Dual regulation of beta-melanotropin receptor function and adenylate cyclase by calcium and guanosine nucleotides in the M2R melanoma cell line

Binding of beta-melanotropin (beta-MSH) and subsequent activation of adenylate cyclase in the M2R mouse melanoma cell line is strongly dependent on the concentration of extracellular free calcium. This effect can be demonstrated both in the intact cell and in a plasma membrane preparation derived therefrom, using an EGTA buffer system. In contrast, stimulation of adenylate cyclase by prostaglandin E1, forskolin, or guanosine 5'-O-(2-thiotriphosphate) is calcium insensitive. It is shown that calcium increases the binding affinity of beta-MSH for its receptor by a factor of 20 (from 400 nM to 20 nM) without affecting maximal hormone binding. At supersaturating concentrations of beta-MSH (greater than 200 nM) binding gradually becomes calcium independent. Hormone-receptor complexes formed in the presence of calcium dissociated rapidly (less than or equal to 2 min) and reversibly upon the elimination of calcium by excess EGTA. Among nine divalent metal cations tested, calcium was found to be the most effective in facilitating hormone binding. Whereas calcium promotes beta-MSH binding, GTP and its stable analogs lead to a reduction in both maximal binding (65%) and affinity (2-fold). These effects are calcium independent, suggesting that the reciprocal control of beta-MSH binding by calcium and guanosine nucleotides is mediated by two separate and independent mechanisms.     

Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of KATP channels

Background

The most important receptor for nitic oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.

Results

We developed a photoaffinity label (³H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of ³H-meta-PAL together with the highly purified sGC leads to a covalent binding to the α₁-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the ³H-meta-PAL labeled sGC was fragmented by CNBr digest. The ³H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236–290 of the α₁-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the ³H-meta-PAL.

Conclusions

Our data demonstrate that the region surrounding the cysteines 238 and 243 in the α₁-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.     

Phorbol ester-induced contractility and Ca2+ influx in human cultured prostatic stromal cells

In this study, we investigated the effects of protein kinase C (PKC)-activating phorbol esters upon Ca(2+) influx and contractility in human cultured prostatic stromal cells. Tissue obtained from patients undergoing transurethral resection of the prostate was used to generate explant cultures of prostatic stromal cells. These cells expressed detectable levels of PKCalpha, delta, gamma, lambda, and zeta, but not epsilon, iota, mu, or theta; isoforms and responded to both phorbol 12,13-diacetate (PDA) and 12-deoxyphorbol 13-tetradecanoate (DPT) with concentration-dependent contractions (pEC50+/-SEM 7.07+/-0.41 and 6.39+/-0.27, respectively). The L-type Ca2+ channel blocker nifedipine (3 microM), and the PKC inhibitors G? 6976, G? 6983 (both 100 nM), myristoylated PKC inhibitor 19-27 (20 microM) and bisindolylmaleimide (1 microM) all abolished PDA-stimulated (1 microM) contractions. Neither PDA nor DPT (at 1 microM) caused translocation of any PKC isoform from the cytosolic to the particulate fraction. Nifedipine (3 microM), myristoylated PKC inhibitor 19-27 (20 microM), and bisindolylmaleimide (1 microM) inhibited PDA-stimulated Ca2+ influx into FURA-2 loaded cells. This study indicates that human cultured prostatic stromal cells respond to phorbol esters with contractions that are dependent upon the influx of Ca2+ through L-type Ca2+ channels and that this effect may be independent of the translocation of PKC from cytosolic to particulate fractions.     

Inhibition of tyrosine kinase and epidermal growth factor receptor internalization by lavendustin A methyl ester in cultured A431 cells

Lavendustin A is a novel microbial secondary metabolite that strongly inhibits tyrosine kinase in vitro. But, since it was found that it did not inhibit tyrosine kinase in situ, possibly because of its poor penetration into the cells, the authors therefore synthesized a methyl ester derivative of lavendustin A. Lavendustin A methyl ester inhibited autophosphorylation and internalization of epidermal growth factor receptor in cultured A431 cells. It also inhibited phosphatidylinositol kinase in vitro and phosphatidylinositol turnover in situ.     

P2 receptor modulation and cytotoxic function in cultured CNS neurons

In this study we investigate the presence, modulation and biological function of P2 receptors and extracellular ATP in cultured cerebellar granule neurons. As we demonstrate by RT-PCR and western blotting, both P2X and P2Y receptor subtypes are expressed and furthermore regulated as a function of neuronal maturation. In early primary cultures, mRNA for most of the P2 receptor subtypes, except P2X(6), are found, while in older cultures only P2X(3), P2Y(1) and P2Y(6) mRNA persist. In contrast, P2 receptor proteins are more prominent in mature neurons, with the exception of P2Y(1). We also report that extracellular ATP acts as a cell death mediator for fully differentiated and mature granule neurons, for dissociated striatal primary cells and hippocampal organotypic cultures, inducing both apoptotic and necrotic features of degeneration. ATP causes cell death with EC(50) in the 20-50 microM range within few minutes of exposure and with a time lapse of at most two hours. Additional agonists for P2 receptors induce toxic effects, whereas selected antagonists are protective. Cellular swelling, lactic dehydrogenase release and nuclei fragmentation are among the features of ATP-evoked cell death, which also include direct P2 receptor modulation. Comparably to P2 receptor antagonists previously shown preventing glutamate-toxicity, here we report that competitive and non-competitive NMDA receptor antagonists inhibit the detrimental consequences of extracellular ATP.Due to the massive extracellular release of purine nucleotides and nucleosides often occurring during a toxic insult, our data indicate that extracellular ATP can now be included among the potential causes of CNS neurodegenerative events.     

Structure-activity relationships of new analogues of arecaidine propargyl ester at muscarinic M1 and M2 receptor subtypes.

1. The potency of arecaidine propargyl ester (APE) and of several analogues containing a modified ester side chain has been assessed at M1 and M2 muscarinic receptor subtypes. APE was shown to act as a potent agonist at ganglionic M1 receptors in the pithed rat, at M2 receptors in guinea-pig isolated atria (-log EC50 = 8.22) and ileum (-log EC50 = 7.77). 2. The arecaidine 2-butynyl and 2-pentynyl esters were approximately equipotent with APE at M1 and M2 receptors, whereas the 2-hexynyl derivative was found to be less potent than APE in atria (-log EC50 = 6.80) and ileum (-log EC50 = 6.70) by about one order of magnitude. The 2-heptynyl and 3-phenyl propargyl esters exhibited no agonist actions in atria and ileum. 3. Shifting the triple bond from the 2 to the 3 position and introducing a bulky group at position 1 of the ester side chain of APE and analogues resulted in competitive antagonists (pA2 ranging from 4.9 to 7.3). 4. APE and its 2-butynyl analogue showed some agonistic selectivity for cardiac M2 receptors (potency ratio, ileum/atria = 2.8 and 4.6 respectively). All antagonists in this series of compounds were not selective in terms of affinity since their pA2 values at cardiac and ileal M2 receptors were similar (potency ratios, ileum/atria = 0.4 to 1.2).     

Heterologous regulation of EGF receptor function in cultured aortic smooth muscle cells

A10 cultured smooth muscle cells from rat embryonic thoracic aorta bound ¹²⁵I-labelled epidermal growth factor (¹²⁵I-EGF), and responded to EGF by an increase in DNA synthesis. Scatchard analysis of binding data obtained at 4° C showed curvilinearity consistent with there being two affinity classes of binding site. The amount of ¹²⁵I-EGF that bound was decreased by treatment of the A10 cells at 37° C with [Arg⁸]vasopressin or with 5-hydroxytryptamine (5-HT). Scatchard analysis of binding (at 4° C after pretreatment at 37° C) revealed this effect to be due to a loss of the high-affinity component of ¹²⁵I-EGF binding, with no change in total receptor number. The presence of vasopressin or 5-HT raised the concentration of EGF required for the stimulation of DNA synthesis. Cultured A10 aortic smooth muscle cells therefore have receptors for EGF that mediate an increase in cell proliferation. EGF receptor function is modified by vasopressin and 5-HT, probably as a consequence of their effects on EGF receptor affinity.     

Angiotensin stimulates Ca2(+)-dependent action potentials in cultured smooth muscle cells

The steady-state angiotensin II response was measured in primary cultures of reaggregated vascular smooth muscle cells derived from rat aorta by use of intracellular microelectrode recording of membrane potentials. Angiotensin II (10(-9)-10(-6) M) produced a depolarization which triggered a single action potential, consisting of a spike plus plateau. In addition, angiotensin II prolonged the action potential plateau and lowered input resistance. The angiotensin II-induced action potentials and the action potential plateau prolongation were inhibited by verapamil. Saralasin blocked the occurrence of angiotensin II-induced action potentials and reversed the increase in action potential duration provoked by angiotensin II. Saralasin, in the absence of angiotensin II, exhibited agonistic activity which was manifest by plateau prolongation. Therefore, angiotensin II, through interaction of the peptide with its receptor, depolarizes cultured vascular smooth muscle cells and prolongs the calcium-dependent action potentials. These effects could be mediated by the known ability of angiotensin II to stimulate production of inositol trisphosphate and diacylglycerol, and activation of protein kinase C.     

星形胶质细胞毒蕈碱样乙酰胆碱受体M2、M4亚型基因克隆及序列分析

目的：克隆胶质细胞M_2、M_4受体亚型基因序列，并比较胶质细胞M_2、M_4受体亚型基因序列和蛋白质序列与神经元细胞M_2、M_4受体基因序列和蛋白质序列间的差异。方法：根据神经元细胞M_2、M_4受体基因序列设计出针对M_2、M_4受体基因序列全长的特异性探针，采用RT-PCR方法扩增胶质细胞M_2、M_4受体亚型基因序列，并对其进行克隆测序。结果：通过RT-PCR方法扩增胶质细胞M_2、M_4受体亚型基因序列，与神经元细胞M_2、M_4受体比较，M_2受体差异碱基17个，发生氨基酸改变的有8个；M_4受体差异碱基3个，发生氨基酸改变的有2个。结论：胶质细胞M_2、M_4受体与神经细胞M_2、M_4受体亚型在基因序列和氨基酸序列上具有明显差异。     

地黄活性成分梓醇对转基因CHO细胞M_2受体的调节作用

目的探讨地黄活性成分梓醇对转染M2受体亚型基因的CHO细胞系(CHOm2细胞)M2受体密度作用。方法采用衰老CHOm2细胞,常规培养后加入不同浓度梓醇及对照生理盐水,放射配基结合分析测定M2受体密度,Lowry法测定蛋白含量;同时观察了梓醇与M受体结合位点的关系及对乙酰胆碱酯酶的作用。结果梓醇在终浓度10-5、10-4mol.L-1明显升高衰老CHOm2细胞M2受体密度(P<0.01),但不占领M受体结合位点,也不抑制乙酰胆碱酯酶活性(P>0.05)。结论梓醇能上调衰老CHOm2细胞M2受体密度,可能是地黄治疗AD的主要有效成分。     

Formaldehyde exposure impairs the function and differentiation of NK cells

We investigated the cytotoxic effects of formaldehyde (FA) on lymphocytes. FA-exposed mice showed a profound reduction not only in the number of natural killer (NK) cells but also in the expression of NK cell-specific receptors, but these mice did not exhibit decreases in the numbers of T or B lymphocytes. FA exposure also induced decreases in NK cytolytic activity and in the expression of NK cell-associated genes, such as IFN-γ, perforin and CD122. To determine the effect of FA on tumorigenicity, C57BL/6 mice were subcutaneously injected with B16F10 melanoma cells after FA exposure. The mass of the B16F10 tumor and the concentration of extravascular polymorphonuclear leukocytes were greater than those in unexposed tumor-bearing control mice. The number and cytolytic activity of NK cells were also reduced in B16F10 tumor-bearing mice exposed to FA. To determine how FA reduces the NK cell number, NK precursor (pNK) cells were treated with FA, and the differentiation status of the NK cells was analyzed. NK cell differentiation was impaired by FA treatment in a concentration-dependent manner. These findings indicate that FA exposure may promote tumor progression by impairing NK cell function and differentiation.     

Bradykinin stimulates production of inositol (1,4,5) trisphosphate in cultured mesangial cells of the rat via a BK2-kinin receptor.

1. Using [125I-Tyr0]-BK, as radiolabelled ligand, and various agonists and antagonists of bradykinin (BK) we identified a single class of specific BK2-binding sites in mesangial cell membranes (Bmax = 73 fmol mg-1 protein and Kd = 3.7 nM). 2. Following the addition of 0.1 microM BK, inositol (1,4,5) trisphosphate (IP3) formation increased within 20 s from a basal level of 64 to a maximal value of 175 pmol mg-1 protein. 3. Incubation in a Ca(2+)-free medium did not change IP3 production but a 5 min preincubation with 1 mM EGTA completely prevented the BK-induced IP3 formation, suggesting that IP3 formation is partly dependent on extracellular calcium. 4. The BK2 antagonist D-Arg-Hyp3-D-Phe7-BK (10 microM) but not the BK1 antagonist (des-Arg9-Leu8-BK) abolished IP3 production in response to 0.1 microM BK. Pretreatment of mesangial cells with pertussis toxin was without effect on BK-induced IP3 formation, whereas phorbol 12-myristate 13-acetate significantly enhanced (by 25%) BK-induced IP3 formation. 5. The present data demonstrate that inositol phosphate breakdown in rat mesangial cells can be mediated via activation of a BK2-kinin receptor and is under negative control of protein-kinase C.     

Trichloroethanol impairs NMDA receptor function in rat mesencephalic and cortical neurones

The effects of 2,2,2-trichloroethanol, the active compound of the sedative-hypnotic chloral hydrate, were investigated on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) in cultured mesencephalic and cortical neurones by means of the fura-2 method. Trichloroethanol inhibited the NMDA response in a concentration-dependent manner in cortical (IC50 = 2.76 mM) and mesencephalic neurones (IC50 = 1.12 mM), with a maximum effect of approximately 85 and 94%, respectively. Ethanol was considerably less potent than trichloroethanol. In conclusion, the trichloroethanol-induced impairment of NMDA receptor function may contribute to the sedative-hypnotic properties of chloral hydrate.     

Expression of D2 receptor isoforms in cultured neurons reveals equipotent autoreceptor function

Alternative splicing of the dopamine D2 receptor gene produces two distinct isoforms referred to as D2long (D2L) and D2short (D2S). In mesencephalic dopamine neurons, inhibition of the firing rate through activation of somatodendritic D2 receptors and blockade of neurotransmitter release through stimulation of terminal D2 receptors represent major roles of D2 autoreceptors. Recently, data obtained from D2L-deficient mice suggested that D2S acts as the preferential D2 autoreceptor. In the present study, we investigate whether this D2 isoform-specific autoreceptor function is linked to differences in the subcellular localization and/or signaling properties of the D2S and D2L using mesencephalic neurons transfected with enhanced green fluorescent protein (EGFP)-tagged receptors. Our results show that EGFP-tagged D2S and D2L are localized to the axonal and somatodendritic compartments of mesencephalic neurons. In addition, we demonstrate that EGFP-tagged D2S and D2L regulate cellular excitability, neurotransmitter release and basal levels of intracellular calcium with similar effectiveness. Overall, our morphological and electrophysiological studies suggest that the major D2 autoreceptor function attributed to D2S is likely explained by the predominant expression of this isoform in dopamine neurons rather than by distinct subcellular localization and signaling properties of D2S and D2L.     

Characterization of a liver metastatic variant of murine K1735M2 melanoma cells

Intraportal vein injection of highly metastatic K1735M2L5 cells consistently resulted in liver metastases (increases in the number of tumor nodules in the liver), whereas inoculation of K1735M2 cells rarely did so. K1735M2L5 cells invaded the basement membrane Matrigel in greater numbers than did K1735M2 cells, suggesting that the metastatic potential of K1735M2L5 cells is partly related to enhanced invasive properties. The adhesion to Matrigel- and laminin-coated substrates was enhanced in K1735M2L5 cells. The up-regulated expression of VLA-4 and VLA-6 on the surface of K1735M2L5 cells was detected by flow cytometry. The RT-PCR and gelatin zymography study revealed that the secretion of MMP-2 was higher in K1735M2L5 cells than in K1735M2 cells. These results indicate that the invasive ability of K1735M2L5 cells may also be attributed to enhanced gelatinolytic activity as well as adhesiveness. K1735M2L5 cells grew more rapidly than K1735M2 cells and showed fibroblastoid morphology with loose cell-cell contacts as compared with K1735M2 cells. Thus, experimental models using highly metastatic K1735M2L5 cells would be useful for analyzing the molecular mechanism of tumor metastasis and for evaluating the efficacy of treatments for metastases which may have already occurred at the time of the diagnosis.     

Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer

1. γ-Aminobutyric acid (GABA), acting via the GABA_A receptor, increased the extracellular acidification rate of rat primary cultured cerebellar granule cells, measured by the Cytosensor microphysiometer.
2. The optimal conditions for the measurement of GABA_A receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9–12×10⁵ cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25°C, with a stimulation time of 33 s.
3. GABA stimulated a concentration-dependent increase in the extracellular acidification rate with an EC₅₀ of 2.0±0.2 μM (mean±s.e.mean, n=7 experiments) and maximal increase (E_max) over basal response of 15.4±1.2%.
4. The sub-maximal GABA-stimulated increase in acidification rate could be potentiated by the 1,4-benzodiazepine, flunitrazepam (100 nM). The 10 nM GABA response showed the maximal benzodiazepine facilitation (GABA alone, 1.4 μV s⁻¹, GABA+flunitrazepam, 3.8 μV s⁻¹, mean increment over basal, n=7).
5. The GABA-stimulated increase in acidification rate was inhibited by the GABA_A antagonist, bicuculline (100 μM) (90% inhibition at 1 mM GABA).
6. The results of this study show that activation of GABA_A receptors in rat cerebellar granule cells caused an increase in the extracellular acidification rate; an effect which was potentiated by benzodiazepines and inhibited by a GABA_A receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABA_A receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABA_A receptor in populations of neurones.

     

R2细胞用于测定人白介素6受体的生物活性

目的　确定可靠的筛选小分子人白介素 6(IL 6 )受体拮抗剂先导化合物的细胞模型。方法分别用间接免疫荧光法和MTT检测法检测了R2细胞的人IL 6受体蛋白表达水平和对重组人白介素 6(rhIL 6 )的反应性。结果　检测到R2细胞具有高丰度的人IL 6受体蛋白表达并对rhIL 6具有极为灵敏的反应性和极高的亲和力 ;用R2细胞检测抗人IL 6单克隆抗体对rhIL 6的拮抗活性 ,发现单抗在 2 0mg·L- 1浓度下对rhIL 6所引起的R2细胞的增殖有很强的抑制作用 ,抑制率达 92 %。结论　R2细胞是可靠的筛选人IL 6受体拮抗剂的细胞模型     

cis-hydroxyproline stimulates the growth of rat mammary carcinoma cells

With the ever-increasing evidence that the extracellular matrix (ECM) can stimulate tumor growth, it follows that inhibiting the synthesis of tumor-derived stroma may be a potential therapeutic target of cancer progression. The proline analog cis-hydroxyproline (CHP), an inhibitor of collagen deposition, was examined for its effects on the growth of clonal tumor cells that differentially produce type IV collagen and laminin. Two separate clones derived from rat mammary carcinoma cells that produce high and low amounts of type IV collagen and laminin were injected into the flanks of nude mice. Tumors in animals receiving CHP treatment grew faster than tumors in control animals receiving saline, although statistically not significant. Furthermore, upon administration of CHP to these clones in culture, increased proliferation rates of both cell types were observed. These results show that CHP is not useful in preventing stromal development and growth of rat mammary tumor xenografts.     

Prostaglandin E2 signaling through E prostanoid receptor 2 impairs proliferative response of double negative regulatory T cells

Limited data are available on the mechanisms that constrain the function of regulatory populations of T cells. Prostaglandin E2 (PGE2) is an endogenous membrane phospholipid metabolite that has important immunomodulatory effects on T cell function. Our previous microarray data indicated that E prostanoid receptor 2 (EP2), a receptor for PGE2, is expressed by regulatory αβTCR⁺ CD4⁻ CD8⁻ NK1.1⁻ double negative T (DN Treg) cell clones but not by their non-regulatory natural mutants. Hence, the hypothesis that PGE2 may influence DN Treg cell proliferation and/or regulatory function was tested in this study. Our data indicate that PGE2 acts via the EP2 receptor on DN Treg cells to inhibit their proliferation, an effect reproduced by the EP2-specific agonist butaprost and abrogated by the EP2 antagonist AH6809. In contrast, PGE2 did not affect the ability of DN Treg cells to kill syngeneic CD8⁺ T cells activated by allogeneic stimulation. Together, these findings suggest a role for PGE2 in limiting the expansion of DN Treg cells.     

Vasopressin stimulates the production of extracellular matrix by cultured rat mesangial cells

1. Mesangial expansion, an indicator of chronic glomerular diseases, occurs as a result of the excessive accumulation of extracellular matrix (ECM) proteins, such as type IV collagen. In order to investigate the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to induce ECM production, an enzyme-linked immunosorbent assay was used to measure type I and IV collagen and fibronectin produced from cultured rat mesangial cells. 2. Addition of AVP (0.01-1000 nmol/L) caused a significant and concentration-dependent production of secreted and cell-associated ECM, type I collagen, type IV collagen and fibronectin by cultured rat mesangial cells. The AVP V(1A) receptor-selective antagonist YM218 (0.01-1000 nmol/L) potently and concentration-dependently inhibited the induced increase in ECM production caused by AVP, but the V(2) receptor-selective antagonist SR 121463A (0.1-1000 nmol/L) did not potently inhibit. 3. Vasopressin inhibited the synthesis of matrix metalloproteinase (MMP)-2, which degrades matrix proteins, including type IV collagen, and stimulated endothelin (ET)-1 secretion from mesangial cells. These effects were potently inhibited by YM218, but not by SR 121463A. 4. In addition, 10 nmol/L ET-1 inhibited the synthesis of MMP-2 and stimulated ECM production in mesangial cells. These effects were completely abolished by the ET(A) receptor-selective antagonist YM598 (1 micromol/L); however, the ET(B) receptor-selective antagonist BQ-788 (1 micromol/L) and the AVP receptor antagonists YM218 and SR 121463A did not inhibit ET-1-induced inhibition of MMP-2 synthesis and ECM production. In addition, AVP-induced inhibition of MMP-2 synthesis and ECM production were partly inhibited by YM598. 5. These findings indicate that AVP may modulate ECM production not only via a direct action on V(1A) receptors, but also through stimulation of ET-1 secretion. Vasopressin may contribute to the glomerular remodelling and ECM accumulation observed in glomerular diseases.