MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Lysophosphatidic Acid-Induced Proliferation-Related Signals in Astrocytes

Abstract: Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 n M LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca²⁺]_i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca²⁺]_i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca²⁺]_i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca²⁺]_i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca²⁺]_i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis.     

Measurement of glial fibrillary acidic protein by a two-site immunoradiometric assay

The two-site immunoradiometric assay (two-site IRMA) for the brain-specific glial fibrillary acidic protein (GFA protein) is carried out by reaction of the GFA protein solution with a solid-phase anti(GFA) followed by a second reaction in which the insoluble product is incubated with purified, radioactive anti-(GFA). Unreacted labeled antibodies remain in solution and are washed away. As the amount of GFA increases, the radioactivity in the solid-phase increases. The most significant assay variables include (a) stability and reactivity of the solid-phase antibody, (b) washing the solid-phase, (c) nonspecific interference by serum proteins, and (d) a paradoxical fall in tube radioactivity which occurs at high dose (the “high-dose hook effect”). The assay becomes more sensitive and precise and the serum effect is minimized when the solid-phase antibody is separated from the matrix by an immunoglobulin “spacer-arm”. For a triplicate determination, the minimal detectable dose averaged $\text{73}\text{pg}\text{200 μ}\text{l}$ incubation. The assay precision enables a 500-fold assay range. GFA activity found in aged crude tissue or tissue-culture extracts, CSF, and used tissueculture media, often did not appear to be immunologically identical to the purified standard GFA protein. This may be explained by the known tendency of GFA protein to aggregate. The assay does not cross-react significantly with other common CNS proteins. Assay of various rat tissues confirms the localization of GFA protein only to the CNS.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.     

Plasma Ca2+ concentration limits melatonin night production in two fish species

In freshwater (FW) rainbow trout Oncorhynchus mykiss of spontaneously low plasma calcium concentrations ([Ca²⁺]_pl), plasma melatonin at night was significantly lower than that measured in FW fish with the highest [Ca²⁺]_pl. In brackish water adapted rainbow trout with originally high [Ca²⁺]_pl, plasma melatonin concentration at night was elevated. In cannulated flounder Platichthys flesus , night plasma melatonin increases (ΔMel) corresponded to [Ca²⁺]_pl. It is postulated that in physiological steady-state conditions, melatonin synthesis capacity is coupled to free calcium concentration in plasma of O. mykiss and P. flesus .     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

ROLE OF MALATE TRANSPORT IN REGULATING METABOLISM IN MITOCHONDRIA ISOLATED FROM RABBIT BRAIN

Abstract— Partly purified chromaffin granules were incubated in vitro with Ca²⁺ (with trace amounts of ⁴⁵Ca²⁺) in concentrations ranging from 4 μm to 1 mm. After incubation the granules were washed with media containing EDTA and then subjected to density gradient centrifugation (1.3 to 2.0 m-sucrose solutions) in order to characterize the particles which had taken up ⁴⁵Ca²⁺. By using marker enzymes and various inhibitors of Ca²⁺ uptake into such cell particles as mitochondria it was established that under the conditions of the experiments chromaffin granules took up Ca²⁺ from the incubation medium. To characterize this uptake a simplified density gradient procedure was tested and found to be suitable. The uptake of Ca²⁺ into chromaffin granules was strongly dependent on temperature. It was not activated by ATP. The uptake was linear up to 10 min. At high calcium concentrations (above 200 μm) the rate of uptake levelled off. The uptake at 37°C was 1 nmol Ca²⁺/mg protein/min at a Ca²⁺ concentration of 500 μm. Mg²⁺ had no influence on Ca²⁺ uptake, whereas Sr²⁺ (1 mm) inhibited it. The methods established in this study should prove useful for a further characterization of this Ca²⁺ uptake into chromaffin granules which is likely to represent a useful model for the Ca²⁺ uptake occurring in the intact gland.     

Effects of Cd2+ and EDTA on young sugar beets (Beta vulgaris). I. Cd2+ uptake and sugar accumulation

Sugar beets ( Beta vulgaris L. cv. Monohill) grown in a complete nutrient solution, were treated with Cd²⁺ (5 or 50 μ M ) and/or EDTA (10 or 100 μ M ) in different combinations. The Cd contents of five-week-old roots and shoots were determined by atomic absorption spectrophotometry, and the sucrose, glucose and fructose contents were measured enzymatically. The Cd²⁺ uptake in both roots and shoots shows a linear relationship to the concentration of free Cd²⁺ in the nutrient solution. This uptake is diminished in the presence of EDTA, suggesting that the Cd-EDTA complex is unable to penetrate the membranes. The contents of glucose, fructose and sucrose in both roots and shoots decrease with increasing uptake of free Cd²⁺. This may be a secondary effect caused by the inhibition of photosynthesis in the presence of Cd²⁺. EDTA reduces the inhibition of Cd²⁺ on sugar formation and accumulation. In the presence of EDTA alone the sugar content increases somewhat. EDTA slightly influences the dry weights of whole plants. The ratio roots:whole plants increases. Cd²⁺ (≤ 50 μ M ) increases the dry matter portion of roots by ca 30%, but not that of shoots.     

Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT₃ receptor, a ligand-gated ion channel. The 5-HT₃ receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K⁺ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca²⁺ concentration ([Ca²⁺]_i), K⁺-induced depolarization resulted in variable but rapid increases in apparent [Ca²⁺]_i among the individual terminals, with some synaptosomes displaying large transient [Ca²⁺]_i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca²⁺]_i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT₃ receptors induced slow increases in [Ca²⁺]_i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca²⁺ entry and inhibited by millimolar external Mg²⁺. We conclude that significant increases in brain nerve terminal Ca²⁺, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.     

An heuristic hypothesis of chilling injury in plants: a role for calcium as the primary physiological transducer of injury

Abstract. It is suggested that increased levels of free cytosolic calcium ([Ca²⁺]_cyt) may serve as the primary physiological transducer of chilling injury in plants. Numerous similarities between the effects of [Ca²⁺]_cyt-raising treatments on plants and the effects of chilling temperatures on chilling-sensitive (CS) plants are noted. It is proposed that chilling temperatures may lead to increases in [Ca²⁺]_cyt in CS plant cells by reducing the rate at which they exclude Ca²⁺ from their cytosol and that rapid cooling (coldshock) may cause rapid increases in [Ca²⁺]_cyt due to the activation of voltage-dependent cation channels. Chill-induced increases in [Ca²⁺]_cyt in the cells of CS plants may reflect either an inherent inability of such plants to maintain homeostatic levels of Ca²⁺ at low temperatures or a stress-induced reaction which has evolved to enable such cells to cope more effectively with the short-term hardships imposed by cold. Previous proposals concerning the physiological transduction of chilling injury are also discussed. It is argued that there is little evidence to suggest that the immediate effects of low temperatures on CS cells include either decreases in ATP levels, general increases in the passive permeability of membranes, or increased rates of fermentation.     

Endoplasmic reticulum Ca(2+) homeostasis and neuronal death

The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca²⁺ signalling by either amplifying Ca²⁺ entry through voltage-gated Ca²⁺ channels by Ca²⁺-induced Ca²⁺ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca²⁺ release (IICR). The ER Ca²⁺ store emerges as a single interconnected pool, thus allowing for a long-range Ca²⁺ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca²⁺ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca²⁺ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca²⁺ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia.     

FAST AXOPLASMIC TRANSPORT OF A CALCIUM-BINDING PROTEIN IN MAMMALIAN NERVE

Abstract— Calcium is transported at a fast rate of 410 mm/day in cat sciatic nerve on injection of ⁴⁵Ca²⁺ into the L7 dorsal root ganglia. Nerve segments corresponding to the crest and the plateau regions of transported activity were analyzed by column chromatography on Sephadex G-100 and Biogel A 5m columns and the fast transported ⁴⁵Ca²⁺ found to be bound to a protein of 15,000 dalton. Using [³H]leucine as a precursor, a labeled calcium binding protein (CaBP) was found located at the same position in elution volumes from the columns as was the protein-bound ⁴⁵Ca^{2 +}. The level of [³H]-labeled CaBP in the crest and plateau regions were compared using column chromatography and polyacrylamide gel electrophoresis techniques and approx 3×4 times more [³H]-labeled activity was found in the crest as compared to the plateau. These findings indicate that Ca²⁺ is fast transported in association with the CaBP. The relation of CaBP to the transport filament model of axoplasmic transport and its possible role in nerve are discussed.     

Influence of aluminium on phosphorus and calcium localization in roots of beech (Fagus sylvatica)

Beech plants ( Fagus sylvatica L. provenance Maramures) were grown in nutrient solution at low pH (4.2) and exposed to different concentrations of AlCl₃. Uptake and leakage of Ca²⁺(⁴⁵Ca²⁺) and H²PO₄-(³²P) were studied. A high external aluminium concentration (1.0m M ) reduced the uptake and export to the shoot of both calcium and phosphate, while 0.1 m M Al increased the phosphorus level in the roots. To determine the impact of aluminium on the localization of calcium and phosphate, leakage of the elements from both intact plants and plants frozen prior to the leakage experiment was studied. The leakage of Ca²⁺ from intact plants was not affected by prior exposure to 0.1 m M Al. Freezing of the beech plants before the leakage experiment increased leakage of calcium slightly more from roots of control plants than for roots exposed to 0.1 m M Al, indicating that even low concentrations of alminium may impede the influx of calcium across the plasma membrane in the roots. The patterns of Ca²⁺ leakage from roots previously exposed to 1.0 m M Al indicated that very little Ca²⁺ was located extracellularly. The extracellular fraction of phosphate increased with increasing Al concentration in the nutrient solution. Low Al concentration (0.1 m M ) only reduced the intracellular phosphate concentration to a minor extent, while 1.0 m M Al profoundly decreased it. It is concluded that 0.1 m M AlCl₃ has a limited effect upon the localization of Ca²⁺ and phosphate in the roots. At higher levels of Al, 0.1–1.0 m M , there is a more dramatic change in nutrient localization in the free space and uptake over the plasma membrane.     

Calcium Mobilization by Ryanodine Promotes the Phosphorylation of Initiation Factor 2α Subunit and Inhibits Protein Synthesis in Cultured Neurons

Abstract: Protein synthesis plays an important role in the viability and function of the cell. There is evidence indicating that Ca²⁺ may be a physiological regulator of the translational process. In the present study, the effect of agents that increase intracellular calcium levels by different mechanisms, as well as repercussion on the rate of protein synthesis, including phosphorylation of initiation factor 2α subunit, and double-stranded RNA-dependent eIF-2α kinase (PKR) activity were analyzed. Glutamate (100 µ M ) and K⁺ (60 m M ), which increase intracellular calcium levels (the former mostly by the influx of extracellular calcium via voltage-sensitive calcium channels, and the latter by receptor-operated calcium channels), and carbachol (1 m M ), as well as glutamate, which mobilizes intracellular calcium from the endoplasmic reticulum via activation of inositol 1,4,5-trisphosphate receptor, did not modify any of the analyzed parameters. Nevertheless, 100 n M ryanodine, which increases intracellular calcium concentration by activating the ryanodine receptor, promoted a significant decrease in the rate of protein synthesis and increased both initiation factor 2α subunit phosphorylation and PKR activity. From our results, we can conclude that inhibition of protein synthesis is dependent on the mobilization of intracellular calcium from internal stores. Moreover, they strongly suggest that this inhibition is only promoted when calcium is increased via ryanodine receptor, and possibly by activation of PKR activity.     

Identification and Characterization of Sulfhydryl-Containing Proteolytic Fragments Involved in the Ca2+-Induced Conformational Change of Beef Brain S-100

Abstract: The Ca²⁺-dependent conformational alteration of the brain-specific S-100 protein was studied by reacting the protein with N -ethyl[2,3-¹⁴C]maleimide in the absence and presence of Ca²⁺ and under denaturing conditions. Peptic hydrolysates of the ¹⁴C-labeled protein were analyzed and fractionated by high-performance liquid chromatography. Labeled peptide fractions were characterized by high-voltage electrophoresis and TLC. A clear distinction could be made between two classes of sulfhydryl-containing fragments: (a) neutral, hydrophobic, and (b) acidic. Ca²⁺ markedly favored ¹⁴C incorporation into the former components, whereas the latter were readily available only under denaturing conditions.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Activation of Ca2+ Transport System of Sea Urchin Sperm by High External pH: 220 kD Membrane Glycoprotein is Involved in the Regulation of the Ca2+ Entry

When sperm of the sea urchin, Hemicentrotus pulcherrimus , were exposed to high pH (9.0) sea water, they showed large increases in intracellular Ca²⁺ ([Ca²⁺]i) and pH (pHi) and underwent the acrosome reaction (AR) without the aid of the egg jelly. Not only [Ca²⁺]i increase but also pHi rise did not occur under Ca²⁺-free conditions. Both the increases in [Ca²⁺]i and pHi and the AR by high pH were inhibited by a Ca²⁺ channel blockers, verapamil and nisoldipine, and by a lectin, wheat germ agglutinin (WGA) which interacts with a 220 kD membrane glycoprotein of sperm. These reagents inhibited also the AR by the egg jelly. The inhibitory effects of WGA were immediately canceled by the addition of N-acetyl-D-glucosamine, a sugar which is known to remove WGA from its binding site. These results suggest that 1) the same Ca²⁺ transport system is activated by high external pH and the egg jelly, 2) increase in [Ca²⁺]i is prerequisite for the stimulation of the H⁺-efflux system(s) and 3) the 220 kD WGA-binding membrane protein functions as a regulator protein of Ca²⁺ transport system.     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.