Synthesis, Fluorescence, and Bioactivity of Novel Isatin Derivatives.

The isatin group is widespread in nature and is considered to be a privileged building block for drug discovery. In order to develop novel SHP1 inhibitors with fluorescent properties as tools for SHP1 biology research, this work designed and synthesized a series of isatin derivatives. The presentive compound 5a showed good inhibitory activity against SHP1PTP with IC50 of 11 ± 3 µM, displayed about 92% inhibitory rate against MV-4-11 cell proliferation at the concentration of 20 µM, exhibited suitable fluorescent properties with a long emission wavelength and a large Stokes shift, and presented blue fluorescent imaging in HeLa cells with low cytotoxicity. This study could offer chemical tool to further understand SHP1 biology and develop novel SHP1 inhibitors in therapy.

Nucleophile-Controlled Trapping of Gold Carbene by Nitriles and Water: Synthesis of 5H-Pyrimido[5,4-b]indoles and 2-Benzylidene-3-indolinones.

A gold-catalyzed, nucleophile-controlled cascade reaction of N-(2-azidophenyl-ynyl)methanesulfonamides with nitriles and water is described that provides structurally diverse 5H-pyrimido[5,4-b]indoles and 2-benzylidene-3-indolinones in good to excellent yields. Mechanistic studies indicate that the ß-sulfonamido-α-imino gold carbene is the key intermediate which is generated through the gold-catalyzed cyclization of N-(2-azidophenyl-ynyl)methanesulfonamides and undergoes formal [4 + 2] cascade annulation with nitriles and intramolecular SN2' type reaction with water, respectively.

Novel imidazo[1,2,4] triazole derivatives: Synthesis, fluorescence, bioactivity for SHP1.

The Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) is a convergent node for oncogenic cell-signaling cascades. Consequently, SHP1 represents a potential target for drug development in cancer treatment. The development of efficient methods for rapidly tracing and modulating the SHP1 activity in complex biological systems is of considerable significance for advancing the integration of diagnosis and treatment of the related disease. Thus, we designed and synthesized a series of imidazo[1,2,4] triazole derivatives containing salicylic acid to explore novel scaffolds with inhibitory activities and good fluorescence properties for SHP1. The photophysical properties and inhibitory activities of these imidazo[1,2,4] triazole derivatives (5a-5y) against SHP1PTP were thoroughly studied from the theoretical simulation and experimental application aspects. The representative compound 5p exhibited remarkable fluorescence response (P: 0.002) with fluorescence quantum yield (QY) of 0.37 and inhibitory rate of 85.21 ± 5.17% against SHP1PTP at the concentration of 100 µM. Furthermore, compound 5p showed obvious aggregation caused quenching (ACQ) effect and had high selectivity for Fe3+ ions, good anti-interference and relatively low detection limit (5.55 µM). Finally, the cellular imaging test of compound 5p also exhibited good biocompatibility and certain potential biological imaging application. This study provides a potential way to develop molecules with fluorescent properties and bioactivities for SHP1.

Exploring the mechanism of the PTP1B inhibitors by molecular dynamics and experimental study.

Protein tyrosine phosphatase 1B (PTP1B) has proven to be an attractive target for the treatment of cancer, diabetes and other diseases. Although many PTP1B inhibitors with various scaffolds have been developed, there is still a lack of PTP1B inhibitor with high specificity and acceptable pharmacological properties. Therefore, it is urgent to develop more methods to explore complex action mode of PTP1B and ligands for designing ideal PTP1B modulators. In this work, we developed a potential molecular dynamics (MD) analytic mode to analyze the mechanism of active compounds 6a and 6e against PTP1B from different perspectives, including the stable ability, interactions and binding site of ligand and protein, the binding energy, relative movement between residues and changes in protein internal interactions. The simulated results demonstrated that compound 6a bound more stably to the active pocket of PTP1B than 6e due to its smaller molecular volume (326 Å3), matched electronegativity, and enhanced the positive correlation motion of residues, especially for WPD loop and P loop. Lastly, compound 6a as a competitive inhibitor for PTP1B was verified by enzyme kinetic assay. This work successfully studied the mechanism of compound 6a against PTP1B from various aspects, enriched the analysis of interaction mode between PTP1B and inhibitors. In summary, we hope that this work could provide more theoretical information for designing and developing more novel and ideal PTP1B inhibitors in the future.

Design, Synthesis and Evaluation of Fluorescent Properties of Benzothiazole Derivatives.

Fluorescence imaging is conducive to establish a bridge between molecular biology and clinical medicine, and provides new tools for disease process research, early diagnosis, and efficacy evaluation, because of the advantages of rapid imaging and nondestructive detection. Herein, a series of fluorescent molecules with thiadiazole, or thiazole, or benzothiazole cores were designed and synthesized to develop more excellent fluorescent molecules in bio-imaging. According to theoretical and experimental methods, we found that benzothiazole derivative 14 B with conjugate expansion by (4-aminophenyl) ethynyl group was the most excellent fluorescent molecule among all the investigated compounds and exhibited low cytotoxicity and strong blue and green fluorescence by confocal cell imaging.

Gold-Catalyzed Cascade Reaction of 2-Alkynyl Aryl Azides with Enecarbamates for Direct Synthesis of α-(3-Indolyl)ketones.

α-(3-Indolyl)ketones are essential building blocks for the generation of biologically active molecules. We described a new method for the direct assembly of α-(3-indolyl)ketones through the cascade reaction of 2-alkynyl aryl azides with enecarbamates, in which the in situ generated α-imino gold carbene intermediate was trapped by enecarbamate to achieve umpolung reactivity of indole at the 3-position.

Safety of Normothermic Cardiopulmonary Bypass in Pediatric Cardiac Surgery: A System Review and Meta-Analysis.

Objectives: Hypothermic cardiopulmonary bypass (HCPB) has been used successfully in cardiac surgery for more than half a century, although adverse effects have been reported with its use. Many studies on temperature management during CPB published to date have shown that normothermic CPB (NCPB) provides more benefits to children undergoing cardiac surgery. The present meta-analysis investigated the effect of NCPB on clinical outcomes based on results of randomized controlled trials and observational studies on pediatric cardiac surgery. Methods: Databases such as PubMed, EMBASE, Cochrane Central Register of Controlled Trials, and Clinical Trials.gov were searched from inception to May 2021 to identify relevant studies published in English. Results: The present meta-analysis included 13 studies characterizing a total of 837 pediatric patients. The random effects model exhibited that the NCPB group had reduced revision for postoperative bleeding [odds ratio (OR): 0.11; 95% confidence interval (CI): 0.01-0.89; I 2 = 0%, P = 0.04], serum lactate 2-4 h after CPB (mean difference: -0.60; 95% CI: -1.09 to -0.11; I 2 = 82%, P = 0.02), serum creatinemia 24 h after CPB (mean difference: -2.73; 95% CI: -5.06 to -0.39; I 2 = 83%, P = 0.02), serum creatinemia 48 h after CPB (mean difference: -2.08; 95% CI: -2.78 to -1.39; I 2 = 0%, P < 0.05), CPB time (mean difference: -19.10, 95% CI: -32.03 to -6.18; I 2 = 96%, P = 0.04), and major adverse events (OR: 0.37; 95% CI: 0.15-0.93; Z = 2.12, P = 0.03) after simple congenital surgery compared with the HCPB group. Conclusion: NCPB is as safe as HCPB in pediatric congenital heart surgery. Moreover, NCPB provides more advantages than HCPB in simple congenital heart surgery.

Synthesis and biological evaluation of 2,5-diaryl-1,3,4-oxadiazole derivatives as novel Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) inhibitors.

The Src homology-2 domain containing-protein tyrosine phosphatase-2 (SHP2) is a convergent node for oncogenic cell-signaling cascades including the PD-L1/PD-1 pathway. As an oncoprotein as well as a potential immunomodulator, SHP2 has now emerged as an attractive target for novel anti-cancer agents. Although significant progress has been made in identifying chemotypes of SHP2 inhibitors, these specific compounds might not be clinically useful to inhibit frequently encountered mutated SHP2 variants. Consequently, it is highly desirable to develop chemically different SHP2 inhibitors sensitive to SHP2 mutants. This work developed a new type of SHP2 inhibitors with 2,5-diaryl-1,3,4-oxadiazole scaffold. The representative compound 6l exhibited SHP2 inhibitory activity with IC50 of 2.73 ± 0.20 µM, showed about 1.56-fold, 5.26-fold, and 7.36-fold selectivity for SHP2 over SHP1, PTP1B and TCPTP respectively. Further investigations confirmed that 6l behaved as mixed-type inhibitor sensitive to leukemia cell TF-1 and inhibited SHP2 mediated cell signaling and proliferation. Molecular dynamics simulation provided more detailed information on the binding modes of compounds and SHP2 protein. These preliminary results could provide a possible opportunity for the development of novel SHP2 inhibitors sensitive to SHP2 mutants with optimal potency and improved pharmacological properties.

Posterior pericardiotomy to prevent new-onset atrial fibrillation after coronary artery bypass grafting: a systematic review and meta-analysis of 10 randomized controlled trials.

BACKGROUND: Atrial fibrillation (AF) is associated with adverse events after cardiac surgery. Multiple studies have reported that posterior pericardiotomy (PP) may be effective for preventing AF after coronary artery bypass grafting (CABG), but some conflicting results have been reported and the quality of evidence from previous meta-analyses has been limited. The present study aimed to systematically evaluate the safety and efficacy of PP for preventing AF after CABG in adults. METHODS: We conducted a quantitative meta-analysis of randomized controlled trials (RCTs) published before May 31, 2021. The primary outcome was AF after CABG under cardiopulmonary bypass. Secondary outcomes included early pericardial effusion, late pericardial effusion, pericardial tamponade, pleural effusion, length of hospital stay, length of intensive care unit (ICU) stay, pulmonary complications, intra-aortic balloon pump use, revision surgery for bleeding, and mortality. RESULTS: Ten RCTs with 1829 patients (910 in the PP group and 919 in the control group) were included in the current meta-analysis. The incidence of AF was 10.3% (94/910) in the PP group and 25.7% (236/919) in the control group. A random-effects model indicated that incidence of AF after CABG significantly lower in the PP group than in the control group (risk ratio = 0.45, 95% confidence interval 0.29-0.64, P < 0.0001). PP also effectively reduced the post-CABG occurrence of early pericardial effusion (RR = 0.28, 95% CI 0.15-0.50; P < 0.05), late pericardial effusion (RR = 0.06, 95% CI 0.02-0.16; P < 0.05), and pericardial tamponade (RR = 0.08, 95% CI 0.02-0.33; P < 0.05) as well as the length of ICU stay (weighted mean difference [WMD] = 0.91,95% CI 0.57-1.24; P < 0.05), while increasing the occurrence pleural effusion (RR = 1.51, 95% CI 1.19-1.92; P < 0.05). No significant differences length of hospital stay (WMD = - 0.45, 95% CI - 2.44 to 1.54, P = 0.66), pulmonary complications (RR = 0.99, 95% CI 0.71-1.39, P = 0.97), revision surgery for bleeding (RR = 0.84, 95% CI 0.43-1.63, P = 0.60), use of IABP (RR = 1, 95% CI 0.61-1.65, P = 1.0), or death (RR = 0.45, 95% CI 0.07-3.03, P = 0.41) were observed between the PP and control groups. CONCLUSIONS: PP may be a safe, effective, and economical method for preventing AF after CABG in adult patients.

Synthesis and biological evaluation of heterocyclic bis-aryl amides as novel Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) inhibitors.

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is a convergent node for oncogenic cell-signaling cascades including the PD-L1/PD-1 pathway. Consequently, SHP2 has emerged as a compelling target for novel anti-cancer agents. Replacing one of phenyl ring in PTP1B inhibitor 1 with heterocyclic ring led to a series of heterocyclic bis-aryl amide derivatives. The representative compound 7b displayed SHP2 inhibitory activity with IC50 of 2.63 ± 0.08 µM, exhibited about 4-fold selectivity for SHP2 over TCPTP and had no detectable activity against SHP1 and PTP1B. These preliminary results could provide a possible opportunity for the development of novel SHP2 inhibitors with optimal potency and improved pharmacological properties.

Improving the accuracy and efficacy of diagnosing polycystic ovary syndrome by integrating metabolomics with clinical characteristics: study protocol for a randomized controlled trial.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine syndrome with poorly understood mechanisms. To provide patients with PCOS with individualized therapy, it is critical to precisely diagnose the phenotypes of the disease. However, the criteria for diagnosing the different phenotypes are mostly based on symptoms, physical examination and laboratory results. This study aims to compare the accuracy and efficacy of diagnosing PCOS by integrating metabolomic markers with common clinical characteristics. METHODS: This is a prospective, multicenter, analyst-blinded, randomized controlled trial. Participants will be grouped into (1) people without PCOS (healthy control group), (2) patients diagnosed with PCOS based on clinical indices (experimental group 1), and (3) patients diagnosed with PCOS based on metabolomic indices (experimental group 2). A total of 276 participants, including 60 healthy people and 216 patients with PCOS, will be recruited. The 216 patients with PCOS will be randomly assigned to the two experimental groups in a 1:1 ratio, and each group will receive a different 6-month treatment. The primary outcome for the experimental groups will be the effect of PCOS treatment. DISCUSSION: The results of this trial should help to determine whether using metabolomic indices is more accurate and effective than using clinical characteristics in diagnosing the phenotypes of PCOS. The results could provide a solid foundation for the accurate diagnosis of different PCOS subgroups and for future research on individualized PCOS therapy. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ID: ChiCTR-INR-1800016346. Registered 26 May 2018.

Down-regulation of DKK1 and Wnt1/ß-catenin pathway by increased homeobox B7 resulted in cell differentiation suppression of intrauterine fetal growth retardation in human placenta.

OBJECTIVE: This study aimed to test the influence of homeobox B7 (HoxB7) on the proliferation, invasion, and migration of human trophoblast cells and to reveal the down-regulation of HoxB7 on the transcriptional suppression of Dick Kopf-related protein1 (DKK1) and of Cysteine-rich glycosylated wingless protein 1 (Wnt1)/ß-catenin in intrauterine fetal growth retardation (FGR). METHODS: Quantitative measurement of HoxB7, DKK1, Wnt1, and ß-catenin was performed in human placentas collected from normal pregnancies and from FGR with quantitative real time PCR (qRT-PCR). Cultured HTR-8/SVneo cells, transfected with a lentiviral plasmid that in-frame expresses human HoxB7 gene, were applied to functional assessment to study the biological impact of HoxB7 gene on DKK1, Wnt1, and ß-catenin. Counting Kit-8, Transwell invasion assays, and flow cytometry were applied for the functional measurements. RESULTS: The expression of HoxB7 was significantly increased, and of DKK1, Wnt1, and ß-catenin was decreased, in FGR placenta tissues and in HTR-8/SVneo cells. Function studies revealed that overexpression of HoxB7 inhibited proliferation, migration, and invasion in HTR-8/SVneo cells. DKK1, Wnt1, and ß-catenin were down-regulated in HTR-8/SVneo cells, inversely correlated with HoxB7 expression. Overexpression of HoxB7 showed a suppressive effect on proliferation, migration, and invasion in the HTR-8/SVneo cells. CONCLUSIONS: Our results indicate that HoxB7 inhibited human trophoblast cell differentiation by down-regulating DKK1 expression and that it may affect transcription of Wnt1/ß-catenin. The activation of HoxB7 might suppress the cell differentiation in HTR-8/SVneo cell cultures. The Wnt/ß-catenin signaling pathway may play a significant role in the pathogenesis of FGR by regulating the invasion and proliferation of trophoblasts.

[Effect of leptin on growth hormone secretion and apoptosis of GH3 cells].

In order to investigate the effect of leptin on the secretion of rat pituitary adenoma GH3 cell and its mechanisms, we observed the effect of leptin on the growth hormone secretion, proliferation and apoptosis of GH3 cells. The results indicated that leptin at 1, 10, and 100 nmol/L could inhibit the basal growth hormone secretion of GH3 cells in a dose dependent manner (P<0.05). Short-term treatment of leptin (10 nmol/L) for 30 min, 1 and 3 h did not affect basal GH secretion. However, treatment of the GH3 cells with leptin (10 nmol/L) for 1 d or longer resulted in an inhibition of GH secretion (P<0.05). We used MTT method and flow cytometery (FCM) to study the effect of leptin on the proliferation and apoptosis of GH3 cells. We found that leptin inhibited proliferation of GH3 cells with a dose-dependent manner. And leptin reduced the proportion of cells in S phase, increased the proportion of cells in G1, and increased the proportion of GH3 cells in 2 and 4 phase. These results demonstrate that leptin inhibits the basal GH secretion of GH3 cells, which may be due to the inhibition of DNA synthesis and advanced apoptosis of GH3 cells.

[Effect of genistein on expression and current of voltage-gated calcium channels in mouse pancreatic beta-cells].

AIM: To determine the effect of long-term inactivation of tyrosine kinases on voltage-gated calcium currents in pancreatic beta-cells and to evaluate the function of tyrosine kinases in pancreatic beta-cells. METHODS: Primarily cultured mouse pancreatic islets and beta-cells were pretreated by 0.1 mmol/L genistein for 12 hours. Voltage-gated calcium currents and action potentials were recorded with patch clamp techniques in the configuration of perforated whole-cell recording. RT-PCR method was used to evaluate the changes in expression of voltage-gated calcium channels alpha1 subunit. RESULTS: After treatment by genistein for 12 hours, the whole-cell voltage-gated calcium currents were significantly diminished (-13.83 +/- 1.515 pA/pF vs. -7.012 +/- 1.502 pA/pF, P < 0.01, n=6). The amplitudes of action potentials in genistein-treated beta-cells were also significantly attenuated (38.50 +/- 7.46 mV vs. 15.95 +/- 4.39 mV, P < 0.01, n=6). The expression of voltage-gated calcium channels alpha1 subunit in mouse islets was significantly decreased to 0.792 +/- 0.078 of that in control conditions (P < 0.01, n=5). CONCLUSION: Genistein treatment decreases expression and current of voltage-gated calcium currents in mouse pancreatic beta-cells, which suggests that inhibition of tyrosine kinases activity plays an important role in the dysfunction of pancreatic beta-cells.

Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways.

Ghrelin is an endogenous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca(2+) concentration ([Ca(2+)]i), which is determined by Ca(2+) influx and release from intracellular Ca(2+) storage sites. Ca(2+) influx is via voltage-gated Ca(2+) channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K(+) channels. The present study investigates the in vitroeffect of ghrelin on membrane voltage-gated K(+) channels in the GH3 rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K(+) currents under voltage-clamp conditions. In the presence of Co(2+) (1 mM, Ca(2+) channel blocker) and tetrodotoxin (1 microM, Na(+) channel blocker) in the bath solution, two types of voltage-gated K(+) currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K(+) current (IA) represented a significant proportion of total K(+) currents in some cells, whereas delayed rectifier K(+) current (IK) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both IA and IK currents to 48% and 64% of control, respectively. Application of apamin (1 microM, SK channel blocker) or charybdotoxin (1 microM, BK channel blocker) did not alter the K(+) current or the response to ghrelin. The ghrelin-induced reduction in K(+) currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K+ current response to ghrelin. These results suggest that ghrelin-induced reduction of voltage-gated K(+) currents in GH3 cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K(+) currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.

[3T3-L1 adipocytes reduces Kir6.2 channel expression in MIN6 insulin-secreting cells in vitro].

Dysfunction of the pancreatic beta-cell is an important defect in the pathophysiological changes of type 2 diabetes, and type 2 diabetes is evidently associated with obesity. But the role of the adipocyte in the dysfunction of the pancreatic beta-cell remains unknown. In the present study, we examined the direct effects of 3T3-L1 adipocytes on the expression of ATP-sensitive potassium channels (K(ATP) channels) in MIN6 insulin-secreting cells. MIN6 cells were divided into two groups as control group, where MIN6 cells were cultured in normal culture medium, and coculture group, where MIN6 cells were cocultured with differentiated 3T3-L1 adipocytes for 1 week. Semi-quantitative RT-PCR was employed to measure the expression of K(ATP) channel subunit Kir6.2 in MIN6 cells. Fura-2 was used to reflect changes in intracellular calcium concentration ([Ca(2+)](i)) in MIN6 cells. The secretary function of MIN6 cells from both groups was estimated by radioimmunoassay method. The results showed that the Kir6.2 cDNA levels corrected by GAPDH cDNA levels after densitometric analysis were 0.989+/-0.035 in control group and 0.726+/-0.087 in coculture group. The expression of Kir6.2 was significantly decreased in MIN6 cells in the coculture group as compared with that in control. MIN6 cells cocultured with 3T3-L1 adipocytes lost the ability to increase [Ca(2+)](i) when stimulated by tolbutamide (0.1 mmol/L), a highly selective KATP channel closer. In contrast, MIN6 cells in control group had typical responses to tolbutamide with a significant increase in [Ca(2+)](i). The magnitudes to basal levels of [Ca(2+)](i) after tolbutamide stimulation were 1.520+/-0.203 in control and 1.114+/-0.097 in coculture group (P<0.05, n=6). MIN6 cells in control showed a significant increase in insulin secretion from 0.38+/-0.099 mU/min to 2.87+/-0.248 mU/min after being stimulated by tolbutamide, whereas MIN6 cells in coculture group did not increase insulin secretion when stimulated by tolbutamide (0.21+/-0.055 mU/min to 0.22+/-0.082 mU/min). It is demonstrated that 3T3-L1 adipocytes decrease the expression of K(ATP) channels in MIN6 cells through secreting certain factors, which impair the secretary function of MIN6 cells. The present results indicate that adipocytes are directly involved in pancreatic beta-cell dysfunction, which may facilitate the development of type 2 diabetes.

[Vasorelaxing effect of idoxifene on human internal mammary arteries].

The purpose of this study was to investigate the vasorelaxing effect and mechanism of idoxifene (a new estrogen receptor modulator) on human internal mammary artery (HIMA). HIMA segments were harvested from men during coronary artery bypass grafting surgery. Patients with diabetes mellitus, hypercholesterolemia, hypertension, or smoking habit were excluded. The vasorelaxing effect of idoxifene on artery rings from HIMA with and without endothelium was measured by means of perfusion in vitro. Cumulative dose-response to idoxifene in the range of 0.01-10 micromol/L was observed in the presence and absence of NO synthase inhibitor L-NAME. It was also studied whether the vasodilation effect of idoxifene on HIMA was blocked by methylene blue (MB), an inhibitor of guanylate cyclase (GC). The results obtained from idoxifene were compared with those from 17beta-estradiol (E(2)). It was found that idoxifene caused a concentration-dependent relaxation on HIMA. The dose range was from 0.03 micromol/L (minimal vasodilatory concentration) to 3 mmol/L (maximal vasodilatory concentration). It was also found that the vasorelaxation effect of idoxifene on HIMA was dependent on endothelium. E(2) (0.1-100 micromol/L) also resulted in an endothelium-dependent vasorelaxation, but the vessels were 15-fold less sensitive to E(2) than to idoxifene in their vasorelaxation responses. The EC(50) for E(2) was 4.65+/-0.34 micromol/L, compared with 0.32+/-0.02 micromol/L for idoxifene. The mean maximal vasodilatory value of E(2) was 88.3+/-5.7%, compared with 88.6+/-7.2% for idoxifene. Pretreatment with L-NAME (100micromol/L) abolished idoxifene-induced vasodilation virtually by blocking nitric oxide production. The vasorelaxing effect of idoxifene disappeared in the presence of MB (10 micromol/L). These findings demonstrate that idoxifene results in an endothelium-dependent vasorelaxation of HIMA, like estrogen. The effect of idoxifene is more potent than that of traditional estrogen, and is possibly mediated by NO-GC-cGMP pathway.

[Three subtypes expressions of leptin receptor in the rat anterior pituitary and influence of leptin on intracellular free Ca2+ of the rat growth hormone cell].

AIM: To observe the expression of Leptin receptors (OB-R) in male rat anterior pituitary, and study the influence of Leptin on the level of intracellular free Ca2+ ([Ca2+]i) in the cultured growth hormone (GH) cell of male rat pituitary. METHODS: RT-PCR method was used to observe the expression of Leptin receptors (OB-R) in male rat anterior pituitary. We used grade centrifuging method to get growth hormone (GH) cell, and [Ca2+]i in GH cell was examined by laser scanning confocal system. RESULTS: OB-R mRNA were expressed in male rat anterior pituitary, including OB-R (common form), OB-Ra (short form) and OB-Rb (long form). There were about 70% or 80% GH cell by grade centrifuging. Leptin at 10(-8)mol/L could decrease the level intracellular free Ca2+ ([Ca2+]i) in cultured GH cell. CONCLUSION: There are three subtypes of Leptin receptors expressions in male rat anterior pituitary, and Leptin could reduce intracellular free Ca2+ level of GH cell markedly.

[Influence of ACh on the level of protein kinase C, intracellular free Ca(2+) and cyclic AMP/cyclic GMP of cultured human pituitary adenoma cells].

We found previously that ACh can significantly inhibit the proliferation of cultured human pituitary adenoma cells. In order to make a further investigation of the mechanism of the inhibitory effect of ACh on the proliferation of pituitary adenoma cells, we observed the levels of protein kinase C (PKC), [Ca(2+)](i) and cAMP/cGMP in cultured pituitary adenoma cells after treatment with ACh. The results demonstrate that (1) compared with control, PMA, a PKC activator, increased the activity of cytoplasm, membrane and total PKC in human pituitary adenoma cells. However, after a 15-min treatment with ACh (10 micromol/L), a significant reduction of the activity of cytoplasm, membrane and total PKC in human pituitary adenoma cells was observed, and the reduction effect could be blocked by atropine. (2) The level of [Ca(2+)](i) of single adenoma cells was found to decrease immediately on the addition of ACh (10 micromol/L), which could also be blocked by atropine. (3) ACh increased the amount of cAMP in the cytoplasm of human pituitary adenoma cells, but had no effect on that of cGMP. These data provide an important clue to explore the molecular mechanisms of the inhibitory effect of ACh on the proliferation of pituitary adenoma cells, and suggest that the modulating effect of ACh on the proliferation of pituitary adenoma cells results from the interactions of several cellular signaling pathways.

Kappa-opioid receptor stimulation contributes to aortic artery dilation through activation of K(ATP) channel in the rats.

To investigate the relaxation effect and underlying mechanism of U50,488H (a selective kappa-opioid receptor agonist) on aorta in the rat, isolated aortic ring was perfused and the tension of the vessel was measured. It was shown: (1) kappa-opioid receptor stimulation with U50,488H relaxed rat aorta dose-dependently; (2) the relaxation effect of U50,488H on aorta was partially endothelium-dependent; (3) the relaxation effect of U50,488H was significantly attenuated in the presence of glybenclamide and glipizide, two ATP-sensitive K(+) channel (K(ATP)) blockers; and (4) the relaxation effect of U50,488H on vessel bore no relationship to muscarinic-receptor, beta-adrenoceptor, prostaglandin and nitric oxide (NO). These results indicate that kappa-opioid receptor stimulation with U50,488H relaxes the aortic artery at least partially via K(ATP) channel in the rat.