The effects of vasopressin in isolated rat hearts

The roles of cGMP, prostaglandins, the entry of extracellular Ca2+ through slow channels, endothelium and V1 receptors in the negative inotropic, chronotropic and coronary vasoconstrictor responses to arginine vasopressin (AVP) have been investigated in isolated perfused rat hearts. The bolus injection of 5 x 10(-5) M AVP produced a significant decrease in contractile force, heart rate and coronary flow. AVP also significantly decreased contractile force, heart rate and coronary flow in hearts pretreated with an inhibitor of soluble guanylate cyclase methylene blue (10(-6) M), an effective drug for removing endothelium saponin (500 micrograms/ml), an inhibitor of cyclooxygenase indomethacin (10(-5) M) or a calcium channel antagonist verapamil (5 x 10(-7) M). The potent V1 receptor antagonist [Deamino-Pen1, Val4, D-Arg8]-vasopressin (9 x 10(-5) M) did not alter effects of AVP but the very potent V1 receptor antagonist [beta-Mercapto-beta, beta-cyclopentamethylene-propionyl1, O-Me-Tyr2, Arg8]-vasopressin (8 x 10(-5) M) abolished these effects. Our results suggest that AVP produces negative inotropic, chronotropic and coronary vasoconstrictor effects in isolated perfused rat hearts. cGMP, prostaglandin release and Ca2+ entry does not involve in the effects of AVP. These effects are endothelium independent and mediated by V1 receptors. The use of V1 receptor antagonist [beta-mercapto-beta, beta-cyclopentamethylene-propionyl1, O-Me-Tyr2, Arg8]-vasopressin may be beneficial for preventing the negative inotropy, chronotropy and coronary vasoconstriction induced by AVP.     

环孢霉素A抑制血管升压素诱导的心脏成纤维细胞增殖和胶原合成

目的探讨环孢霉素A(CsA)对血管升压素(AVP)诱导心脏成纤维细胞(CFs)增殖和胶原合成作用的影响。方法采用胰酶消化法培养新生Sprague-Dawley大鼠CFs，无血清培养基培养24 h以上的细胞在AVP刺激的基础上给予不同浓度的CsA。采用MTT法测定细胞数目，应用流式细胞仪分析细胞周期，羟基脯氨酸比色法测定细胞培养上清液羟基脯氨酸含量，钙调神经磷酸酶(CaN)活性通过分光光度计测定。结果环孢霉素A可剂量依赖性地抑制AVP诱导的心脏成纤维细胞数目的增加，0.05，0.5及5 μmol·L^-1 CsA对CFs的MTT吸收度值的抑制率分别为12%，24%和29%(均P<0.05)；细胞周期分析显示，0.5 μmol·L^-1 CsA可降低AVP诱导的S期百分数和增殖指数(P<0.05)；0.5及5 μmol·L^-1 CsA可降低细胞培养上清液羟基脯氨酸含量，抑制率分别为29%和33%，有统计学差异(均P<0.05)；CsA可完全阻断AVP诱导CFs细胞内CaN活性，而CsA对基础状态下的CaN活性和细胞存活率无明显影响。结论CsA通过阻断CaN信号通路抑制AVP诱导的CFs增殖和胶原合成，可抑制心肌纤维化进程，为心肌纤维化的防治提供了新的治疗靶点。     

Effects of arginine vasopressin on growth of rat cardiac fibroblasts: role of V1 receptor

The abnormal proliferation of cardiac fibroblasts is involved in the pathophysiologic process of left ventricular hypertrophy (LHV) associated with essential hypertension. Arginine vasopressin (AVP) has been reported to contribute significantly to the pathogenesis of hypertension. In this study, the authors investigated the effects of AVP and its V1 receptor antagonist [d(CH2)5Tyr2(Me)]AVP on the growth of rat cardiac fibroblasts. Cardiac fibroblasts of neonatal Sprague-Dawley rats were isolated, and growth-arrested cardiac fibroblasts were stimulated with 2.5% fetal calf serum in the presence and absence of AVP (0.001, 0.01, 0.1, and 1 microM) and [d(CH2)5Tyr2(Me)]AVP (0.1 microM). DNA synthesis was measured by [3H]thymidine incorporation. Thiazolyl blue assay and flow cytometry techniques were adopted to measure cell numbers and analyze cell cycle, respectively. Arginine vasopressin (0.1 and 1 microM) significantly increased DNA synthesis in cardiac fibroblasts. Moreover, AVP (0.1 and 1 microM) significantly increased the number of cardiac fibroblasts. Analysis of cell cycle showed that AVP (0.1 microM) increased S-stage percentage and proliferation index (PI). The V1 receptor antagonist [d(CH2)5Tyr2(Me)]AVP (0.1 microM) significantly inhibited DNA synthesis in cardiac fibroblasts. The cell number, S-stage percentage, and PI induced by AVP (0.1 microM) were significantly decreased by [d(CH2)5Tyr2(Me)]AVP (0.1 microM). These findings suggest that AVP might promote the proliferation of rat cardiac fibroblasts, which seems to be mediated via the V1 receptor. Arginine vasopressin may be involved in the pathophysiologic process of LVH by promoting cardiac fibroblast proliferation.     

Involvement of adenosine receptor, potassium channel and protein kinase C in hypoxic preconditioning of isolated cardiomyocytes of adult rat.

A possible mechanism for hypoxic preconditioning of adult rat cardiomyocytes was pharmacologically investigated. Isolated cardiomyocytes in all experimental groups were incubated for 120 min under hypoxic conditions followed by 15-min reoxygenation (sustained H/R). Sustained H/R decreased rod-shaped cells. Exposure of the cardiomyocytes to 20-min of hypoxia/30-min reoxygenation (hypoxic preconditioning) attenuated the sustained H/R-induced decrease in rod-shaped cells. The effects of hypoxic preconditioning were abolished by treatment with the protein kinase C (PKC) inhibitor polymyxin B, but abolished by neither the adenosine A1/A2-antagonist sulfophenyl theophylline (SPT) nor the ATP-sensitive potassium channel (K(ATP) channel) blocker glibenclamide. In another series of experiments, cardiomyocytes were incubated without hypoxic preconditioning in the presence of either the PKC activator PMA, adenosine or K(ATP)-channel opener nicorandil and then subjected to sustained H/R. Treatment of the cells with PMA, adenosine or nicorandil mimicked the effects of hypoxic preconditioning. The effects of treatment with adenosine and nicorandil were abolished by polymyxin B treatment. Combined treatment with both SPT and glibenclamide abolished the effects of hypoxic preconditioning, whereas it failed to abolish PMA-induced cytoprotection. These results suggest that the activation of PKC in hypoxic preconditioned cardiomyocytes coupled independently with stimulation of adenosine receptor or opening of K(ATP) channel, either of which is fully enough to exert the cytoprotective effects.     

Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27-dependent G1/S cell cycle arrest in conjunction with NF-κB activation

2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21^Cip1/Waf1 and up-regulating p16^INK4A. Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27^Kip1. Bcl-2 and p27^Kip1 were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-κB activity and p50 levels were increased by 2-ME2 and suppression of NF-κB signaling reduced p27^Kip1 expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27^Kip1 in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27^Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.     

Effect of YM218, a nonpeptide vasopressin V(1A) receptor-selective antagonist, on rat mesangial cell hyperplasia and hypertrophy

Mesangial cell growth constitutes a key feature of progressive glomerular injury. Vasopressin (AVP), a potent peptide vasoconstrictor, acts on mesangial cells through the V(1A) receptors, inducing contraction and cell proliferation. This study examined the effects of YM218, a nonpeptide AVP V(1A) receptor-selective antagonist, on the mitogenic and hypertrophic effects of AVP in rat mesangial cells. When added to mesangial cells whose growth was arrested, AVP concentration-dependently induced hyperplasia and hypertrophy. YM218 potently prevented AVP-induced hyperplasia and hypertrophy of these cells. Furthermore, AVP stimulated endothelin (ET)-1 secretion from mesangial cells in a concentration-dependent manner and this effect was potently inhibited by YM218. ET-1 also induced hyperplasia and hypertrophy in mesangial cells and this effect was completely abolished by ET(A) receptor-selective antagonist YM598. In addition, AVP-induced hyperplasia and hypertrophy were partly inhibited by YM598. These results suggest that AVP may modulate mesangial cell growth not only by its direct action but also through the stimulation of ET-1 secretion. YM218 displays high potency in inhibiting the AVP-induced physiologic responses of mesangial cells via the V(1A) receptors and is a potent pharmacologic probe for investigating the physiologic and pathophysiologic roles of AVP in several renal diseases.     

Vasopressin-evoked [Ca2+]i responses in neonatal rat cardiomyocytes.

The presence of arginine vasopressin (AVP) V1 receptors on neonatal rat cardiomyocytes (NRCs) linked to processes capable of elevating intracellular free calcium ([Ca2+]i) is now firmly established. This study examined the sources and signaling involved in [Ca2+]i elevations evoked by AVP in NRCs. AVP promoted increases in both [Ca2+]i and 1,4,5-inositoltrisphosphate (IP3) levels in NRCs. The degree of [Ca2+]i elevation was less than that of angiotensin II, but greater than that of endothelin-1. Extracellular Mg2+ depletion led to diminution of the maximal [Ca2+]i response, with a rightward shift in the concentration-response curves to AVP. The phospholipase C inhibitors, D-609, NCDC, or U73122, and the IP3 receptor blocker, heparin, abolished the [Ca2+]i response to AVP. Neither cyclooxygenase inhibition with indomethacin nor PKC inhibition with staurosporine had any effect. Neither ryanodine nor caffeine, which deplete sarcoplasmic reticulum (SR) Ca2+ stores, nor ruthenium red, which inhibits both SR and mitochondrial Ca2+ stores, affected [Ca2+]i responses to AVP. The SR Ca2+ pump inhibitor, cyclopiazonic acid, abolished, and removal of extracellular Ca2+ attenuated, the response to AVP. These data indicate that activation of cardiac V1 receptors by AVP results in mobilization of Ca2+ from a distinct, non-SR, nonmitochondrial, intracellular Ca2+ pool that is Ca2+ pump replenished and IP3 sensitive. This process occurs secondary to phospholipase C (PLC)-mediated generation of IP3, requires the presence of Mg2+ and extracellular Ca2+, and occurs in a manner independent of PKC and cyclooxygenase activation. Such mechanisms of Ca2+ mobilization might indicate a distinct role for AVP in cardiac physiology and disease.     

Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy.

The partial cross-utilization of molecules and pathways involved in opposing processes like cell survival, proliferation and cell death, assures that mutations within one signaling cascade will also affect the other opposite process at least to some extent, thus contributing to homeostatic regulatory circuits. This review highlights some of the connections between opposite-acting pathways. Thus, we discuss the role of cyclins in the apoptotic process, and in the regulation of cell proliferation. CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), and the Cip1/Waf1/Kip1-2-family (p21(Cip1/Waf1), p27(Kip1), p57(Kip2)) are shown both in the context of proliferation regulators and as contributors to the apoptotic machinery. Bcl2-family members (i.e. Bcl2, Bcl-X(L) Mcl-1(L); Bax, Bok/Mtd, Bak, and Bcl-X(S); Bad, Bid, Bim(EL), Bmf, Mcl-1(S)) are highlighted both for their apoptosis-regulating capacity and also for their effect on the cell cycle progression. The PI3-K/Akt cell survival pathway is shown as regulator of cell metabolism and cell survival, but examples are also provided where aberrant activity of the pathway may contribute to the induction of apoptosis. Myc/Mad/Max proteins are shown both as a powerful S-phase driving complex and as apoptosis-sensitizers. We also discuss multifunctional proteins like p53 and Rb (RBL1/p107, RBL2/p130) both in the context of G1-S transition and as apoptotic triggers. Finally, we reflect on novel therapeutic approaches that would involve redirecting over-active survival and proliferation pathways towards induction of apoptosis in cancer cells.     

曲古抑菌素A对结肠癌细胞细胞周期影响的机制研究

目的 研究组蛋白去乙酰化酶（HDAC）的抑制剂曲古抑菌素A（TSA）对结肠癌细胞株SW480细胞周期、凋亡的影响,初步探讨TSA作用细胞周期的可能机制,为HDAC抑制剂用于结肠癌治疗提供理论依据.方法 培养人结肠癌细胞系SW480,采用HDAC抑制刺TSA干预细胞,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用Western blot对细胞周期相关的基因进行检测.结果 TSA处理细胞后,流式细胞计数分析显示,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyelinE、cyclinA聚集,而对凋亡无明显的影响.Western blot显示,TSA能够上调p21wafl/Cipl、p27Kipl的表达,下调CDK2、cyclinE以及cyclinA的表达.结论 在结肠癌细胞中,TSA能够通过上调p21Wafl/Cip1、p27Kip1的表达以及下调CDK2、cyclinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长.     

Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(0)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(0)/G(1) blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(0)/G(1) and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEC-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta-dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(0)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms.     

Ameliorative and exacerbating effects of [pGlu(4),Cyt(6)]AVP((4-9)) on impairment of step-through passive avoidance task performance by group II metabotropic glutamate receptor-related drugs in mice

To examine the effect of the arginine-vasopressin fragment, [pGlu(4),Cyt(6)]AVP((4-9)) (AVP4-9), on group II metabotropic glutamate receptor (mGluR2/3) agonist and antagonist induced impairment of passive avoidance (PA) task performance, AVP4-9 or phorbol 12-myristate 13-acetate (PMA) was administered in the presence of mGluR2/3-related drugs that induced the impairment of the step-through-type PA task performance. The PA task performance was evaluated in terms of the latency (the time that elapsed prior to entry into the dark compartment) at 24 h after the electrical stimulation. The subcutaneous injection of AVP4-9 at 1 mug/kg had the greatest facilitative effect on the performance, and the facilitative effect of AVP4-9 was inhibited by NPC-15437, a specific protein kinase C (PKC) inhibitor. The injection of AVP4-9 ameliorated PA task performance impairment induced by DCG-IV, an mGluR2/3 agonist. Intracisternal injection of PMA, a PKC activator, also ameliorated the DCG-IV-induced impairment. High doses of AVP4-9 exacerbated the PA task performance impairment induced by LY341495 (an mGluR2/3 antagonist), and PMA injection (1 mug) also exacerbated the impairment induced by the antagonist. These results suggest that an increase in the activity of the PKC-signaling pathway may not always facilitate PA task performance; therefore, AVP4-9 can either enhance or inhibit memory performance in mice.     

Effect of bradykinin and PMA on the synthesis of proteoglycans during the cell cycle of endothelial cells in culture

Bradykinin (BK) and phorbol 12-myristate-13-acetate (PMA) were used in the present work to study the biosynthesis of proteoglycans (PG) during the cell cycle of endothelial cells. PMA, an activator of PKC, stimulated the synthesis of heparan sulfate proteoglycan (HSPG) secreted to the medium of endothelial cells mainly during the G(1) phase of the cell cycle [J. Cell. Biochem. 70 (1998) 563]. BK is a vasoactive peptide that increases calcium levels inside the cells indirectly stimulating PKC. Treatment of the endothelial cells with BK, as well as PMA, stimulated the synthesis of HSPG secreted to the medium and produced an antimitogenic effect on the cell cycle. These results led to the conclusion that PKC is directly involved in the synthesis of HSPG secreted to the medium. Also, comparing the effect showed by BK with PMA, one may suggest that different PKC isoforms are involved in these two processes and that their isoforms are mainly Ca(2+) dependent.     

JM91, a newly synthesized indoledione derivative, inhibits rat aortic vascular smooth muscle cells proliferation and cell cycle progression through inhibition of ERK1/2 and Akt activations

The increased potential for growth of vascular smooth muscle cells (VSMCs) is a key abnormality in the development of atherosclerosis and postangioplasty restenosis. Platelet-derived growth factor (PDGF)-BB is a potent mitogen for VSMCs that plays an important role in the intimal accumulation of VSMCs. This study examined the effect of JM91, a newly synthesized indoledione derivative, on the proliferation of PDGF-BB-stimulated rat aortic VSMCs. The antiproliferative effect of JM91 on rat aortic VSMCs was examined by cell counting and [(3)H]thymidine incorporation assay. The pre-incubation of JM91 (0.5-3.0 microM) significantly inhibited the proliferation and DNA synthesis of 25 ng/mL PDGF-BB-stimulated rat aortic VSMCs in a concentration-dependent manner. JM91 inhibited the PDGF-BB-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt kinase, while had no effect on PLCgamma1 and PDGF-Rbeta activation. In addition, treatment with JM91 (0.5-3.0 microM) induced cell-cycle arrest in the G(1) phase, which was associated with the down-regulation of cyclins and CDKs. These findings suggest that the inhibitory effects of JM91 against proliferation, DNA synthesis and cell cycle progression of PDGF-BB-stimulated rat aortic VSMCs are mediated by the suppression of the ERK1/2 and PI3K/Akt signaling pathways. Furthermore, JM91 may be a potential antiproliferative agent for the treatment of atherosclerosis and angioplasty restenosis.     

Activation of protein kinase C selectively inhibits the gamma-aminobutyric acidA receptor: role of desensitization.

The effects of protein kinase C (PKC) activators on gamma-aminobutyric acidA (GABAA) receptor function were studied by two-electrode voltage-clamp in Xenopus oocytes expressing brain mRNA or subunit cDNAs and in isolated mouse brain cerebellar membrane vesicles (microsacs), using 36Cl- uptake. Both oocytes and microsacs showed transient (desensitizing) and sustained (nondesensitizing) GABAA receptor responses. In oocytes expressing brain mRNA, the PKC activator phorbol myristoyl acetate (PMA), but not the inactive analog phorbol 12-monomyristate, inhibited both transient and sustained GABA-gated chloride currents. The inhibition by PMA was concentration dependent, with an EC50 of approximately 5 nM, and resulted in a decrease in the efficacy, but not the potency, of GABA. Additionally, PMA inhibited GABA-gated chloride currents in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. The effect of PMA on recombinant receptors was significantly antagonized by PKC inhibitory peptide (PKCI). In the microsac preparation, the PKC activators (-)-7-octylindolactam V and PMA inhibited the sustained phase of 36Cl- flux without altering the transient phase. The action of PMA was blocked by kinase inhibitors and by depletion of Mg-ATP and was mimicked by protein phosphatase inhibitors. These results demonstrate that activation of PKC inhibits GABAA receptor function, and the results from the microsac experiments suggest that PKC-dependent phosphorylation preferentially inactivates a nondesensitized form or state of the receptor.     

精氨酸升压素(4-8)对原代培养星状胶质细胞及胚胎神经元细胞中MAPK活性的影响(英文)

目的:研究精氨酸升压素(4-8)(AVP_(4-8))对来自大鼠大脑皮层和海马的原代培养星状胶质细胞及胚胎神经元细胞中促细胞分裂素活化的蛋白激酶(MAPK)活性变化的影响.同时探讨了这种影响经由的信号传递途径中可能牵涉的蛋白激酶.方法:来自大鼠脑的原代细胞培养于无血清培养基(胚胎神经元细胞)或于给药前无血清饥饿24 h,转入加药物的无钙镁磷酸缓冲液中温育,测定MAPK激酶活性.结果:(1)AVP_(4-8)能刺激原代培养的星状胶质细胞中MAPK活性但对胚胎神经元中MAPK活性没有影响.AVP_(4-8)在星状胶质细胞中诱导的MAPK活性增强作用可被AVP_(4-8)的拮抗剂ZDC(c)PR阻断.(2)MEK(MAPK/ERK激酶)的选择性抑制剂PD98059以及PKC专一性抑制剂GF109203X能消除星状胶质细胞中AVP_(4-8)诱导的活性增强作用.结论:AVP_(4-8)能以受体特异的方式激活星状胶质细胞中的MAPK活性,MEK和PKC这两个激酶参与了这个MAPK信号传递途径.AVP_(4-8)不影响原代培养胚胎神经元中的MAPK活性.     

Differential roles of p21(Waf1) and p27(Kip1) in modulating chemosensitivity and their possible application in drug discovery studies

In this study, the differential role of the cyclin-dependent kinase (CDK) inhibitors p21(Waf1) and p27(Kip1) in cell cycle regulation was proposed for use in screening natural or synthetic compounds for cell cycle-dependent (particularly M phase-dependent) antineoplastic activity. p21(Waf1) or p27(Kip1) was ectopically expressed with an ecdysone-inducible mammalian expression system in a human colon adenocarcinoma cell line. Induction of p21(Waf1) or p27(Kip1) expression inhibited the activities of CDK2 and completely arrested cells at G(1) phase of the cell cycle by p27(Kip1) and at G(1) and G(2) phases by p21(Waf1). We examined the sensitivity of these cells to several antineoplastic agents known to be cell cycle-dependent or -independent. Substantially increased resistance to cell cycle-dependent antineoplastic agents was found in the cells when the expression of p21(Waf1) or p27(Kip1) was induced. In contrast, only a desensitization to cell cycle-independent antineoplastic agents was found in the cells arrested by p21(Waf1) or p27(Kip1). Because p21(Waf1) induces an additional block at G(2) phase that inhibits cell entry into M phase, we further examined the difference between p21(Waf1)- and p27(Kip1)-induced cells in their sensitivity to D-24851, a novel M phase-dependent compound. We found that induction of p21(Waf1) after exposure of the cells to D-24851 conferred stronger resistance than did induction of p27(Kip1). Taken together, our results suggest that the differential effect of p21(Waf1) and p27(Kip1) on cell cycle regulation may be advantageous for screening chemical libraries for novel antineoplastic candidates that are cell cycle-dependent, and M phase-dependent in particular.     

Downregulation of vasopressin V(1A) receptors and activation of mitogen-activated protein kinase in rat mesangial cells cultured under high-glucose conditions

SUMMARY: In the present study we examined the effects of high extracellular glucose concentrations on vasopressin (AVP) V(1A) receptor kinetics and signal transduction in cultured rat mesangial cells. Scatchard analysis of [(3) H]-AVP binding to mesangial cell plasma membranes showed that although high glucose (30?mmol/L) decreased V(1A) receptor numbers relative to cells cultured in normal glucose (10?mmol/L), receptor affinity was not affected. This V(1A) receptor downregulation was associated with an attenuated increase in AVP-stimulated cytosolic free calcium concentrations ([Ca(2+) ](i) ). In addition, high glucose increased both the basal and AVP-stimulated activity of the classic mitogen-activated protein kinase, namely extracellular signal-regulated kinase (ERK). Furthermore, high glucose induced activation of protein kinase C (PKC) in mesangial cells that could be inhibited by coincubation with the PKC inhibitor staurosporine (10?nmol/L). Staurosporine also markedly attenuated the high glucose-induced downregulation of V(1A) receptors on mesangial cells and blocked the depressed [Ca(2+) ](i) response and increased ERK activity induced by AVP. The results indicate that high extracellular glucose downregulates V(1A) receptors on rat mesangial cells and modulates their signal transduction properties via PKC activation.     

Binding and signal transduction characteristics of the nonpeptide vasopressin V1A receptor-selective antagonist YM218 in cultured rat mesangial cells

Vasopressin (AVP) causes mesangial cell contraction, proliferation and hypertrophy. The present study investigated the effects of YM218, a potent, nonpeptide AVP V(1A) receptor-selective antagonist, on rat mesangial cells using binding, signal transduction and cell growth assays. Specific binding of (3)H-AVP to rat mesangial cell plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with the expected V(1A) receptor profile. YM218 showed high affinity for V(1A) receptors, exhibiting a K(i) value of 0.19 nmol/l. AVP concentration-dependently increased intracellular Ca(2+) ([Ca(2+)](i)) levels, stimulated mitogen-activated protein (MAP) kinase and induced hyperplasia. Conversely, YM218 potently suppressed [Ca(2+)](i) elevation, activation of MAP kinase and hyperplasia induced by AVP. These results indicate that YM218 displays both high affinity for rat mesangial cell V(1A) receptors and high potency in inhibiting AVP-induced signal transduction and growth response. Therefore, YM218 is a useful pharmacologic tool for investigating the physiologic and pathophysiologic roles of AVP in kidney, and may have clinical application in the prevention or regression of mesangial cell growth.     

Neurohypophyseal hormone receptors in the septum are implicated in social recognition in the rat

The effects on social recognition memory of (Arg(8))-vasopressin (AVP-(1-9), [pGlu(4), Cyt(6)]AVP-(4-8) and oxytocin locally administered into the rat's septum were investigated. In the behavioural paradigm used, a juvenile intruder was presented to an adult resident male rat twice for 5 min, with an inter-exposure interval of 120 min. Peptide-free residents investigated the juvenile during the second encounter as long as during the first encounter. Intraseptal injection just after the first encounter with graded doses of (Arg(8))-vasopressin, [pGlu(4),Cyt(6)]AVP-(4-8) or oxytocin caused a decrease of social investigation, as compared to placebo treatment, indicating facilitation of social recognition. The least effective dose was 100pg, 200pg and 300pg respectively. The action of vasopressin was blocked by both d(CH(2))(5)[Tyr(Me)(2)]AVP and d(CH(2))(5)[D-Ile(2)Ile(4)]AVP, V(1) and V(2) vasopressinergic receptor antagonists, but not by desGly(NH(2))(9)-d(CH(2))(5)[Tyr(Me)(2)Thr(4)]-OVT, an oxytocinergic receptor antagonist. None of the antagonists blocked the oxytocin-facilitating action on social recognition. They also did not affect social recognition when injected alone. The effects of vasopressin seem to be mediated by vasopressinergic receptors dissimilar to those found in the periphery, while the receptors involved in the action of oxytocin remain to be elucidated.