Stimulation of the P2Y Purinergic Receptor on Type 1 Astroglia Results in Inositol Phosphate Formation and Calcium Mobilization

Cultured astroglia express purinergic receptors that initiate phosphoinositide metabolism and calcium mobilization. Experiments were conducted to characterize the purinergic receptor subtype on type 1 astroglia responsible for stimulation these second-messenger systems. Inositol phosphate (IP) accumulation and calcium mobilization were measured after stimulation with ATP or purinergic receptor subtype-selective ATP analogues. ATP (10(-5) M) increased IP accumulation severalfold. Dose-effect assays monitoring astroglial IP accumulation revealed the order of potency that defines the P2Y receptor: 2-methylthioadenosine 5'-triphosphate greater than ATP greater than alpha beta-methyleneadenosine 5'-triphosphate greater than beta gamma-methyleneadenosine 5'-triphosphate. The influence of ATP on intracellular calcium levels in individual type 1 astroglia was examined using the calcium indicator dye, fura-2. Dose-effect experiments indicated that ATP was equally potent for generating inositol phosphates and increasing cellular calcium. The most prevalent response (87% of total responses) to ATP consisted of a rapid increase in calcium to a peak level that was approximately five times greater than the prestimulation level. This peak was followed by a decline to a plateau level that was significantly above baseline. This plateau phase of the calcium increase was maintained for at least 5 min in the presence of ATP and was dependent on external calcium. Many (23%) astroglia exhibited spontaneous calcium oscillations whose frequency and magnitude increased after the addition of 10(-5) M ATP. Immunocytochemical staining indicated that the responses occurred in glial fibrillary acidic protein positive cells. We conclude that type 1 astroglia express the P2Y purinergic receptor which regulates IP production and calcium mobilization.     

Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts

Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC₅₀) and maximal, saturable binding (B_max) were estimated from Scatchard analyses and were found to be 24.2 ± 3.3 pM and 56500 ± 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of 100 nM to a peak level of 600–800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC₅₀ of 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of ⁴⁵Ca from cells loaded to isotopic equilibrium (3 h) with ⁴⁵Ca. The intracellular second messenger, IP₃, also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with ⁴⁵Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 ± 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP₃-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.     

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.     

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na⁺-K⁺-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser⁴⁷³ phosphorylation, as evidenced by an increase in phospho-Akt Ser⁴⁷³ band density. Ouabain-stimulated Akt Ser⁴⁷³ phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser⁴⁷³ phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser⁴⁷³ phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in ⁸⁶Rb uptake but did not significantly alter Na⁺-K⁺-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na⁺-K⁺-ATPase-mediated ion transport. Na⁺-K⁺-ATPase; opossum kidney cells     

TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange and regulation of cytoplasmic concentration of calcium in rat cerebellar neurons treated with glutamate

In the present work, the forward and/or reversed Na+/Ca2+ exchange in cerebellar granular cells was suppressed by substitution of Na+o by Li+ before, during, and after exposure to glutamate for varied time and also using the inhibitor KB-R7943 of the reversed exchange. After glutamate challenge for 1 min, Na+o/Li+ substitution did not influence the recovery of low [Ca2+]i in a calcium-free medium. A 1-h incubation with 100 microM glutamate induced in the neurons a biphasic and irreversible [Ca2+]i rise (delayed calcium deregulation (DCD)), enhancement of [Na+]i, and decrease in the mitochondrial potential. If Na+o had been substituted by Li+ before the application of glutamate, i.e. the exchange reversal was suppressed during the exposure to glutamate, the number of cells with DCD was nearly fourfold lowered. However, addition of the Na+/K+-ATPase inhibitor ouabain (0.5 mM) not preventing the exchange reversal also decreased DCD in the presence of glutamate. Both exposures decreased the glutamate-caused loss of intracellular ATP. Glucose deprivation partially abolished protective effects of the Na+o/Li+ substitution and ouabain. KB-R7943 (10 microM) increased 7.4-fold the number of cells with the [Ca2+]i decreased to the basal level after the exposure to glutamate. Thus, reversal of the Na+/Ca2+ exchange reinforced the glutamate-caused perturbations of calcium homeostasis in the neurons and slowed the recovery of the decreased [Ca2+]i in the post-glutamate period. However, for development of DCD, in addition to the exchange reversal, other factors are required, in particular a decrease in the intracellular concentration of ATP.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

Purinergic agonists stimulate the secretion of endothelin-1 in rat thyroid FRTL-5 cells

The aim of the present study was to investigate the mechanisms regulating endothelin-1 (ET-1) secretion in rat thyroid FRTL-5 cells. ET-1 was found to be secreted after stimulation with adenosine and ATP. The release of ET-1 was sensitive to pertussis toxin, indicating a role of G-proteins in the stimulus-secretion coupling. The stimulation evoked by ATP or adenosine was inhibited by the P₁-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and in the presence of adenosine deaminase the adenosine- and ATP-mediated ET-1 secretion was abolished. These evidences suggest a role of a P₁-adenosine receptor in the secretion of ET-1. Increasing cyclic AMP with forskolin decreased the adenosine-mediated secretion. In addition, the intracellular calcium chelator BAPTA or inhibition of calcium entry with Ni²⁺ prevented the response. Protein kinase C (PKC) is also partly involved in ET-1 secretion in FRTL-5 cells. Activation of PKC with the phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated the secretion of ET-1 in a time- and dose-dependent manner. Furthermore, downregulation of PKC decreased the secretion of ET-1 stimulated by adenosine. In conclusion, ET-1 secretion in FRTL-5 cells is stimulated via a pertussis toxin-sensitive P₁-receptor pathway which is modulated by several signal transduction mechanisms including cAMP, Ca²⁺, and PKC. © 1996 Wiley-Liss, Inc.     

ET-1 increases distensibility of acutely loaded myocardium: a novel ETA and Na+/H+ exchanger-mediated effect

This study investigated, in rabbit papillary muscles (n = 61) and human auricular strips (n = 7), effects of endothelin-1 (ET-1; 0.1-10 nM) on diastolic myocardial properties. ET-1 (1 nM) was also given in the presence of selective ET(A) or ET(B) antagonism, nonselective ET(A)/ET(B) antagonism, and Na(+)/H(+) exchanger inhibition. Effects of 6.3 mM Ca(2+) were also studied. ET-1 dose dependently increased inotropism. In contrast to baseline, in the presence of ET-1, resting tension (RT) decreased, after an isometric twitch, 3.4 +/- 1.4, 6.9 +/- 1.5, and 12.5 +/- 3.1% with 0.1, 1, and 10 nM, respectively, reflecting an increase in myocardial distensibility. ET-1 effects were abolished with selective ET(A) as well as with nonselective ET(A)/ET(B) antagonism, whereas they were still present with ET(B) antagonism. Na(+)/H(+) exchanger inhibition abolished ET-1 effects on distensibility, whereas it only partially inhibited positive inotropic effect. Ca(2+) increased inotropism to a similar extent to ET-1 (1 nM) but did not affect distensibility. ET-1 therefore increased diastolic distensibility of acutely loaded human and nonhuman myocardium. This effect is mediated by ET(A) receptors, requires Na(+)/H(+) exchanger activation, and cannot be elicited by Ca(2+).     

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.     

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

Agonist-Specific Calcium Signaling and Phosphoinositide Hydrolysis in Human SK-N-MCIXC Neuroepithelioma Cells

Abstract: Fura-2 digital imaging microfluorimetry was used to evaluate the Ca²⁺ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca²⁺_i) but of different magnitudes (ET-1 = ATP> NE). The Ca²⁺ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca²⁺ influx. The Ca²⁺ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca²⁺ signal, which was characteristic of the response to Oxo-M, was observed in <40% of cells stimulated with ET-1 and absent from Ca²⁺ signals elicited after NE addition. Removal of extracellular Ca²⁺ enhanced the rate of decay of Ca²⁺ signals generated by ATP, ET-1, or Oxo-M and, when evident, abolished the sustained phase of Ca²⁺ influx. In the absence of extracellular Ca²⁺, NE elicited asynchronous multiple Ca²⁺ transients. In either the absence or presence of extracellular Ca²⁺,>94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were ～74 and ～48%. Sequential addition of agonists to cells maintained in a Ca²⁺-free buffer indicated that each ligand mobilized Ca²⁺ from a common intracellular pool. When monitored as a release of a total inositol phosphate fraction, all four agonists elicited similar (four- to sixfold) increases in phosphoinositide hydrolysis. However, the addition of ET-1 or ATP resulted in larger increases in the net formation of inositol 1,4,5-trisphosphate than did either Oxo-M or NE. These results indicate that, in SK-N-MCIXC cells, the characteristics of both Ca²⁺ signaling and inositol phosphate production are agonist specific.     

Effects of endothelins on signal transduction and proliferation in human melanocytes

We demonstrate here that human melanocytes could be regulated by endothelin (ET) derivatives, potent vasoconstrictive peptides synthesized by endothelial cells, to stimulate their proliferation and melanization via a receptor-mediated signal transduction pathway. Receptor-binding assay using [125I]ET indicated that unlabeled ET-1 or ET-2 competitively inhibited each binding of labeled ETs to melanocytes with a concentration for half-maximal inhibition (IC50) of 0.7 or 0.9 nM, respectively. The dissociation constant (Kd) and the number of sites of the specific bindings of ET-1 and those of ET-2 were almost the same (Kd: 1.81 nM, binding sites: 7.0-8.0 x 10(4) per cell). Upon incubation with cultured cells, the mass contents of inositol 1,4,5-trisphosphate and intracellular calcium level were substantially increased by 10 nM ET-1, ET-2, and ET-3, but not by big-ET with maximal response at 80-130-s postincubation. The addition of ET-1 and ET-2 at 1-50 nM concentrations caused human melanocytes to significantly stimulate DNA [( 3H]thymidine incorporation) and melanin synthesis (3H2O release and [14C] thiouracil incorporation). Furthermore, ETs exhibited an additive stimulatory effect on basic fibroblast growth factor-stimulated DNA synthesis. In a long-term serum-free culture system, the strongest stimulation of growth by 10 nM ET-1 or ET-2 was observed in the presence of 10 nM cholera toxin and 0.2% bovine pituitary extract, resulting in a 4.5-fold increase in cell number for 12 culture days. These findings strongly suggest involvement of ET in the mechanism regulating proliferation and melanization of human melanocytes.     

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line.

Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.     

Vagally mediated cholestatic and choleretic effects of centrally applied Endothelin-1 through ETA receptors

The role of Endothelin-1 (ET-1) in the central nervous system is not fully understood yet although several studies strongly support its neuromodulatory role. A high density of endothelin receptors is present in the dorsal vagal complex that is the major site for the regulation of the digestive function. Therefore in the present study we sought to establish the role of ET-1 in the central regulation of bile secretion in the rat. Intracerebroventricular ET-1 injection exhibited opposite behaviors on spontaneous bile secretion according to the dose administered. Lower doses of ET-1 (1 fM) increased bile flow and bicarbonate excretion whereas higher doses (1 nM) decreased bile flow and bile acid output. Both the choleretic and the cholestatic effects of ET-1 were abolished in animals pretreated with icv BQ-610 (selective ETA antagonist) but not with BQ-788 (selective ETB antagonist). In addition, truncal vagotomy but not adrenergic blockade abolished ET-1 effects on bile secretion. Brain nitric oxide was not involved in ET-1 response since L-NAME pretreatment failed to affect ET-1 actions on the liver. Portal venous pressure was increased by centrally administered ET-1 being the magnitude of the increase similar with low and high doses of the peptide. These results show that centrally applied ET-1 modified different bile flow fractions independent of hemodynamic changes. Lower doses of ET-1 increased bile acid independent flow whereas higher doses decreased bile acid dependent flow. Vagal pathways through the activation of apparently distinct ETA receptors mediated the cholestatic as well as the choleretic effects induced by ET-1. Present findings show that ET-1 participates in the central regulation of bile secretion in the rat and give further insights into the complexity of brain-liver interaction.