Epidermal growth factor and calcitriol synergistically induce osteoblast maturation

Calcitriol (1,25(OH)₂D₃) plays a key role in the differentiation of osteoblasts, the cells responsible for the formation and maintenance of healthy bone matrix. Recently it has emerged that calcitriol influences the trafficking or stability of epidermal growth factor (EGF) receptors. However, how these agents might work together in regulating growth and differentiation has not been examined. Using the human osteoblast cell line, MG63, we were able to induce a profound differentiation response by treating these cells with a combination of calcitriol (100 nM) and EGF (10 ng/ml). Co-stimulation of MG63 osteoblasts with calcitriol and EGF led to synergistic increases in osteocalcin and alkaline phosphatase (ALP), proteins expressed by differentiating cells. Inhibition of differentiation was accomplished by MEK and protein kinase C (PKC) inhibitors. Other ligands known to signal via receptor tyrosine kinases could not substitute for EGF in the maturation response. These novel findings may help identify new processes that drive osteoblast differentiation.     

Evidence for the participation of protein kinase C and 3′,5′-cyclic AMP-dependent protein kinase in the stimulation of muscle cell proliferation by 1,25-dihydroxy-vitamin D3

Treatment with 1,25-dihydroxy-vitamin D₃ (1,25(OH)₂D₃) (1–12 h, 10⁻¹⁰ M) stimulates DNA synthesis in proliferating myoblasts, with an early response at 2–4 h of treatment followed by a maximal effect at 10 h. To investigate the mechanism involved in the mitogenic action of the hormone we studied the possible activation of intracellular messengers by 1,25(OH)₂D₃. The initial phase of stimulation of [³H]thymidine incorporation into DNA by the sterol was mimicked by the protein kinase C activator tetradecanoylphorbol acetate (TPA) in a manner which was dose dependent and specific as the inactive analog 4-phorbol was without effect. Maximal responses to TPA (100 nM) were obtained at 4 h. Staurosporine, a protein kinase C inhibitor, blocked the effect of 1,25(OH)₂D₃ on myoblast proliferation at 4 h. In addition, a fast (1–5 min) elevation of diacylglycerol levels and membrane-associated protein kinase C activity was observed in response to 1,25(OH)₂D₃. The adenylate cyclase activator forskolin (20 μM) and dibutyryl-cAMP (50 μM) increased DNA synthesis reproducing the second 1,25(OH)₂D₃-dependent stimulatory phase at 10 h. Inhibitors of protein kinase A blocked the increase in muscle cell DNA synthesis induced by 1,25(OH)₂D₃ at 10 h. Significant increases in cyclic AMP levels were detected in myoblasts treated with the sterol for 1–10 h. The calcium channel antagonist nifedipine (5–10 μM) abolished both the effects of 4-h treatment with 1,25(OH)₂D₃ or TPA and 10-h treatment with 1,25(OH)₂D₃ or dibutyryl-cAMP. Similar to the calcium channel agonist Bay K8644, 1,25(OH)₂D₃ stimulated myoblast ⁴⁵Ca uptake and its effects were blocked by nifedipine. Our results suggest that activation of calcium channels by phosphorylation via protein kinases C and A and is involved in the mitotic response of myoblasts to 1,25(OH)₂D₃.     

Demonstration of a high affinity receptor for 1,25-dihydroxyvitamin D3 in rat pancreas

The existence of a high affinity receptor for 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in rat pancreas was biochemically demonstrated in this study. In order to study the properties of this putative receptor, we took advantage of the analysis of low ionic strength chromatin-localized 1,25(OH)₂D₃ receptor. Using this method, the susceptibility of receptor protein to enzymatic degradation was so decreased, and the contamination by plasma vitamin D binding protein (DBP) component was so efficiently eliminated that a specific, saturable binding for 1,25(OH)₂D₃ could be demonstrated in the saturation analysis and the peak for the receptor was consistently apparent in the sucrose density gradient analysis. The equilibrium dissociation constant (Scatchard K_d) was found to be 3.7 ± 1.5 × 10^-10 (M), and the concentration of specific binding sites was calculated to be 1.22 ± 0.40 (fmol/mg protein). The number of specific binding sites in the rat pancreas was only 0.44% of that present in rat intestine (277 ± 19 (fmol/mg protein)) and 6.7% of that in rat kidney (18.1 ± 1.0 (fmol/mg)). However, when a correction is made for the 1,25(OH)₂D₃ receptor distribution in the tissues and expressed as the receptor concentration per receptor-containing cells, the rat pancreatic receptor level was calculated to be about 30% of the rat intestine. Sucrose density gradient sedimentation of this receptor yielded a value of 3.2 ± 0.1 (S) for the sedimentation coefficient and this peak was displaceable by a 100-fold excess of nonradioactive 1,25(OH)₂D₃. These data provide evidence for the presence of a specific 1,25(OH)₂D₃ receptor in mammalian pancreas, and we suggest, in conjunction with the facts that vitamin D₃ and its active metabolites play a physiological role on insulin secretion from rat pancreatic β-cells, that this receptor might be involved in the mechanisms of action of vitamin D₃ on insulin secretion from rat pancreas via a genomic effect.     

Age and gender effects on 1,25-dihydroxyvitamin D3-regulated gene expression

Several factors involved in regulation of bone mineral metabolism were compared in male and female Fischer 344 rats of different ages (1, 2.5, 6, and 18 months). Plasma 1,25-(OH)₂D₃ concentrations decreased with age in rats of both genders. Abundance of calbindin-D_28K and its mRNA in kidney and calbindin-D_9K and its mRNA in duodenum also decreased with age in both male and female rats. Renal 24-hydroxylase activity and 24-hydroxylase mRNA content were elevated significantly in 18-month-old males and females, compared with younger ages. These data suggest that increased renal catabolism of 1,25-(OH)₂D₃ may be responsible for low plasma 1,25-(OH)₂D₃ concentrations observed in older animals. Plasma PTH and 1,25-(OH)₂D₃ concentrations, renal 24-hydroxylase enzyme activity and 24-hydroxylase mRNA content, duodenal 24-hydroxylase mRNA abundance, and duodenal calbindin-D_9K and calbindin-D_9K mRNA content were greater in males than in females at 2.5 months of age. Lower plasma 1,25-(OH)₂D₃ concentrations in females seem to explain observed gender differences in expression of 1,25-(OH)₂D₃-stimulated genes. The combined effects of these gender differences at ages when peak bone density is being developed may contribute to the greater incidence of osteoporosis in females than in males.     

1,25（OH）2D3通过STAT5抑制Th17细胞分化的调控作用研究*

目的 研究1.25二羟基维生素D₃[1,25（OH）₂D₃]抑制辅助性T细胞17（Th17）的分化与STAT5调控的关系。方法 通过分选出的CD4⁺T细胞,在1,25（OH）₂D₃和/或STAT5抑制剂的作用下,采用ELISA法检测抑制剂处理后细胞培养上清液Th17细胞因子（IL-17A、IL-22）水平的变化;采用细胞免疫荧光技术检测STAT5的磷酸化水平,采用Western blot技术检测STAT5蛋白表达水平。结果 1,25（OH）₂D₃组细胞培养上清IL-17A和IL-22水平（12.5±0.5 ng/ml,48.5±0.9 pg/ml）明显低于对照组（22.7±0.5 ng/ml,73.8±1.9 pg/ml）,而STAT5抑制剂组IL-17A和IL-22水平明显升高（33.5±0.7 ng/ml,89.1±1.4 pg/ml）,1,25（OH）₂D₃与STAT5抑制剂联合作用细胞IL-17A和IL-22水平（18.5±0.7 ng/ml,54.1±1.6 pg/ml）显著高于1,25（OH）₂D₃组,但低于STAT5抑制剂组,差异有统计学意义（P<0.01）;1,25（OH）₂D₃组细胞p-STAT5表达显著强于对照组,1,25（OH）₂D₃联合STAT5抑制剂组p-STAT5表达量低于对照组,而STAT5抑制剂组细胞p-STAT5表达量最低;1,25（OH）₂D₃组STAT5蛋白表达明显升高,而1,25（OH）₂D₃联合STAT5抑制剂组或STAT5抑制剂组STAT5蛋白表达明显降低,以STAT5抑制剂组细胞表达最弱,差异均具有统计学意义（P<0.01）。结论 1,25（OH）₂D₃通过STAT5信号通路能抑制Th17细胞分化。     

Monocytic activation of protein tyrosine kinase,protein kinase A and protein kinase C induced by porins isolated from Salmonella enterica serovar Typhimurium

OBJECTIVES: In the present study a monocytic cell line, U937, was used to investigate the possible involvement of protein tyrosine kinases (NT-PTKs), protein kinase A (PKA) and protein kinase C (PKC) in cell signaling pathways following Salmonella enterica serovar Typhimurium porin stimulation. METHODS: Different concentrations of porins and lipopolysaccharide (LPS) were analysed to evaluate changes in PTK activity by a non radioactive tyrosine kinase assay and in PKA and PKC phosphorylation by Western blotting analysis. The inhibitors of PTK, PKA and PKC activation used, were: 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase activity; dihychloride (H-89), a selective inhibitor of PKA which is useful to discriminate between the effects of PKC and PKA; diacylglycerol kinase inhibitor II (R59949), which is useful for elucidating roles of PKC; calphostin C, a specific inhibitor of PKC. RESULTS: Porins of the outer membrane of the ST were isolated to be used as a stimulus in the performed experiments. Following porin treatment, a dose-dependent increase in PTK, PKA and PKC activation was observed. U937 monocytes pretreated with inhibitors induced an evident decrease in PTK activity and PKA and PKC phosphorylation pattern in porin stimulated monocytes. CONCLUSIONS: Our data support the important role played by NT-PTK, PKA and PKC in transducing the activating signal in macrophages stimulated with porins through the activation of the mitogen-activated protein kinase (MAPK) pathway that participate in the regulation of gene expression.     

Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium

OBJECTIVE: In human heart failure beta-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases. METHODS: Isometric force was measured at different [Ca(2+)] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA. RESULTS: Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca(2+) sensitivity (pCa(50)). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca(2+) sensitivity to a larger extent in failing (DeltapCa(50)=0.19+/-0.03) than in non-failing (DeltapCa(50)=0.08+/-0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa(50). Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. CONCLUSION: In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca(2+) sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.     

Activation of a beta-adrenergic-sensitive signal transduction pathway by the secosteroid hormone 1,25-(OH)2-vitamin D3 in chick heart.

In recent studies we have established that 1 alpha, 25-dihydroxy-vitamin D3[1,25(OH)2D3] rapidly stimulates dihydropyridine-sensitive calcium channel-mediated Ca2+influx in chick cardiac muscle by a non-genomic action which is accompanied by PKA-dependent phosphorylation of a 45 kDa microsomal membrane protein. To investigate the signal transduction pathway activated by 1,25(OH)2D3 in heart, we have compared the effects of the secosteroid hormone with those of the beta-adrenergic agonist isoproterenol (IPT) by employing cultured chick embryonic cardiac cells (myocytes) and thin-slice preparations of differentiated adult heart muscle. The increases in 45Ca2+ uptake and intracellular calcium ([Ca2+]i), cyclic AMP accumulation and changes in microsomal protein phosphorylation evoked by 1,25(OH)2D3 could be reproduced by IPT. When combined treatments with the sterol and the beta-adrenergic agonist were performed, no additive stimulation of these parameters was observed, suggesting that a common signal transduction pathway mediates the effects of 1,25(OH)2D3 and IPT. The participation of a guanine nucleotide binding protein (G protein) in the 1, 25(OH)2D3-induced changes in heart was investigated. AlF4(-), an activator of G proteins, and cholera and pertussis toxins, like 1, 25(OH)2D3 increased 45Ca2+ uptake by myocytes. AlF4(-) did not further stimulate the effects of 1,25(OH)2D3 thereby showing that a G protein is involved in the hormone action. Moreover, 1,25(OH)2D3 potentiated pertussis toxin but was unable to modify choleric toxin-dependent myocyte Ca2+ influx. Altogether, these results provide evidence indicating that the non-genomic action of 1,25(OH)2D3 on cardiac muscle calcium influx involves modulation of the beta-adrenergic-sensitive adenylyl cyclase/cAMP/PKA pathway coupled to a Gs protein.     

Evidence on the participation of the 3',5'-cyclic AMP pathway in the non-genomic action of 1,25-dihydroxy-vitamin D3 in cardiac muscle.

Several studies have suggested that vitamin D plays a role in cardiovascular function. It has been recently shown that in vitro treatment of vitamin D-deficient chick cardiac muscle with physiological concentrations of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) induces a rapid (1-10 min) increase of tissue 45Ca uptake which can be suppressed by Ca channel blockers. The hormone simultaneously stimulated heart microsomal membrane protein phosphorylation. Experiments were performed to investigate the existence of a relationship between these changes and to obtain information about the mechanism involved in 1,25(OH)2D3-induced modifications in cardiac protein phosphorylation. Dibutyryl cyclic AMP (10 microM) and forskolin (10 microM), known activators of the cAMP pathway, produced time courses of changes in 45Ca uptake by chick heart tissue similar to 1,25(OH)2D3 (10(-10) M). Analogously to the hormone, the effects of both compounds were abolished by nifedipine (30 microM) and verapamil (10 microM). In agreement with these observations, 1,25(OH)2D3 significantly increased (34-70%) heart muscle cAMP levels within 1-10 min of treatment. In addition, 1,25(OH)2D3 and forskolin caused similar changes in cardiac microsomal membrane protein phosphorylation (e.g. stimulation in 43 kDa and 55 kDa proteins). These changes were also evidenced by direct exposure of isolated heart microsomes to 1,25(OH)2D3, suggesting a direct membrane action of the hormone. The fast effects of 1,25(OH)2D3 on dihydropyridine-sensitive cardiac muscle Ca uptake could be reproduced in primary-cultured myocytes isolated from chick embryonic heart. Furthermore, the effects of the hormone could be suppressed by a specific protein kinase A inhibitor. These results suggest that 1,25(OH)2D3 affects heart cell calcium metabolism through regulation of Ca channel activity mediated by the cAMP pathway.     

Effect of vitamin D deficiency and 1,25-dihydroxycholecalciferol treatment on epidermal calcium-binding protein (ECaBP) RNA activity

We have previously reported that the amount of epidermal calcium binding protein (ECaBP) in the skin decreases in the absence of vitamin D. Since vitamin D influences epidermal differentiation, and the synthesis of ECaBP may vary with cell differentiation, it was necessary to know whether vitamin D acts directly on the translational or post-translational level of ECaBP synthesis or indirectly by its action on epidermopoiesis. The cell-free translation technique was used to demonstrate the presence of mRNA coding for ECaBP. The activity of this mRNA has been evaluated in the skin of vitamin D-fed and in vitamin D-deficient rats with or without treatment with 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃). Vitamin D deficiency decreased the ECaBP mRNA activity. The latter was selectively increased in animals given a single dose of 1,25(OH)₂D₃. These results suggest that 1,25(OH)₂D₃ stimulates the production of ECaBP mRNA or stabilizes this mRNA.     

Effect of cyclic adenosine 3',5'-monophosphate and protein kinase A on ligand-dependent transactivation via the vitamin D receptor

We examined the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and protein kinase A (PKA) on the ligand-dependent transactivation mediated via the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor (VDR). A human VDR expression plasmid was transfected into HeLa, Saos-2 and MG63 cells with a luciferase reporter gene construct containing the vitamin D responsive element. With the addition of 0.5 mM 8 bromo-cAMP, the response to 1,25(OH)2D3 was suppressed to 61 and 78% in the HeLa and Saos-2 cells, respectively. The suppressive effect of 8 bromo-cAMP was observed without the introduction of the VDR expression plasmid in the MG63 cells. In the HeLa cells the co-expression of PKA reduced the ligand-inducible transactivation to 61% and the fold induction by 1,25(OH)2D3 to 89% of that without PKA. The CREB binding protein (CBP) was recently reported to integrate the intracellular signals via the cAMP/PKA cascade and nuclear hormone receptors. However, the suppressive effect of cAMP was not influenced by the co-expression of CBP. Lastly, we introduced point mutations at possible PKA phosphorylation sites into the VDR expression vector at serine-172 and threonine-175, but both mutant receptors still exhibited reduced transactivation with the co-expression of PKA. These results indicate that the phosphorylation of proteins other than the VDR may also be involved in the inhibitory effect mediated by the cAMP/PKA cascade.     

1,25（OH）2D3通过调节miR-146a水平抑制大鼠肝纤维化的体内和体外观察

目的 体内体外观察1,25（OH）₂D₃通过调节miR-146a水平抑制大鼠肝纤维化的作用机制。方法 建立CCl₄诱导的大鼠肝纤维化模型,体外转染肝星状细胞（HSCs）miR-146a 模拟剂/抑制剂,观察1,25（OH）₂D₃处理对动物肝组织变化和HSC增殖和凋亡的影响。采用qPCR法检测肝组织miR-146a水平,采用CCK8法检测细胞增殖,使用流式细胞术检测HSC凋亡。结果 在干预8 w末,1,25（OH）₂D₃干预组大鼠肝纤维化程度明显减轻; 1,25（OH）₂D₃干预组大鼠肝组织miR-146a水平为（0.70± 0.03）,显著高于橄榄油组【（0.33±0.17,P<0.05）】; 1,25（OH）₂D₃组细胞增殖率为58.8%,较DMSO组下降了15.9%,转染miR-146a 模拟剂组大鼠HSC增殖率为46.5%,较对照组下降了53.3%,转染miR-146a 抑制剂组HSC增殖率为132.8%,较对照物组升高了32.8%（P<0.05）,1,25（OH）₂D₃干预组细胞凋亡率为12.6%,较DMSO组增加了5.2%,转染miR-146a 模拟剂组细胞凋亡率为16.8%,较对照组细胞凋亡率增加了8.2%,转染miR-146a 抑制剂组细胞凋亡率为6.3%,较对照组细胞凋亡率减少了2.2%（P<0.05）,提示1,25（OH）₂D₃具有抑制HSC增殖、促进凋亡作用。结论 1,25（OH）₂D₃可能通过调节miR-146a水平抑制HSC活化和抑制大鼠肝纤维化。     

Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins

The bioactive component of mildly oxidized low-density lipoproteins, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), activates tissue factor expression and monocyte adhesion to endothelial cells (EC) from systemic circulation, but blocks expression of inflammatory adhesion molecules (VCAM, E-selectin) and neutrophil adhesion associated with EC acute inflammatory response to bacterial lypopolysacharide (LPS). Due to constant exposure to oxygen free radicals, lipids in the injured lung are especially prone to oxidative modification and increased OxPAPC generation. In this study, we focused on OxPAPC-mediated intracellular signaling mechanisms that lead to physiological responses in pulmonary endothelial cells. Our results demonstrate that OxPAPC treatment activated in a time-dependent fashion protein kinase C (PKC), protein kinase A (PKA), Raf/MEK1,2/Erk-1,2 MAP kinase cascade, JNK MAP kinase and transient protein tyrosine phosphorylation in human pulmonary artery endothelial cells (HPAEC), whereas nonoxidized PAPC was without effect. Pharmacological inhibition of PKC and tyrosine kinases blocked activation of Erk-1,2 kinase cascade upstream of Raf. OxPAPC did not affect myosin light chain (MLC) phosphorylation, but increased phosphorylation of cofillin, a molecular regulator of actin polymerization. Finally, OxPAPC induced p60Src-dependent tyrosine phosphorylation of focal adhesion proteins paxillin and FAK. Our results suggest a critical involvement of PKC and tyrosine phosphorylation in OxPAPC-induced activation of Erk-1,2 MAP kinase cascade associated with regulation of specific gene expression, and demonstrate rapid phosphorylation of cytoskeletal proteins, which indicates OxPAPC-induced EC remodeling.     

Acute signaling by the LH receptor is independent of protein kinase C activation

LH receptor activation leads to the phosphorylation/activation of p42/44 MAPK in preovulatory granulosa cells. As the LH receptor can activate both adenylyl cyclase and phospholipase C, we hypothesized that the LH receptor could elicit phosphorylation of p42/44 MAPK through activation of protein kinase A (PKA) and/or protein kinase C (PKC). Preovulatory granulosa cells in serum-free primary cultures were treated with ovulatory concentrations of human chorionic gonadotropin (hCG), an LH receptor agonist, with or without various inhibitors. The PKA inhibitor H89 as well as the myristoylated PKA inhibitor peptide PKI strongly inhibited hCG-stimulated p42/44 MAPK phosphorylation, whereas the PKC inhibitor GF109203X had no effect on p42/44 MAPK phosphorylation. LH receptor-stimulated phosphorylation of cAMP response element-binding protein (CREB), histone H3, and MAPK kinase (MEK) was also strongly inhibited by H89 and not by GF109203X. The extent of PKC activation was assessed in preovulatory granulosa cells using three criteria: translocation of PKC isoforms to the membrane fraction, phosphorylation of a known PKC substrate, and autophosphorylation of PKC delta on an activation-related site. By all three criteria PKCs were partially activated before hCG stimulation, and hCG treatment failed to elicit further PKC activation, in vitro or in vivo. Taken together, these results indicate that, under primary culture conditions where physiological levels of signaling proteins are present, hCG signals to activate MEK, p42/44 MAPK, CREB, and histone H3 in a predominantly PKA-dependent and PKC-independent manner. Unexpectedly, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation.     

Rapid uncoupling of serotonin-1A receptors in rat hippocampus by 17beta-estradiol in vitro requires protein kinases A and C

17beta-Estradiol decreases R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding [an index of serotonin-1A (5-HT(1A)) receptor coupling] through the activation of estrogen receptors. We hypothesize that this occurs as a result of activation of protein kinase A (PKA) and/or protein kinase C (PKC) and phosphorylation of 5-HT(1A) receptors. Hippocampus from ovariectomized rats was incubated with 17beta-estradiol in HEPES buffer (37 degrees C). Cytosolic and membrane fractions were prepared to assess PKA and PKC activities, respectively. In separate experiments, membranes were prepared to measure R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding. 17beta-Estradiol (50 nM) increased PKA and PKC activities approximately 2- to 3-fold. PKC activity was elevated at 10, 30 and 60 min, whereas PKA activity was increased at 10 and 30 min. The ability of 17beta-estradiol to increase PKA and PKC was blocked by the estrogen receptor antagonist ICI 182,780 (1 microM). A selective PKA inhibitor (KT 5720, 60 nM) blocked 17beta-estradiol-stimulated PKA but NOT PKC activity. Conversely, the PKC inhibitor calphostin C (100 nM) blocked the increase in PKC activity produced by 17beta-estradiol but NOT the PKA response. The protein kinase inhibitors individually blocked the effects of 17beta-estradiol on R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding. By contrast, preincubation with the protein synthesis inhibitor cycloheximide (200 microM) or the mitogen activated protein (MAP) kinase kinase inhibitor PD 98059 (50 microM) was without effect. Incubation of hippocampus with 17beta-estradiol (50 nM, 60 min) caused the phosphorylation of a protein consistent with the 5-HT(1A) receptor. These studies demonstrate that 17beta-estradiol acts on estrogen receptors locally within the hippocampus through nongenomic mechanisms to activate PKA and PKC, phosphorylate 5-HT(1A) receptors and uncouple them from their G proteins.     

Calcium-calmodulin and calcium-phospholipid dependent phosphorylation of membranous fraction proteins related to the tropic regulation by estradiol in the corpus luteum

Estradiol assumes a major role in the regulation of growth, vascularization, and progesterone synthesis in the midpregnant rat corpus luteum. To explore whether molecular events triggered by estradiol could be mediated, at least in part, by protein phosphorylation, we investigated whether estradiol treatment in vivo affects endogenous luteal protein phosphorylation systems detectable in vitro. Luteal nuclear, mitochondrial, and microsomal fractions were obtained by differential centrifugation from rats hypophysectomized and hysterectomized on day 12 of pregnancy and treated with or without estradiol for 72 h. Using [gamma-32P]ATP as phosphate donor, proteins were phosphorylated in the presence or absence of either calcium (Ca), Ca plus calmodulin, or Ca plus phospholipid. Phosphoproteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. The Coomassie blue stained proteins and phosphoprotein profiles were markedly different in the various fractions. Estradiol treatment in vivo caused an increase in the basal endogenous phosphorylation of several proteins in vitro. It also substantially enhanced the protein kinase C (PKC) and Ca-calmodulin kinase-dependent phosphorylation of selected proteins in subcellular fractions. The Ca-calmodulin kinase catalyzed phosphorylation of microsomal 56 and 60 kilodalton (kDa) proteins was remarkably increased by estradiol. Proteins (56 and 60 kDa) were also phosphorylated when Ca-calmodulin was added to the nuclear fraction, however, this phosphorylation did not appear to be affected by estradiol treatment. A major PKC substrate in the nuclear fraction was an 80 kDa protein whose phosphorylation was increased remarkably by estradiol treatment. In the mitochondrial fraction the most striking effect of estradiol was a marked increase in PKC-mediated phosphate transfer into a 76 kDa substrate. To determine whether estradiol action on protein phosphorylation was related to its tropic effect in the corpus luteum, the hormone was administered to day 10 hypophysectomized and hysterectomized pregnant rats. In this rat model, where estradiol has no stimulatory effect on either luteal steroidogenesis or growth, neither endogenous nor kinase-mediated phosphorylation was affected by this steroid. In summary, the present investigation has revealed that in vivo treatment with estradiol affects the PKC and the Ca-calmodulin dependent in vitro phosphorylation of selected proteins localized in different subcellular compartments and further suggests that phosphorylation systems are potential control points for estradiol regulation of rat corpus luteum function.     

Melatonin restores impaired contractility in aged guinea pig urinary bladder

Abstract:  Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin-treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca²⁺]_i) was estimated by epifluorescence microscopy of fura-2-loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca²⁺]_i in response to the stimuli, suggesting a reduced Ca²⁺sensitivity. Indeed, the agonist-induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca²⁺ sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and with the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca²⁺ handling and Ca²⁺ sensitizations pathways. Moreover, the indoleamine restored age-induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging-related urinary bladder contractile impairment.