Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]_e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca²⁺ influx may mediate this effect. We show that increase in [Os]_e (by 18–125%) differentially suppresses L-type and T-type Ca²⁺ channel currents (I_L and I_T, respectively); I_L was more sensitive than I_T. Hyperosmotic suppression of I_L depended on the magnitude of increase in [Os]_e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of I_L and I_T persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca²⁺ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca²⁺ influx involves Ca²⁺ channel proteins. We therefore recorded the activity of L-type Ca²⁺ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]_e reduced the activity of Ca²⁺ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca²⁺ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca²⁺ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

Patch-Clamp Study on a Ca2+-Regulated K+ Channel in the Tonoplast of the Brackish Characeae Lamprothamnium succinctum

Cytoplasmic drops were prepared from internodal cells of thebrackish Characeae Lamprothamnium succinctum. Applying the patch-clamptechnique to single drops covered with tonoplast, we demonstratedthe presence of Ca²⁺-regulated K⁺ channels in the tonoplast.In a cell-attached mode, the selectivity of such channels forK⁺ was about 50 times that for Na⁺. This channel showed a tendencyto rectify in an outward direction. In the negative region ofthe pipette voltage, the conductance of this channel was 50pS, while it was 100 pS in the positive voltage region. Whenthe pipette voltage was increased above 50 mV, two conductancelevels were found in the cell-attached mode as well as in theexcised patch (cytoplasmic-side-out patch), which was obtainedby pulling the patch pipette from the cytoplasmic drop underconditions of low levels of Ca²⁺. Using the excised patch, wecontrolled the level of Ca²⁺ on the cytoplasmic side of thechannels. At a low level of Ca²⁺ (pCa=8) on the cytoplasmicside, the open frequency was very low and the opening time wasshort. An increase in Ca²⁺ on the cytoplasmic side (pCa = 5)increased both the frequency and the duration of opening. However,the conductance of the channels did not change. This regulationby Ca²⁺ of the K⁺ channels was reversible, that is, additionof EGTA on the cytoplasmic side inactivated the channels. Thepresent study demonstrates a direct action of Ca²⁺ on the K⁺channels. The physiological role of the K⁺ channel in the regulationof turgor in Lamprothamnium is discussed. (Received January 9, 1989; Accepted March 8, 1989)     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation

Pulmonary vasoconstriction and vascularmedial hypertrophy greatly contribute to the elevated pulmonaryvascular resistance in patients with pulmonary hypertension. A rise incytosolic free Ca²⁺ ([Ca²⁺]_cyt)in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E_m) regulates[Ca²⁺]_cyt by governing Ca²⁺influx through voltage-dependent Ca²⁺ channels. Thusintracellular Ca²⁺ may serve as a shared signaltransduction element that leads to pulmonary vasoconstriction andvascular remodeling. In PASMC, activity of voltage-gated K⁺(Kv) channels regulates resting E_m. In thisstudy, we investigated whether changes of Kv currents[I_K(V)], E_m, and[Ca²⁺]_cyt affect cell growth by comparingthese parameters in proliferating and growth-arrested PASMC. Serumdeprivation induced growth arrest of PASMC, whereas chelation ofextracellular Ca²⁺ abolished PASMC growth. Resting[Ca²⁺]_cyt was significantly higher, andresting E_m was more depolarized, inproliferating PASMC than in growth-arrested cells. Consistently, wholecell I_K(V) was significantly attenuated in PASMCduring proliferation. Furthermore, E_mdepolarization significantly increased resting[Ca²⁺]_cyt and augmented agonist-mediatedrises in [Ca²⁺]_cyt in the absence ofextracellular Ca²⁺. These results demonstrate that reducedI_K(V), depolarized E_m, and elevated [Ca²⁺]_cyt may play a criticalrole in stimulating PASMC proliferation. Pulmonary vascular medialhypertrophy in patients with pulmonary hypertension may be partlycaused by a membrane depolarization-mediated increase in[Ca²⁺]_cyt in PASMC.

     

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

A sustained increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca²⁺ stores by thapsigargin (TG), induced an increase in the [Ca²⁺]_i and cell death. However, the TG-induced death was not related to the [Ca²⁺]_i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca²⁺-dependent anti-apoptotic mechanism. After TG treatment, Ca²⁺-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca²⁺-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca²⁺]_i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca²⁺-dependent MAPK activation counteracts death signaling; removal of Ca²⁺ activated a second caspase-independent death pathway. sarco(endo)plasmic reticulum calcium ion adenosine triphosphatase; cytosolic calcium ion concentration; mitogen-activated protein kinase     

Ca2+ Regulation of Outward Rectifying K+ Channel in the Plasma Membrane of Tobacco Cultured Cells in Suspension: a Role of the K +Channel in Mitigation of Salt-Stress Effects by External Ca2+

The patch-clamp technique was used to study effect of the Ca²⁺on K⁺ channels in the plasma membrane of protoplasts isolatedfrom tobacco (Nicotiana tabacum L., cv. Bright Yellow) culturedcells in suspension. The outward rectifying whole-cell K⁺ currentswere not affected by in-tracellular Ca²⁺, but they were reducedwith increasing extracellular Ca²⁺. Neither extracellular norintracellular Ca²⁺ affected the permeability ratios (p_K+/P_Na+)of the plasma membrane. These results suggest that the inhibitionof outward-rectifying K⁺ channels by extracellular Ca²⁺may partiallycontribute towards the mitigation of detrimental effects ofsalinity on growth by extracellular Ca²⁺. (Received January 19, 1998; Accepted July 30, 1998)     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca²⁺ overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca²⁺-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules <800 Da to enter the cell. The cells then lyse not by rupture of the plasmalemma but through the activation of a "death" channel that lets large proteins (e.g., 140–160 kDa) leave the cell. These changes in permeability are accompanied by the formation of membrane blebs. In this study, we took advantage of the well-known differences in affinity of various Ca²⁺-binding proteins for Ca²⁺ and Sr²⁺ vs. Ba²⁺ to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single-cell level (time-lapse videomicroscopy), we found that the replacement of Ca²⁺ with either Sr²⁺ or Ba²⁺ delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated green fluorescent protein. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba²⁺ not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca²⁺-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba²⁺. Taken together, these results suggest that intracellular high-affinity Ca²⁺-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise. necrosis; vital dyes; membrane blebs; time-lapse videomicroscopy; fura 2     

Effects of Calcium and Abscisic Acid on Volume Changes of Guard Cell Protoplasts of Commelina

Calcium ions contracted guard cell protoplasts (GCP) of Commelinacommunis L., being particularly effective within the concentrationrange of 0 to 0.2 mol m^–3. Abscisic acid (ABA) in thepresence of EGTA, which chelates free Ca²⁺ in the medium, contractedGCP to a similar extent to Ca²⁺ alone or Ca²⁺ and ABA together.Similarly, ABA in the absence of free Ca²⁺ (i.e. an ABA/EGTAtreatment) inhibited K⁺-induced swelling of contracted GCP,as did Ca²⁺ alone or ABA and Ca²⁺ together. Lanthanum, a Ca²⁺channel blocker, prevented the contraction of GCP by Ca²⁺ buthad no effect if ABA was also present with Ca²⁺. The inhibitionof swelling of GCP by Ca²⁺ was also prevented by the presenceof lanthanum or verapamil (another Ca²⁺ channel blocker). These results indicate that Ca²⁺ and ABA can act independentlyof each other in contracting swollen GCP and in preventing K⁺-inducedswelling of contracted GCP of C. communis. If swelling and contractionof GCP are equivalent to stomatal opening and closure, respectively,the results do not support the hypothesis that ABA opens Ca²⁺channels in the plasma membrane of guard cells allowing Ca²⁺to enter the cells and, as a second messenger, to set in motionclosing processes. Key words: Abscisic acid, calcium, guard cell protoplasts, stomata     

A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line

Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn²⁺, and 50 µM Cu²⁺. Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC₅₀ for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca²⁺ and 2 mM Mg²⁺, and 33 µM in low-Mg²⁺, nominally Ca²⁺-free saline. Overall, the results are most consistent with activation of a P2X₇ receptor by ATP^4–. However, low permeability to N-methyl-D-glucamine⁺ and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X₇ receptor activation. ATP stimulation of internalization occurs in Na⁺-free, Ca²⁺-free, or low-Mg²⁺ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X₇ receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

Voltage-gated Ca2+ currents in rat gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells are histamine-containingendocrine cells in the gastric mucosa that maintain a negative membranepotential of about 50 mV, largely due to voltage-gated K⁺ currents [D. F. Loo, G. Sachs, and C. Prinz. Am. J. Physiol. 270 (Gastrointest Liver Physiol. 33):G739-G745, 1996]. The current study investigated thepresence of voltage-gated Ca²⁺channels in single ECL cells. ECL cells were isolated from rat fundicmucosa by elutriation, density gradient centrifugation, and primaryculture to a purity >90%. Voltage-gatedCa²⁺ currents were measured insingle ECL cells using the whole cell configuration of the patch-clamptechnique. Depolarization-activated currents were recorded in thepresence of Na⁺ orK⁺ blocking solutions and additionof 20 mM extracellular Ca²⁺. ECLcells showed inward currents in response to voltage steps that wereactivated at a test potential of around 20 mV with maximalinward currents observed at +20 mV and 20 mM extracellular Ca²⁺. The inactivation rate of thecurrent decreased with increasingly negative holding potentials and wastotally abolished at a holding potential of 30 mV. Addition ofextracellular 20 mM Ba²⁺ insteadof 20 mM Ca²⁺ increased thedepolarization-induced current and decreased the inactivation rate. Theinward current was fully inhibited by the specific L-typeCa²⁺ channel inhibitor verapamil(0.2 mM) and was augmented by the L-typeCa²⁺ channel activator BAY K 8644 (0.07 mM). We conclude that depolarization activateshigh-voltage-activated Ca²⁺channels in ECL cells. Activation characteristics,Ba²⁺ effects, and pharmacologicalresults imply the presence of L-type Ca²⁺ channels, whereasinactivation kinetics suggest the presence of additional N-typechannels in rat gastric ECL cells.

     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL     

Are second messengers crucial for opening the pore associated with P2X7 receptor?

Stimulation of the P2X₇ receptor by ATP induces cell membrane depolarization, increase in intracellular Ca²⁺ concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X₇, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X₇ pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X₇ pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X₇ receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca²⁺ as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca²⁺ and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X₇ pore depends on second messengers such as Ca²⁺ and MAP kinases. electrophysiology; pore formation     

Differentiation-dependent changes in the membrane properties of fiber cells isolated from the rat lens

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd³⁺. Isolated fiber cells were viable in physiological [Ca²⁺] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 µm) contained variable combinations of Ba²⁺-and tetraethylammonium-sensitive K⁺ currents. Longer fiber cells (150–650 µm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na⁺ with N-methyl-D-glucamine, indicating that the lens contains both Gd³⁺-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K⁺ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate. electrophysiology; potassium channel; anion channel; nonselective cation channel     

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores

IntracellularCa²⁺ release channels such asryanodine receptors play crucial roles in theCa²⁺-mediated signaling thattriggers excitation-contraction coupling in muscles. Although theexistence and the role of these channels are well characterized inskeletal and cardiac muscles, their existence in smooth muscles, andmore particularly in the myometrium, is very controversial. We have nowclearly demonstrated the expression of ryanodine receptorCa²⁺ release channels in ratmyometrial smooth muscle, and for the first time, intracellularCa²⁺ concentration experimentswith indo 1 on single myometrial cells have revealed the existence of afunctional ryanodine- and caffeine-sensitive Ca²⁺ release mechanism in 30% ofrat myometrial cells. RT-PCR and RNase protection assay on wholemyometrial smooth muscle demonstrate the existence of all threeryr mRNAs in the myometrium:ryr3 mRNA is the predominant subtype,with much lower levels of expression forryr1 andryr2 mRNAs, suggesting that theryanodine Ca²⁺ release mechanismin rat myometrium is largely encoded byryr3. Moreover, using intracellularCa²⁺ concentration measurementsand RNase protection assays, we have demonstrated that the expression,the percentage of cells responding to ryanodine, and the function ofthese channels are not modified during pregnancy.