Anabolic-androgenic steroids: In cell culture

Summary Testosterone and related steroids at physiological concentrations positively stimulate in cell culture a number of reactions in a variety of tissues from different species of animals. Cells maintained in cell culture provide a means to study toxic effects in target organs and also the mechanism of action of these steroids.     

血管紧张素II诱导培养的成年大鼠心肌细胞凋亡

研究血管紧张素Ⅱ（ＡｎｇⅡ）诱导培养的成年大鼠心肌细胞（ＡＲＶＭｓ）凋亡。酶灌流消化法分离培养ＡＲＶＭｓ,不同处理后,光镜观察形态改变,琼脂糖凝胶电泳定性分析ＤＮＡ降解程度。结果发现培养的ＡＲＶＭｓ经ＡｎｇⅡ１０μｍｏｌ／Ｌ处理４８ｈ后,大部分细胞变圆,胞浆浓缩;电泳显示核酸断裂片段“梯形”结构,上述改变在７２ｈ更为明显。上述作用可被氯沙坦、维拉帕米和ｓｔａｕｒｏｓｐｏｒｉｎｅ所取消。这表明Ａｎｇ     

Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in -adrenergic receptor (-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). -AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed -AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited -AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with ₁-integrins after -AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of ₁-integrin signaling using laminin inhibited the increased association of MMP-2 with ₁-integrins. -AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) -AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits -AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with ₁ integrins and poly-ADP-ribose-polymerase cleavage. integrins; poly-ADP-ribose-polymerase     

Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes

Adrenomedullin (ADM) acts as an autocrine or a paracrine factor in the regulation of cardiac function. The intracellular mechanisms involved in the direct effect of ADM on adult rat ventricular myocytes (ARVMs) are still to be elucidated. In ARVMs from normal rats, ADM produced an initial (< 30 min) increase in cell shortening and Ca2+ transients and a marked decrease in both on prolonged incubation (> 1 h). Both effects were sensitive to ADM antagonist ADM-(22-52). Treatment with SQ-22536, an inhibitor of adenylate cyclase, blocked the positive inotropic effect of ADM and potentiated its negative inotropic effect. The negative inotropic effect was sensitive to inhibition by pertussis toxin (PTX), an inhibitor of Gi proteins and KT-5720, an inhibitor of PKA. The observations suggest a switch from Gs-coupled to PTX-sensitive, PKA-dependent Gi coupling by ADM in ARVMs. The ADM-mediated Gi-signaling system involves cAMP-dependent pathways because SQ-22536 further increased the negative inotropic actions of ADM. Also, because ADM is overproduced by ARVMs in our rat model of septic shock, ARVMs from LPS-treated rats were subjected to treatment with ADM-(22-52) and PTX. The decrease in cell shortening and Ca2+ transients in LPS-treated ARVMs could be reversed back with ADM-(22-52) and PTX. This indicates that ADM plays a role in mediating the negative inotropic effect in LPS-treated ARVM through the activation of Gi signaling. This study delineates the intracellular pathways involved in ADM-mediated direct inotropic effects on ARVMs and also suggests a role of ADM in sepsis.     

Stretch-activated whole cell currents in adult rat cardiac myocytes

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 microM Gd(3+) but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately -6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 - 5.0 SL (microm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.     

Structural adaptation in adult rabbit ventricular myocytes

The mechanism of induction of cardiomyocyte (CM) dedifferentiation, as seen in chronic hibernating myocardium, is largely unknown. Recently, a cellular model was proposed consisting of long-term cocultures of adult rabbit CMs and cardiac fibroblasts in which typical structural characteristics of hibernation-like dedifferentiation could be induced. Only CMs in close contact with fibroblasts underwent these changes. In this study, we further investigated the characteristics of the fibroblast-CM interaction to seek for triggers and phenomena involved in CM dedifferentiation. Adult rabbit CMs were cocultured with cardiac or 3T3 fibroblasts. Heterocellular interactions and the structural adaptation of the CMs were quantified and studied with vital microscopy and electron microscopy. Immunocytochemical analysis of several adhesion molecules, i.e., N-cadherin, vinculin, β1-integrin, and desmoplakin, were examined. Upon contact with CMs, fibroblasts attached firmly and pulled the former cells, resulting in anisotropic stretch. Quantification of the attachment sites revealed a predominant binding of the fibroblast to the distal ends of the CM in d 1 cocultures and a shift towards the lateral sides of the CMs on d 2 of coculture, suggesting a redistribution of CM membrane proteins. Immunocytochemical analysis of cell adhesion proteins showed that these were upregulated at the heterocellular contact sites. Addition of autologous and nonautologous fibroblasts to the CM culture similarly induced a progressive and accelerated structural adaptation of the CM. Dynamic passive stretch invoked by the fibroblasts and/or intercellular communication involving cell adhesion molecule expression at the interaction sites may play an important role in the induction of hibernation-like dedifferentiation of the cocultured adult rabbit CMs. These authors contributed equally to this study.     

Relatively low-dose cyclophosphamide is likely to induce apoptotic cell death in rat thymus through Fas/Fas ligand pathway.

Cyclophosphamide (CPA) is widely used as an efficiently antineoplastic drug, but also causes immunosuppression as its adverse-side effect. To understand the effect of low- or relative low-dose CPA on the immune system, apoptotic cell death in rat thymus, either exposed to different doses of CPA (0, 2, 7, 20 and 70 mg/kg) for 12 h or exposed to 70 mg/kg for different times (4-48 h), was investigated by DNA fragmentation (DNA ladder) detection and in situ morphological examination using hematoxylin and eosin (H and E) staining. Immunohistochemical staining for Fas protein expression in the thymus of rats exposed to CPA was performed. Results showed that exposure of rats to CPA 0-70 mg/kg for 12 h did not cause significant decrease in the ratio of thymus weight to body weight. However, the ratio of thymus weight to body weight was decreased significantly at 48 h after exposure to 70 mg/kg CPA. Exposure to 20 and 70 mg/kg CPA for 12 h caused a visible DNA ladder in gel electrophoresis. DNA ladder formation was increased progressively in the groups from 8 h to optimal magnitude at 12-24 h and then disappeared at 48 h after 70 mg/kg CPA. This pattern was confirmed by a quantitative evaluation of the apoptotic cells using H and E staining. Expression of Fas protein was enhanced in the thymus of rats exposed to 70 mg/kg CPA for 4-8 h as compared to control rats. These results are different from previous studies on high dose CPA and the induction of the apoptotic cell death in thymus by low or relative low doses of CPA might be a result of Fas/Fas-ligand interactions.     

Salicylic acid alters endothelin-1 binding in intact adult rat ventricular myocytes.

Endothelin receptors ET(A)R and ET(B)R form tight receptor-ligand complexes that complicate our understanding of the physiological, pharmacological, and biochemical properties of these receptors. Although radioligand-binding studies have demonstrated the binding of endothelin-1 (ET-1) to ET(A)R to be essentially irreversible, ET(A)R internalize in a ligand-dependent manner, release ET-1, and then recycle to the cell surface. Salicylic acid (SA) reduces ET-1 binding (IC(50) = 10 mmol/L) to recombinant ET(A)R in isolated membranes by promoting dissociation of [(125)I]ET-1. In the present study, SA (5 mmol SA/L) did not alter [(125I)]ET-1 binding to intact adult rat ventricular myocytes. The lack of effect was not due to internalization of receptor-ligand complexes. However, 100 mmol SA/L significantly reduced [(125)I]ET-1 binding to both intact myocytes and isolated membranes. SA induced the phosphorylation p42/44 extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and an unidentified 40-kDa protein on the activating threonine-glutamic acid-tyrosine (T-E-Y) motif. ERK phosphorylation was reduced by a MAP kinase kinase (MEK) inhibitor, PD98059. Phosphorylation of p40 was reduced by the p38 MAP kinase inhibitor SB203580, but not PD98059. However, inhibition of ERK or p38 MAP kinases did not alter the ability of 100 mmol SA/L to induce dissociation of [125I]ET-1. These results suggest that, in the ventricular myocyte, salicylic acid alters the kinetics of ET-1 binding. The results also suggest an allosteric binding site may be present that modulates the dissociation of ET-1 receptor-ligand complexes in response to an as-of-yet unidentified mediator.     

Characterization of calcium-dependent forms of protein kinase C in adult rat ventricular myocytes

The presence and subcellular localization of the Ca2+-dependent protein kinase C (PKC) isoforms and were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was measured in cytosolic and particulate fractions prepared from control myocytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-oleoyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studies detected no Ca2+-/phospholipid-dependent histone kinase activity in either subcellular fraction. To reproducibly observe Ca2+-/phospholipid-dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-dependent protein kinase were essential. MonoQ chromatography of cytosolic and particulate fractions resulted in three peaks of Ca2+/phospholipid-dependent protein kinase activity. In the cytosolic fraction a large peak of activity eluted at 230-300 mM NaCl. Isoform-specific antisera indicated both PKC and PKC were present. In the particulate fraction two peak of Ca2+-/phospholipid-dependent protein kinase activity, both containing PKCa immunoreactivity, were observed. The larger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at lower salt concentrations contained a Ca2+-/phospholipid-independent histone kinase activity. This peak of kinase activity contained PKC immunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenzyme of PKC whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, the peak of kinase activity eluting at 230-300 mM NaCl contained PKC in the form of an 80-kDa doublet; this suggested the presence of autophosphorylated PKC. Incubation of the myocytes with PMA, but not OAG, resulted in translocation of PKC from the cytosolic to the particulate fraction. Curiously, a transient decrease in PKC activity was observed in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated adult rat cardiac ventricular myocytes contain both PKC and PKC, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG. (Mol Cell Biochem 166: 11-23, 1997)     

Genistein induced apoptotic cell death in adult T-cell leukemia cells through estrogen receptors

Adult T-cell leukemia (ATL) occurs in human T-lymphotropic virus type I-infected individuals and is endemic to the southwestern area of Kyushu in Japan. Here, we found that nM levels of genistein and 17β-estradiol had cytotoxic effects on ATL cells and activated caspase-3. The estrogen receptor antagonist ICI182780 negated the cytotoxic effect of genistein. In addition, G protein-coupled estrogen receptor agonist G-1 also had a cytotoxic effect on ATL cells. This is the first report suggesting that estrogen receptors are a molecular target for ATL therapy.     

Silver nanoparticles induce apoptotic cell death in Candida albicans through the increase of hydroxyl radicals

Silver nanoparticles have been shown to be detrimental to fungal cells although the mechanism(s) of action have not been clearly established. In this study, we used Candida albicans cells to show that silver nanoparticles exert their antifungal effect through apoptosis. Many studies have shown that the accumulation of reactive oxygen species induces and regulates the induction of apoptosis. Furthermore, hydroxyl radicals are considered an important component of cell death. Therefore, we assumed that hydroxyl radicals were related to apoptosis and the effect of thiourea as a hydroxyl radical scavenger was investigated. We measured the production of reactive oxygen species and investigated whether silver nanoparticles induced the accumulation of hydroxyl radicals. A reduction in the mitochondrial membrane potential shown by flow cytometry analysis and the release of cytochrome c from mitochondria were also verified. In addition, the apoptotic effects of silver nanoparticles were detected by fluorescence microscopy using other confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, DNA and nuclear fragmentation, and the activation of metacaspases. Cells exposed to silver nanoparticles showed increased reactive oxygen species and hydroxyl radical production. All other phenomena of mitochondrial dysfunction and apoptotic features also appeared. The results indicate that silver nanoparticles possess antifungal effects with apoptotic features and we suggest that the hydroxyl radicals generated by silver nanoparticles have a significant role in mitochondrial dysfunctional apoptosis.     

Tunicamycin and brefeldin a induce in plant cells a programmed cell death showing apoptotic features

Summary The recent identification ofDAD (defender against apoptotic death) gene in plants suggests that the N-linked glycosylation of proteins could be an important control point of plant programmed cell death. In this paper we describe the effects of Tunicamycin, an inhibitor of N-linked protein glycosylation, and Brefeldin A, an inhibitor of protein trafficking from the Golgi apparatus, on sycamore (Acer pseudoplatanus L.) cell cultures. These two chemicals proved able to induce a strong acceleration of the cell death; changes in cell and nucleus morphology; an increase in DNA fragmentation, detectable by a specific immunological reaction; and the presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Moreover, Brefeldin A, but not Tunicamycin, strongly stimulated the production of hydrogen peroxide. These results indicate that also in plants chemicals interfering with the activities of endoplasmic reticulum and of Golgi apparatus strongly induce a form of programmed cell death showing apoptotic features.     

Protection of adult rat cardiac myocytes from ischemic cell death: role of caveolar microdomains and delta-opioid receptors

The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of delta-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-beta-cyclodextrin (MbetaCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MbetaCD and MbetaCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 +/- 3 vs. 59 +/- 3% dead cells in the presence and absence of 1 muM SNC, respectively, P < 0.01) or by use of the IPC protocol (35 +/- 4 vs. 62 +/- 3% dead cells, P < 0.01). MbetaCD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MbetaCD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage.     

Cell swelling impairs dye coupling in adult rat ventricular myocytes. Cell volume as a regulator of cell communication

The influence of cell swelling on cell communication was investigated in cardiomyocytes isolated from the ventricle of adult rats. Measurements of dye coupling were performed in cell pairs using intracellular dialysis of Lucifer Yellow CH. The pipette was attached to one cell of the pair and after a gig ohm seal was achieved, the membrane was ruptured by a brief suction allowing the dye to diffuse from the pipette into the cell. Fluorescence of the dye in the injected as well as in non-dialyzed cell of the pair was continuously monitored. The results indicate that in cell pairs exposed to hypotonic solution the cell volume was increased by about 60% within 35 min and the dye coupling was significantly reduced by cell swelling. Calculation of gap junction permeability (P _j) assuming an the intracellular volume accessible to intracellular diffusion of the dye as 12% of total cell volume, showed an average P _j value of 0.16 ± 0.04 × 10^?4 cm/s (n = 35) in the control and 0.89 ± 1.1 × 10^?5 cm (n = 40) for cells exposed to hypotonic solution (P < 0.05). Similar results were found assuming intracellular volumes accessible to the dye of 20 and 30% of total cell volume, respectively. Cell swelling did not change the rate of intracellular diffusion of the dye. The results which indicate that cell volume is an important regulator of gap junction permeability, have important implications to myocardial ischemia and heart failure as well as to heart pharmacology because changes in cell volume caused by drugs and transmitters can impair cell communication with consequent generation of slow conduction and cardiac arrhythmias.     

Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes

Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ET(A))/ET type B (ET(B)) receptor family. ET(A), the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ET(A) in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ET(A) protein levels. In these T tubule-compromised myocytes, an overexpression of ET(A) using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ET(A) downregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ET(A) and intact T tubules (preserved with CD). Therefore, the activation of ET(A) localized in T tubules was associated with a strong PIE, whereas the activation of ET(A) in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ET(A) is upregulated and T-tubule morphology is altered.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

TRPV1 stimulation triggers apoptotic cell death of rat cortical neurons

Transient receptor potential vanilloid 1 (TRPV1) functions as a polymodal nociceptor and is activated by several vanilloids, including capsaicin, protons and heat. Although TRPV1 channels are widely distributed in the brain, their roles remain unclear. Here, we investigated the roles of TRPV1 in cytotoxic processes using TRPV1-expressing cultured rat cortical neurons. Capsaicin induced severe neuronal death with apoptotic features, which was completely inhibited by the TRPV1 antagonist capsazepine and was dependent on extracellular Ca²⁺ influx. Interestingly, nifedipine, a specific L-type Ca²⁺ channel blocker, attenuated capsaicin cytotoxicity, even when applied 2-4 h after the capsaicin. ERK inhibitor PD98059 and several antioxidants, but not the JNK and p38 inhibitors, attenuated capsaicin cytotoxicity. Together, these data indicate that TRPV1 activation triggers apoptotic cell death of rat cortical cultures via L-type Ca²⁺ channel opening, Ca²⁺ influx, ERK phosphorylation, and reactive oxygen species production.