Effect of genistein on expression and current of voltage-gated calcium channels in mouse pancreatic beta-cells / 中国应用生理学杂志

<p><b>AIM</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Effect of leptin on growth hormone secretion and apoptosis of GH3 cells / 生理学报

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.

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 / 中国应用生理学杂志

<p><b>AIM</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Vasorelaxing effect of idoxifene on human internal mammary arteries / 生理学报

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.

3T3-L1 adipocytes reduces Kir6.2 channel expression in MIN6 insulin-secreting cells in vitro / 生理学报

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.

Expression of estrogen receptors alpha and beta in human tongue squamous cancer cell and influence of beta-estradiol on the proliferation of tongue cancer cell / 中国应用生理学杂志

<p><b>AIM</b>To observe the expression of estrogen receptors alpha and beta in human tongue squamous cancer line Tca8113 cell, and to study the influence of beta-estradiol (beta-E2) on the proliferation and cell cycle of cultured Tca8113 cell.</p><p><b>METHODS</b>Immunocytochemistry and RT-PCR methods were used to observe the expression of estrogen receptors (ER) in human tongue squamous carcinoma line Tca8113 cell. 3H-TdR incorporation and cell cycle analysis were used to examine the change of proliferation and DNA synthesis of Tca8113 cell.</p><p><b>RESULTS</b>ER-alpha and ER-beta mRNA were expressed in human tongue squamous cancer cell, and the expression of ER-beta was weaker than that of ER-alpha. beta-Estradiol at 10(-8) mol/L - 10(-6) mol/L could increase the proliferation of human tongue squamous carcinoma cell in a dose dependent manner (P < 0.01). beta-E2 (10(-6) mol/L) could increase the proportion of cells in S phase and G2 phase from 23.5% up to 37.7%. The effect of estradiol on the proliferation of cultured human tongue squamous cancer line Tca8113 cell could be inhibited by Tamoxifen.</p><p><b>CONCLUSION</b>There are ER-alpha and ER-beta expression in human tongue squamous cancer line Tca8113 cell, and beta-estradiol promotes the proliferation and cell cycle of cultured human Tca8113 cell.</p>

Effect of tamoxifen on proliferation of cultured breast cancer and cervical carcinoma cell lines / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effects of tamoxifen on proliferation of human breast cancer Bcap-37 cells and cervical carcinoma HeLa cells and to explore it's possible mechanism.</p><p><b>METHODS</b>The techniques of cell culture, growth curves, flow cytometry and laser scanning confocal microscope were used.</p><p><b>RESULTS</b>Tamoxifen (10(-6) mol/L) shifted the growth curve of Bcap-37 cells downward, and shifted the growth curve of HeLa cells upward. Tamoxifen (10(-8) - 10(-6) mol/L) inhibited the proliferation of Bcap-37 cells in a dose-dependent manner, but stimulated the proliferation of HeLa cells in a dose-dependent manner. Bcap-37 cells appeared apoptosis when treated with tamoxifen (10(-6) mol/L), and the same dose stimulated the proliferation of HeLa cells at GI/S phases. The apoptotic rate of Bcap-37 cells was 97.5%. It blocked G1 phase of HeLa cells from 55.5% to 32.8%, and increased the S phase from 29.0% to 49.4%. Tamoxifen (10(-6) mol/L) also increased the releasing of calcium in Bcap-37 and HeLa cells.</p><p><b>CONCLUSION</b>Tamoxifen can significantly influence the proliferation of breast cancer and cervical carcinoma cells possibly by affecting cell cycle and stimulating the releasing of Ca2+ in the cells.</p>

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

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.

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 / 生理学报

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.

Expression of interleukin-2 receptors on breast cancer cell line MCF-7 cells / 中国应用生理学杂志

<p><b>AIM</b>To investigate the expression of interleukin-2 receptors (IL-2Rs) on MCF-7 cells, estradiol's regulation of IL-2Rs expression and the influence of IL-2 on the proliferation of MCF-7 cells.</p><p><b>METHODS</b>Immunocytochemistry and flow cytometric analysis were used to investigate the expression of interleukin-2 receptors (IL-2Rs) by using of specific IL-2R polyclonal antibody; MTT method and 3H-TdR incorporation method were used to examine the changes of proliferation of MCF-7 cells.</p><p><b>RESULTS</b>IL-2Ralpha, beta, gamma like immunoreactive substances can be found on MCF-7 cells and the IL-2Rgamma immunostaining was more strong than the other two. Estradiol of 10(-6) mol/L can increase the percentage of immunoreactive cells of IL-2Ralpha, beta and the expression of IL-2Rgamma. Exogenous addition of recombinant IL-2 of 100 U/ml to 1 000 U/ml can significantly increase the proliferation of MCF-7 cells.</p><p><b>CONCLUSION</b>MCF-7 cell can express IL-2R and estradiol can regulate their expression, IL-2 can influence the proliferation of MCF-7 cells.</p>

The effect of acetylcholine on the proliferation and apoptosis of three kinds of cultured human pituitary adenoma cells / 生理学报

In order to elucidate the effect of acetylcholine (ACh) on the occurrence and development of human pituitary adenoma, it was firstly observed whether there exists choline acetyl transferase (ChAT) which is necessary for the synthesis of acetylcholine in the cells of human pituitary adenoma, and then MTT method, (3)H TdR incorporation, cell cycle analysis and TUNEL were employed to estimate the influence of ACh on the proliferation, DNA synthesis and apoptosis of three kinds of human pituitary adenoma (human prolactinoma, somatotropinoma and non-functional tumor) cells cultured in vitro. The results showed that (1) the positive staining of ChAT was obviously observed in the cells of the three kinds of human pituitary adenoma, however, it was lower than that in normal human pituitary gland; (2) ACh had a similar effect on the proliferation of the three kinds of human pituitary adenoma cells. ACh at 0.1-10 micromol/L decreased the (3)H TdR incorporation and the MTT A value in a dose-dependent manner. At the same time, ACh decreased the ratio of S or G(2) phase pituitary adenoma cells significantly, but increased the ratio of G(1) phase pituitary tumour cells markedly; (3) the effect of acetylcholine on the proliferation of human pituitary adenoma cells was inhibited by atropine, but not by tubocurarine; (4) ACh had no effect on the apoptosis of human pituitary adenoma cells cultured in vitro. These data suggest that ACh may have a significant modulating effect on the proliferation of pituitary adenoma cells by means of paracrine or autocrine, and the effect is mediated by muscarinic receptor.