 77% (n=372) had used selfmedication. The most frequently used medicines were nonsteroid antiinflammatory drugs (61%), antibiotics (34%) and glucocorticoids (2%). No association was found between selfmedication and patients' having health coverage (ᵪ2=13; p=0.08). No significant association was found between educational level and selfmedication (ᵪ2=10; p=0.22). Nevertheless, the lowest percentages of selfmedication were found in subjects with complete university studies (77%; CI95: 60% to 89%), while the highest percentages were found in subjects with incomplete primary education (89%; CI95: 69% to 97%), complete primary education (92%; CI95: 82% to 96%) and incomplete secondary educations (90%; CI95: 84% to 94%).High levels of selfmedication were found in the study population. Although no association was found between educational level and selfmedication behavior, the percentage of selfmedication was higher among patients with lower educational levels. The high level of selfmedication highlights the importance of conducting campaigns to raise awareness about the adequate use of medicines (AU)

Los objetivos del presente trabajo fueron: Determinar qué porcentaje de pacientes que concurrió por primera vez al Servicio de Urgencias de la Facultad de Odontología de la Universidad de Buenos Aires consumió medicamentos para aliviar o tratar su dolencia. Determinar qué porcentaje de pacientes fueron automedicados, y cuáles fueron los medica mentos más utilizados. Determinar si existe relación entre la automedicación y el nivel de estudio y entre la automedicación y la presencia de cobertura médica. Se realizó un estudio observacional y transversal. Se relevaron 567 historias clínicas de pacientes que concurrieron entre marzo 2015 y septiembre 2016 y se valoraron los siguientes parámetros: sexo, edad, origen de la consulta, medicación, dosis, intervalo, duración, e indicación. Se indagó el nivel educacional alcanzado y la existencia de cobertura médica. Se calcularon intervalos de confianza al 95% para porcentajes mediante el método score de Wilson. Se realizaron análisis inferenciales mediante la prueba Chicuadrado (ᵪ2). Se fijó un nivel de significación del 5%. El 85% (n=481) de los pacientes había consumido al menos un medicamento. El 77% (n=372) de los pacientes estaba autome dicado. Los medicamentos más utilizados fueron antiinflamatorios no esteoroideos (61%), antibióticos (34%) y glucocorticoides (2%). No se encontró asociación entre la automedicación y la presencia de cobertura médica (ᵪ2=13; p=0,08). No se encontró asociación significativa entre el nivel de estudios y la automedicación (ᵪ2=10; p=0,22). Sin embargo, los sujetos con estudio universitario completo presentaron el menor porcentaje de automedicación (77%; IC95: 60% a 89%), mientras que los mayores porcentajes se encontraron en sujetos con primario incompleto (89%; IC95: 69% a 97%), primario completo (92%; IC95: 82% a 96%) y secundario incompleto (90%; IC95: 84% a 94%). Se encontraron niveles elevados de automedicación en la población estudiada. Si bien no se observó asociación entre nivel educativo y la conducta de automedicación, fue mayor el porcentaje de automedicación en pacientes con menor nivel educativo. La alta presencia de automedicación refuerza la importancia de realizar campañas de concientización sobre el consumo adecuado de medicamentos (AU)

Cisplatin inhibits testosterone synthesis by a mechanism that includes the action of reactive oxygen species (ROS) at the level of P450scc.

Cisplatin (Cs) is a chemotherapeutic agent able to generate reactive oxygen species (ROS) which are linked to several side effects of the drug. Even when it is known that Cs produces Leydig cell dysfunction, it is unknown whether this particular side effect is mediated by ROS. The aim of this study was to evaluate the in vitro effects of Cs on testosterone production and the participation of ROS in this effect. We demonstrate that Cs promotes the generation of ROS in a time-, and concentration-dependent fashion, not only in mouse testicular interstitial cells but also in MA-10 Leydig cells. Also, Cs inhibits testosterone synthesis in a concentration-dependent fashion (5-50 µM for 4 h) and to a similar extent, in cells exposed to human chorionic gondadotropin hormone (hCG), to an analog of the second messenger cAMP (8Br-cAMP) or to a freely diffusible cholesterol analog (22R-hydroxycholesterol). However, this treatment does not inhibit the conversion of pregnenolone to testosterone. These data suggest that Cs exerts its inhibitory action on testosterone synthesis by an action at the level of P450scc. We also demonstrated that an antioxidant impairs the inhibitory effect of Cs on the conversion of the cholesterol analog into pregnenolone and that Cs does not change the expression level of P450scc mRNA. Therefore, it is concluded that Cs inhibits testosterone synthesis by a mechanism that includes the inhibition of P450scc by ROS.

MAPK phosphatase-1 (MKP-1) expression is up-regulated by hCG/cAMP and modulates steroidogenesis in MA-10 Leydig cells.

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.

Role of 20-HETE in D1/D2 dopamine receptor synergism resulting in the inhibition of Na+-K+-ATPase activity in the proximal tubule.

Previous studies propose 20-hydroxyeicosatetraenoic acid (20-HETE), a major arachidonic acid metabolite of cytochrome P-450 (CYP), as a possible mediator of Na(+)-K(+)-ATPase inhibition by dopamine (DA). The aim of this study was to investigate the intracellular mechanisms involved in this effect and to elucidate the DA receptor associated with the 20-HETE pathway in the rat kidney. DA (10(-5) M) inhibited Na(+)-K(+)-ATPase activity in microdissected tubular segments to 59.4 +/- 3.8% of control activity. This response was suppressed by the CYP4A inhibitor 17-octadecynoic acid (10(-6) M), which had no effect per se, thus confirming the participation of CYP arachidonic acid metabolites in DA-induced Na(+)-K(+)-ATPase inhibition. We next examined whether 20-HETE is involved in the signaling pathways triggered by either D(1) or D(2) receptors. Neither fenoldopam nor quinpirole (D(1) and D(2) agonists, respectively, both 10(-5) M) modified Na(+)-K(+)-ATPase activity when tried alone. However, coincubation of a threshold concentration of 20-HETE (10(-9) M) with fenoldopam resulted in a synergistic inhibition of Na(+)-K(+)-ATPase activity (66 +/- 2% of control activity), while 20-HETE plus quinpirole had no effect. Furthermore, 20-HETE (10(-9) M) synergized with forskolin (10(-5) M) and with the diacylglycerol analog 1-oleoyl-2-acetoyl-sn-glycerol (OAG; 10(-11) M; 62.0 +/- 5.3 and 69.9 +/- 2.0% of control activity, respectively), indicating a cooperative role of 20-HETE with the D(1)-triggered pathways. In line with these results, no additive effect was observed when OAG and 20-HETE were combined at concentrations which per se produced maximal inhibition (10(-6) M). These results demonstrate that the inhibition of Na(+)-K(+)-ATPase activity by DA in the proximal tubule may be the result of the synergism between 20-HETE and the D(1) signaling pathway.

Insulin enhances L-dopa renal proximal tubule uptake: a regulatory mechanism impaired in insulin resistance.

A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on L-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 microU/ml) increased L-dopa uptake into PT cells by about 50% (705+/-186 vs.1117+/-140 pmol L-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity L-dopa transport sites (L-dopa 0.2 microM) (0.59+/-0.05 vs. 0.82+/-0.09 pmol L-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1-10 microM) or colchicine (50-100 microM), whereas it was abolished by cytochalasin D or latrunculin B (both 1 microM). This suggests that the process is independent of Na(+),K(+)-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. L-dopa transport by the low-affinity transport sites (L-dopa 5 microM) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal L-dopa reabsorption as compared to control animals (81+/-4 vs. 51+/-9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal L-dopa reabsorption.

ACTH regulation of mitochondrial acyl-CoA thioesterase activity in Y1 adrenocortical tumour cells.

We have previously purified and cloned a phosphoprotein, Arachidonic acid-Related Thioesterase Involved in Steroidogenesis (ARTISt), involved in steroid synthesis through Arachidonic Acid (AA) release. Arachidonic acid-related thioesterase involved in steroidogenesis resulted to be a member of a new family of acyl-CoA thioesterases. The protein was identified by its biocapacity to increase mitochondrial steroidogenesis in a cell free bioassay. In the present study we measure the activity of ARTISt using arachidonoyl-CoA (AA-CoA) as substrate. We demonstrate that ACTH significantly stimulates endogenous mitochondrial thioesterase activity as early as 5 min after ACTH stimulation of Y1 cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH activation of the enzyme protected ARTISt to the inhibitory effect of NDGA. These results show that an enzyme that release AA from AA-CoA can be regulated in intact cells by steroidogenic hormones.

Protein serine/threonine phosphatase 2A activity is inhibited by cAMP in MA-10 cells.

PP1 and PP2A are members of the protein serine/threonine phosphatases (PPs) family and their activities have been proposed as a requirement for hormone- and cAMP-regulated steroid synthesis. These findings raise the question whether the PPs activity is increased by hormonal action in steroidogenic systems. Thus, the aim of the study was to evaluate the action of cAMP on the activity of PP1 and PP2A in MA-10 Leydig cells. Our results demonstrate that 8Br-cAMP stimulation produces a transient inhibition of PP2A activity. In contrast, PP1 activity remains unchangeable. As reported in other steroidogenic cells, cAMP-induced steroidogenesis in MA-10 cells is reduced by Cantharidin (Can) and also by Calyculin A (CA), two chemically unrelated PP1/PP2A inhibitors (data not shown). Taking into account the inhibitory effect of cAMP treatment on PP2A activity, the latest findings result paradoxical. Therefore, we next evaluated the action of these compounds on total protein synthesis. Can 10(-5) M and CA 10(-7) M markedly reduced total protein synthesis (35 and 50% respectively) in MA-10 cells, measured by 35S-methonine incorporation. These results suggest that hormone-dependent steroidogenesis is working through inhibition of PP2A-dependent dephosphorylation and the effect of PP1/PP2A inhibitors on steroidogenesis may be due to a general inhibition of protein synthesis rather than to a specific action on StAR protein induction.

Rapid association of protein kinase C-epsilon with insulin granules is essential for insulin exocytosis.

Glucose-dependent exocytosis of insulin requires activation of protein kinase C (PKC). However, because of the great variety of isoforms and their ubiquitous distribution within the beta-cell, it is difficult to predict the importance of a particular isoform and its mode of action. Previous data revealed that two PKC isoforms (alpha and epsilon) translocate to membranes in response to glucose (Zaitzev, S. V., Efendic, S., Arkhammar, P., Bertorello, A. M., and Berggren, P. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9712-9716). Using confocal microscopy, we have now established that in response to glucose, PKC-epsilon but not PKC-alpha associates with insulin granules and that green fluorescent protein-tagged PKC-epsilon changes its distribution within the cell periphery upon stimulation of beta-cells with glucose. Definite evidence of PKC-epsilon requirement during insulin granule exocytosis was obtained by using a dominant negative mutant of this isoform. The presence of this mutant abolished glucose-induced insulin secretion, whereas transient expression of the wild-type PKC-epsilon led to a significant increase in insulin exocytosis. These results suggest that association of PKC-epsilon with insulin granule membranes represents an important component of the secretory network because it is essential for insulin exocytosis in response to glucose.

Concerted regulation of free arachidonic acid and hormone-induced steroid synthesis by acyl-CoA thioesterases and acyl-CoA synthetases in adrenal cells.

Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a Km of 2 micro m. Antibodies raised against recombinant acyl-CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC50 for NDGA was reduced from 50 micro m to 25, 7.5 and 4.5 micro m in the presence of 0.1, 0.5 and 2 micro m triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells.