Influence of the genetic background on the reproductive phenotype of mice lacking Cysteine-Rich Secretory Protein 1 (CRISP1).

Epididymal sperm protein CRISP1 has the ability to both regulate murine CatSper, a key sperm calcium channel, and interact with egg-binding sites during fertilization. In spite of its relevance for sperm function, Crisp1-/-mice are fertile. Considering that phenotypes can be influenced by the genetic background, in the present work mice from the original mixed Crisp1-/- colony (129/SvEv*C57BL/6) were backcrossed onto the C57BL/6 strain for subsequent analysis of their reproductive phenotype. Whereas fertility and fertilization rates of C57BL/6 Crisp1-/- males did not differ from those reported for mice from the mixed background, several sperm functional parameters were clearly affected by the genetic background. Crisp1-/- sperm from the homogeneous background exhibited defects in both the progesterone-induced acrosome reaction and motility not observed in the mixed background, and normal rather than reduced protein tyrosine phosphorylation. Additional studies revealed a significant decrease in sperm hyperactivation as well as in cAMP and protein kinase A (PKA) substrate phosphorylation levels in sperm from both colonies. The finding that exposure of mutant sperm to a cAMP analog and phosphodiesterase inhibitor overcame the sperm functional defects observed in each colony indicated that a common cAMP-PKA signaling defect led to different phenotypes depending on the genetic background. Altogether, our observations indicate that the phenotype of CRISP1 null males is modulated by the genetic context and reveal new roles for the protein in both the functional events and signaling pathways associated to capacitation.

Nitric oxide reduces paracellular resistance in rat thick ascending limbs by increasing Na+ and Cl- permeabilities.

About 50% of the Na+ reabsorbed in thick ascending limbs traverses the paracellular pathway. Nitric oxide (NO) reduces the permselectivity of this pathway via cGMP, but its effects on absolute Na+ ([Formula: see text]) and Cl- ([Formula: see text]) permeabilities are unknown. To address this, we measured the effect of l-arginine (0.5 mmol/l; NO synthase substrate) and cGMP (0.5 mmol/l) on [Formula: see text] and [Formula: see text] calculated from the transepithelial resistance (Rt) and [Formula: see text]/[Formula: see text] in medullary thick ascending limbs. Rt was 7,722 ± 1,554 ohm·cm in the control period and 6,318 ± 1,757 ohm·cm after l-arginine treatment (P < 0.05). [Formula: see text]/[Formula: see text] was 2.0 ± 0.2 in the control period and 1.7 ± 0.1 after l-arginine (P < 0.04). Calculated [Formula: see text] and [Formula: see text] were 3.52 ± 0.2 and 1.81 ± 0.10 × 10-5 cm/s, respectively, in the control period. After l-arginine they were 6.65 ± 0.69 (P < 0.0001 vs. control) and 3.97 ± 0.44 (P < 0.0001) × 10-5 cm/s, respectively. NOS inhibition with Nω-nitro-l-arginine methyl ester (5 mmol/l) prevented l-arginine's effect on Rt Next we tested the effect of cGMP. Rt in the control period was 7,592 ± 1,470 and 4,796 ± 847 ohm·cm after dibutyryl-cGMP (0.5 mmol/l; db-cGMP) treatment (P < 0.04). [Formula: see text]/[Formula: see text] was 1.8 ± 0.1 in the control period and 1.6 ± 0.1 after db-cGMP (P < 0.03). [Formula: see text] and [Formula: see text] were 4.58 ± 0.80 and 2.66 ± 0.57 × 10-5 cm/s, respectively, for the control period and 9.48 ± 1.63 (P < 0.007) and 6.01 ± 1.05 (P < 0.005) × 10-5 cm/s, respectively, after db-cGMP. We modeled NO's effect on luminal Na+ concentration along the thick ascending limb. We found that NO's effect on the paracellular pathway reduces net Na+ reabsorption and that the magnitude of this effect is similar to that due to NO's inhibition of transcellular transport.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

Curcumin effects on Leydig cell functions and potential therapeutic uses.

Curcumin has been ascribed with countless therapeutic effects, but its impact on testicular function has been scarcely researched. Leydig cells comprise the androgen-secreting population of the testis and may give rise to Leydig cell tumours (LCTs). Due to their steroid-secreting nature, LCTs entail endocrine, reproductive, and psychological disorders. Approximately 10% are malignant and do not respond to chemotherapy and radiotherapy. The aim of this study was to assess curcumin's impact on Leydig cells' functions and its potential effect on LCT growth. In vitro assays on MA-10 Leydig cells showed that curcumin (20-80 µmol/L) stimulates acute steroidogenesis, both in the presence and absence of db-cAMP. This effect is accompanied by an increase in StAR expression. Regarding curcumin's in vitro cytostatic capacity, we show that 40-80 µmol/L curcumin reduces MA-10 Leydig cells' proliferative capacity, which could be explained by the arrest in G2/M and the reduced viability due to the activation of the apoptotic pathway. Finally, CB6F1 mice were inoculated with MA-10 cells to generate ectopic LCT in both flanks. They received i.p. injections of 20 mg/kg curcumin or vehicle every other day for 15 days. We unveiled curcumin's capacity to inhibit LCT growth as evidenced by reduced tumour volume, weight, and area under the growth curves. No detrimental effects on general health parameters or testicular integrity were observed. These results provide novel evidence of curcumin's effects on the endocrine cell population of the testis and propose this natural compound as a therapeutic agent for LCT.

Prevalence of hypersomatotropism and hyperthyroidism in cats with diabetes mellitus from referral centers in Buenos Aires (2020-2022).

OBJECTIVES: The aim of this study was to estimate the prevalence of hypersomatotropism (HST) and hyperthyroidism in cats with diabetes mellitus (DM) from referral centers in Buenos Aires, Argentina. METHODS: This was a prospective study. Systematic screening of serum insulin-like growth factor 1 (IGF-1) and total thyroxine was performed in all cats diagnosed with DM at referral centers in Buenos Aires between February 2020 and February 2022. RESULTS: In total, 154 diabetic cats were evaluated (99 males and 55 females; median age 12 years [range 3-21]; mean body weight 5 kg [range 2-12]). Altogether, there were 115 (75%) domestic shorthairs and one domestic longhair; the remaining 38 cats were purebred (mainly Siamese, n = 25 [16%]). Twenty (12.9%) cats had IGF-1 concentrations >1000 ng/ml, and three (1.9%) had IGF-1 concentrations between 800 and 1000 ng/ml along with pituitary enlargement on CT, resulting in a 14.9% HST prevalence rate in diabetic cats. Intracranial imaging was performed in all cats with HST; median pituitary dorsoventral height was 5.8 mm (range 3.1-9.5). Fourteen of 23 (61%) cats had phenotypic changes consistent with acromegaly at the time of diagnosis of HST. Four of 154 (2.5%) cats had concurrent hyperthyroidism. CONCLUSIONS AND RELEVANCE: To date, this is the first study outside of Europe to have evaluated the prevalence of HST and hyperthyroidism in cats with DM. In Buenos Aires referral centers, feline HST is the most common concurrent endocrinopathy in cats with DM but with a lower prevalence than has previously been reported. Hyperthyroidism is a rare concurrent endocrinopathy in diabetic cats from referral centers in Buenos Aires.

Homeostasis of extracellular ATP in human erythrocytes.

We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ß-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ((10)Panx1) decreased [ATP]e by 75-84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ∼1 pmol × (10(6) cells)(-1). A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (10(6) cells)(-1), followed by a slower exponential decay (t(½) = 3.7 min) to a constant value of 1.3 pmol × (10(6) cells)(-1). Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (∼28 fmol × (10(6) cells min)(-1)), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.

Effect of bitter compounds on amylase secretion in murine submandibular glands: Signaling pathway mechanisms.

BACKGROUND: Amylase is synthesized in submandibular glands (SMG) and released into the oral cavity to degrade carbohydrates in the mouth. Bitter taste receptors (T2R) belong to the G-protein coupled receptor (GPCR) family and are expressed in the taste cells and also in the digestive tract. METHODS: The activity of amylase secreted by murine SMG was measured, detecting maltose by Bernfeld's method. Amylase and T2R6 were detected by imunohistochemistry and Western blot. The expression of Ggustducin, Gi, and phospholipase Cß2 was also studied by Western blot. cAMP levels were measured by radioimmunoassay and inositol monophosphate production was quantified by ELISA. RESULTS: Theophylline, denatonium and cycloheximide exerted a dose-dependent inhibition on amylase secretion. This effect was reverted by preincubating SMG with an anti-Gαi antibody. cAMP production was increased by the same compounds, an effect that was also abrogated by an anti-Gαi antibody. Bitter compounds reduced inositol monophosphate formation in SMG and H-89, a protein kinase A inhibitor, reverted this action, revealing that this protein kinase down regulates phospholipase C activity. GENERAL SIGNIFICANCE: We demonstrated that theophylline, denatonium and cycloheximide inhibit salivary amylase secretion, activating an intracellular signaling pathway that involves cAMP and phospholipase C, that cross talks via protein kinase A.

Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi.

Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi.

Therapy for feline secondary hypertriglyceridemia with fenofibrate.

OBJECTIVES: The aim of this study was to assess the short-term safety and efficacy of fenofibrate in controlling secondary hypertriglyceridemia in cats. METHODS: This was a prospective cohort study. Seventeen adult cats with hypertriglyceridemia (serum triglycerides [TG] >160 mg/dl) were enrolled. Cats received a median dose of 5 mg/kg (range 3.2-6) fenofibrate (q24h PO) for 1 month. Serum TG, total cholesterol (TC), creatine kinase and liver enzymes (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were evaluated before (t0) and after 1 month (t1) of fenofibrate treatment. RESULTS: The causes of secondary hypertriglyceridemia were diabetes mellitus (DM; 29.4%), obesity (29.4%), hyperadrenocorticism (HAC) and DM (11.7%), HAC without DM (5.9%), hypersomatotropism (HST) and DM (5.9%), hypothyroidism (5.9%), long-term treatment with glucocorticoids (5.9%) and chylothorax (5.9%). Serum TG (t0 median 920 mg/dl [range 237-1780]; t1 median 51 mg/dl [range 21-1001]; P = 0.0002) and TC (t0 median 278 mg/dl [range 103-502]; t1 median 156 mg/dl [range 66-244]; P = 0.0001) concentrations showed a significant decrease after 1 month of fenofibrate treatment. Fifteen cats normalized their TG concentration at t1 (88.2%). Of the eight cats that were hypercholesterolemic at t0, six (75%) normalized their TC concentrations at t1. One of 17 cats (5.9 %) presented with diarrhea; the remaining 16 did not show any adverse effects. CONCLUSIONS AND RELEVANCE: DM and obesity are the most common endocrine causes of secondary hyperlipidemia, although it can also be found in cats with HAC, HST or hypothyroidism. This study suggests that fenofibrate treatment was associated with reduction and normalization of TG and TC concentrations in cats with moderate and severe hypertriglyceridemia, regardless of the cause of secondary hypertriglyceridemia. Further work should focus on controlled studies with a greater number of cases.

Cabergoline treatment in cats with diabetes mellitus and hypersomatotropism.

OBJECTIVES: The aim of this study was to evaluate the safety and efficacy of cabergoline to control hypersomatotropism (HST) and diabetes mellitus (DM) in cats. METHODS: This was a prospective cohort study. Twenty-three cats with HST and concurrent DM were enrolled. Cats received a dose of 10 µg/kg cabergoline q48h PO for 6 months. Serum insulin-like growth factor 1 (IGF-1) and fructosamine concentrations, insulin dose and Insulin Resistance Index (IRI) were measured at the time of diagnosis of HST and at the start of cabergoline treatment (t0), and 3 months (t1) and 6 months (t2) during cabergoline treatment. RESULTS: A decrease and normalization of serum IGF-1 concentration was observed in 35% and 26% of cats, respectively. Median IGF-1 (t0: 1350 ng/ml [range 832-1501]; t1: 1284 ng/ml [range 365-1501]; t2: 1240 ng/ml [range 263-1501]; P = 0.016) decreased significantly. Twelve cats underwent diagnostic imaging of the pituitary area. The median pituitary height at t0 of cats that experienced an IGF-1 reduction (n = 5/12) was significantly lower compared with those that did not experience an IGF-1 reduction (n = 7/12) (3.2 mm [range 3.1-3.7] vs 6 mm [range 3.5-9.5]; P = 0.011). Median fructosamine (t0: 628 µmol/l [range 400-963]; t1: 404 µmol/l [range 249-780]; t2: 400 µmol/l [range 260-815]; P <0.0001), insulin dose (t0: 1.3 IU/kg [range 0.5-4.6]; t0: 0.5 IU/kg [range 0-2.3]; t2: 0.4 IU/kg [range 0-2.1]; P <0.0001) and IRI (t0: 800 µmolIU/kgl [range 257-2700]; t1: 300 µmolIU/kgl [range 0-1498]; t2: 250 µmolIU/kgl [range 0-1498]; P <0.0001) decreased significantly during cabergoline treatment. Eight cats achieved diabetic remission between months 1 and 6 of cabergoline treatment (median time to achieve remission: 3 months [range 1-6]). Three cats experienced asymptomatic hypoglycemia. CONCLUSIONS AND RELEVANCE: Cabergoline was effective in normalizing IGF-1 concentration in 26% of cats. Cabergoline improved diabetes control and was associated with remission of DM in 35% of cases. Cabergoline could be a treatment option for cats with HST and DM, especially in those cases with a relatively small pituitary tumor.

Classical membrane progesterone receptors in murine mammary carcinomas: agonistic effects of progestins and RU-486 mediating rapid non-genomic effects.

In this article, we demonstrate the expression of functional progesterone binding sites at the cell membrane in murine mammary carcinomas that are stimulated by progestins and inhibited by antiprogestins. Using confocal immunofluorescence, ligand binding and cell compartment-specific western blots, we were able to identify the presence of the classical progesterone receptors. Medroxyprogesterone acetate (MPA) and RU-486 (1 × 10(-11) and 1 × 10(-8) M) behaved as agonists activating extracellular signal-regulated kinases (ERKs) and progestin-regulated proteins, except for Cyclin D1 and Tissue factor which failed to increase with 1 × 10(-8) M RU-486, an experimental condition that allows PR to bind DNA. These results predicted a full agonist effect at low concentrations of RU-486. Accordingly, at concentrations lower than 1 × 10(-11) M, RU-486 increased cell proliferation in vitro. This effect was abolished by incubation with the ERK kinase inhibitor PD 98059 or by OH-tamoxifen. In vivo, at a daily dose of 1.2 µg/kg body weight RU-486 increased tumor growth, whereas at 12 mg/kg induces tumor regression. Our results indicate that low concentrations of MPA and RU-486 induce similar agonistic non-genomic effects, whereas RU-486 at higher concentrations may inhibit cell proliferation by genomic-induced effects. This suggests that RU-486 should be therapeutically administered at doses high enough to guarantee its genomic inhibitory effect.

Impairment of renal steroidogenesis at the onset of diabetes.

Accumulating evidence indicates the association between changes in circulating sex steroid hormone levels and the development of diabetic nephropathy. However, the renal synthesis of steroid hormones during diabetes is essentially unknown. Male Wistar rats were injected with streptozotocin (STZ) or vehicle. After one week, no changes in functional or structural parameters related to kidney damage were observed in STZ group; however, a higher renal expression of proinflammatory cytokines and HSP70 was found. Expression of Steroidogenic Acute Regulatory protein (StAR) and P450scc (CYP11A1) was decreased in STZ kidneys. Incubation of isolated mitochondria with 22R-hydroxycholesterol revealed a marked inhibition in CYP11A1 function at the medullary level in STZ group. The inhibition of these first steps of renal steroidogenesis in early STZ-induced diabetes led to a decreased local synthesis of pregnenolone and progesterone. These findings stimulate investigation of the probable role of nephrosteroids in kidney damage associated with diabetes.

Proliferative actions of muscarinic receptors expressed in macrophages derived from normal and tumor bearing mice.

Macrophages (Mps) are essential cellular components of the innate immune system. They are released from the bone marrow as immature monocytes and after circulating in the blood stream, migrate into tissues to undergo final differentiation into resident Mps. In general terms Mps behavior in breast tumors, was described as being either for or against tumor growth. Under certain well defined circumstances Mps are able to kill cells in two ways: direct tumor cytotoxicity or antibody dependent cytotoxicity. We had previously demonstrated that peritoneal Mps from LMM3 mammary tumor bearing mice (TMps) enhanced in vivo the LMM3 induced angiogenesis, promoting tumor growth while Mps from normal BALB/c mice (NMps) did not. In this work, we demonstrate that Mps, expressing functional muscarinic acetylcholine receptors, are able to proliferate in vitro in response to the muscarinic agonist carbachol. These peritoneal cells use two distinct metabolic pathways: TMps are primed by tumor presence and they proliferate mainly by activating arginase pathway and by producing high levels of prostaglandin E(2) via M(1)-M(3) receptors activation. In NMps, carbachol stimulates M(2) receptors function, triggering protein kinase C activity and induces moderate prostaglandin E(2) liberation via M(1) receptor.

Cell cycle inhibitor, p19INK4d, promotes cell survival and decreases chromosomal aberrations after genotoxic insult due to enhanced DNA repair.

Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.

Direct effect of melatonin on Syrian hamster testes: melatonin subtype 1a receptors, inhibition of androgen production, and interaction with the local corticotropin-releasing hormone system.

Besides the hypothalamus and pituitary, melatonin action at the testicular level has been recently suggested. Therefore, we investigated in the Syrian hamster, a well-characterized seasonal breeder, melatonin action on Leydig cells, testicular expression of melatonergic receptors, and possible interactions between melatonin receptors and the previously identified testicular serotoninergic and CRH systems. In isolated Leydig cells from active testes of adult hamsters kept in a long-day (14 h light, 10 h dark) photoperiod and from regressed testes of adult animals exposed to a short-day photoperiod during 16 wk (6 h light, 18 h dark), melatonin significantly reduced human chorionic gonadotropin-stimulated production of cAMP and the main androgens: testosterone and androstane-3alpha,17beta-diol, respectively, and decreased the expression of steroidogenic acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase. In Leydig cells exposed to a short-day photoperiod during 16 wk, melatonin stimulated the conversion of testosterone into 5alpha-reduced androgens by inducing 5alpha-reductase isoform 1, and controlled androstane-3alpha,17beta-diol production by inhibiting 3alpha-hydroxysteroid dehydrogenase expression. Melatonin subtype (mel1a) receptors were detected in Leydig cells. Although the local serotonin system did not mediate melatonin action on androgen production, melatonergic effect on steroidogenesis involved the interaction between mel1a receptors and the inhibitory CRH system. Moreover, melatonin significantly increased CRH mRNA levels and production in hamster Leydig cells expressing CRH subtype 1 receptors. Our studies indicate that melatonin may act as a local inhibitor of human chorionic gonadotropin-stimulated cAMP and androgen production through mel1a receptors, down-regulation of steroidogenic acute regulatory protein, and key steroidogenic enzymes expression and its interaction with the local CRH system.

Gonadotropin-releasing hormone signaling pathways in an experimental ovarian tumor.

Previous results showed that GnRH signaling is altered in cells from rat luteinized ovarian tumors (tumor group) because it did not activate the phospholipase C pathway, in contrast to control ovarian cells from superovulated prepubertal rats (SPO). In the present work, alternate GnRH-induced second messengers such as phospholipase A(2) and phospholipase D activation, cAMP production, ERK1/2 phosphorylation, and the presence of G proteins were evaluated to determine GnRH mechanism of action in tumor cells. G proteins examined were present in both cell types. Buserelin, a GnRH agonist, (1, 10, and 100 ng/ml) increased phosphatidylethanol in SPO, indicating phospholipase D activation. Only 100 ng/ml buserelin induced a significant response in the tumor group. Buserelin (100 ng/ml) increased (3)H-arachidonic acid in culture media in SPO, indicating phospholipase A(2) activation; no effect was observed in the tumor group. Buserelin (100 and 1000 ng/ml) induced pertussis toxin-insensitive cAMP increases in both cell types, with similar potencies. In the tumor group, buserelin (100 ng/ml) inhibited human chorionic gonadotropin-induced cAMP and progesterone; this effect was protein kinase C (PKC) dependent (inhibited by GF109203X, a PKC inhibitor). Buserelin (100 and 1000 ng/ml) induced ERK1/2 phosphorylation in both cell kinds. Buserelin-induced ERK1/2 activation was G(i/0) independent and PKC dependent. Only in the tumor group, buserelin-induced ERK1/2 activation was cAMP dependent (abolished by SQ 22536, the adenylyl cyclase inhibitor). Furthermore, dibutyryl cAMP-induced ERK1/2 activation in the tumor group was PKC dependent (inhibited by GF109203X). In conclusion, activation of phospholipases in tumor cells does not seem to mediate GnRH effects. GnRH signaling seems to involve adenylyl cyclase activation, PKC stimulation, and ERK1/2 phosphorylation.

Protection against radiation-induced damage of 6-propyl-2-thiouracil (PTU) in thyroid cells.

Many epidemiologic studies have shown that the exposure to high external radiation doses increases thyroid neoplastic frequency, especially when given during childhood or adolescence. The use of radioprotective drugs may decrease the damage caused by radiation therapy and therefore could be useful to prevent the development of thyroid tumors. The aim of this study was to investigate the possible application of 6-propyl-2-thiouracil (PTU) as a radioprotector in the thyroid gland. Rat thyroid epithelial cells (FRTL-5) were exposed to different doses of γ irradiation with or without the addition of PTU, methimazole (MMI), reduced glutathione (GSH) and perchlorate (KClO4). Radiation response was analyzed by clonogenic survival assay. Cyclic AMP (cAMP) levels were measured by radioimmunoassay (RIA). Apoptosis was quantified by nuclear cell morphology and caspase 3 activity assays. Intracellular reactive oxygen species (ROS) levels were measured using the fluorescent dye 2',7'-dichlorofluorescein-diacetate. Catalase, superoxide dismutase and glutathione peroxidase activities were also determined. Pretreatment with PTU, MMI and GSH prior to irradiation significantly increased the surviving cell fraction (SF) at 2 Gy (P < 0.05), while no effect was observed with KClO4. An increase in extracellular levels of cAMP was found only in PTU treated cells in a dose and time-dependent manner. Cells incubated with agents that stimulate cAMP (forskolin and dibutyril cAMP) mimicked the effect of PTU on SF. Moreover, pretreatment with the inhibitor of protein kinase A, H-89, abolished the radioprotective effect of PTU. PTU treatment diminished radiation-induced apoptosis and protected cells against radiation-induced ROS elevation and suppression of the antioxidant enzyme's activity. PTU was found to radioprotect normal thyroid cells through cAMP elevation and reduction in both apoptosis and radiation-induced oxidative stress damage.

Melatonin inhibits glucocorticoid-dependent GR-TIF2 interaction in newborn hamster kidney (BHK) cells.

The antagonism exerted by melatonin on the glucocorticoid response has been well established, being strongly dependent on the cellular context. Previously, we found that melatonin inhibits glucocorticoid receptor (GR) dissociation from the chaperone hetero-complex and nuclear translocation on mouse thymocytes. Here, by performing confocal fluorescence microscopy and the Number and Brightness assay we show that in newborn hamster kidney cells (BHK21) melatonin neither affects GR nuclear translocation nor GR homodimerization. Instead, co-immunoprecipitation studies suggest that physiological concentrations of melatonin impair GR interaction with the transcriptional intermediary factor 2 (TIF2). This melatonin effect was not blocked by the MT(1)/MT(2) receptor antagonist luzindole. Curiously, luzindole behaved as an antiglucocorticoid per se by impairing the glucocorticoid-dependent MMTV-driven gene expression affecting neither GR translocation nor GR-TIF2 interaction.

CDK2 and PKA mediated-sequential phosphorylation is critical for p19INK4d function in the DNA damage response.

DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, ß-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with (32)P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.

Localization and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1) in the adult rat kidney.

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22R-hydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3ß-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla. In conclusion, we have established the expression and localization of StAR and CYP11A1 protein, the ability of synthesizing pregnenolone and a region-specific content of sex hormones in the adult rat kidney. These data clearly show that the kidney is a steroid hormones synthesizing organ. It is proposed that the existence in the kidney of complete steroidogenic machinery would respond to a physiological significance.