Ca2+, calmodulin, and protein dephosphorylation are required for GA-induced gene expression in petunia corolla

Gibberellic acid (GA₃) induces the expression of different genes, including chalcone synthase ( chs ) and gip , in detached petunia corollas. To initiate a study on gibberellin (GA)-signal transduction in this tissue, we examined the effect of agents that inhibit or promote specific steps in signal-transduction pathways. The calcium chelator 1,2- bis ( o -aminophenoxy)ethane N,N,N ' ,N '-tetraacetic acid (BAPTA) had no effect on GA-induced gene expression, while the calcium-channel blocker, ruthenium red (RR), inhibited the activation of the genes. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) inhibited the induction of chs and gip by the hormone, and its analog, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5), had lower effect. The activation of chs and gip by GA₃ was completely blocked by the protein phosphatase inhibitor, okadaic acid (OA), and partially inhibited by the protein kinase inhibitor, 1-(5-isoquinoline-sulfonyl)- 2-methylpiperazine dihydrochloride (H-7). We suggest that Ca²⁺ from intracellular sources, calmodulin and protein dephosphorylation and phosphorylation are involved in GA-induced gene expression in petunia corollas.     

Possible roles of calcium and ammonium in the development of bitter pit in apple

Apple trees ( Malus pumila Mill . var. domestica Fuji/ Malus prunifolia rootstock) showed a high susceptibility to bitter pit when supplyed with ammonium salt instead of nitrate (control) in the nutrient solution. When apple fruit was affected by bitter pit, a lower calcium as well as a higher nitrogen and ammonium-nitrogen contents was observed in the fruit flesh near the calyx end. The activity of the mitochondrial Ca²⁺-uptake of the fruit flesh near the calyx end was higher when the tree was grown with ammonium salt than when grown with nitrate. Both the activities of succinate: cytochrome c oxidoreductase and the mitochondrial Ca²⁺-uptake per g of tissue were higher in affected fruit than in healthy fruit. Each of chlorpromazine, N-(6-aminohexyl)-5-chloro-l-napthalenesulfonamide (W-7) and N-(6-aminohexyl)-l-naphthalenesulfonamide (W-5), calmodulin antagonists, was infiltrated into the fruit for 20 min under reduced pressure (about 1 × 10⁴ Pa). Few days later, numerous bitter pit-like spots were observed in both fruit treated with W-7 and chlorpromazine, while only a few spots were observed after the infiltration with W-5, a less potent calmodulin antagonist. A possible mechanism for the occurrence of bitter pit is discussed.     

Inhibitory Effects of Diltiazem and Verapamil, Calcium Antagonists, on Fertilization-Induced Increase in the Phosphorylase Reaction in Echiuroid Eggs

The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus . The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose-1-phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased ⁴⁵Ca²⁺ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca²⁺ level increases associated with fertilization-induced Ca²⁺ influx and that this stimulates Ca²⁺ dependent protein kinase to phosphorylate phosphorylase b , resulting in an increased rate of the phosphorylase reaction.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

Evidence for a Role of Calmodulin in Calcium-Induced Noradrenaline Release from Permeated Synaptosomes: Effects of Calmodulin Antibodies and Antagonists

Abstract: The nervous tissue-specific protein B-50 (GAP-43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin-binding proteins. To investigate to what extent calmodulin and the interaction between B-50 and calmodulin are involved in the mechanism of Ca²⁺-induced noradrenaline release, we introduced polyclonal anti-calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W-7, calmidazolium, and polymyxin B into streptolysin-O-permeated synaptosomes prepared from rat cerebral cortex. Anti-calmodulin antibodies, which inhibited Ca²⁺/calmodulin-dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca²⁺-induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W-7, and calmidazolium inhibited Ca²⁺-induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca²⁺-induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B-50 and calmodulin, thus lending further support to the hypothesis that B-50 serves as a local Ca²⁺-sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca²⁺/calmodulin-dependent enzymes involved in the regulation of one or more steps in the release mechanism.     

Effects of calmodulin antagonists on auxin-stimulated proton extrusion in Avena sativa coleoptile segments

The effect of the 5 calmodulin (CaM) antagonists trifluoperazine (TFP). compound 48/80, N-(6-aminohexyl)-naphthalenesulfonamtde (W-5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium on auxin-dependent medium acidification was investigated in abraded segments of Avena sativa L. cv. Victory I. Buffering capacity, Asn content, and changes in pH of bathing solutions were measured in the presence of these inhibitors. When coleoptiles were treated with TFP or compound 48/80, the Asn content and the buffering capacity increased, thus suggesting that plasma membrane permeability was modified. On the contrary. the effect of calmidazolium, W-5. and W-7 on Asn release and buffering capacity was rather low; only small effects being observable at the highest concentration employed. Calmidazolium and W-7 strongly inhibited auxin-dependent medium acidification. W-5 did not affect medium acidification. The specificity of these CaM antagonists and their effects on medium acidification are discussed. The data adduced is consistent with the working hypothesis which postulates an essential role for the Ca²⁺-CaM system on auxin-dependent medium acidification.     

Intracellular Microinjection of Anticalmodulin Drugs Does not Inhibit the Cortical Reaction Induced by Fertilization, Ionophore A 23187 or Injection of Calcium Buffers in Sea Urchin Eggs

The drugs, fluphenazine, chlorpromazine, dibucaine, propranolol, vinblastine and W7[N-(6-arninohexyl)-5 chloro-1-napthalene-sulfonamide], which have been shown to prevent formation of the ternary activated complex of Ca⁺⁺-calmodulin with several soluble or membrane proteins, inhibit the cortical reaction induced by fertilization, by ionophore A 23187 or by the microinjection of Ca⁺⁺ buffers when applied from outside to sea urchin eggs. In contrast, direct intracellular microinjection of these drugs, even at concentrations much exceeding their I₅₀ for external application, does not suppress elevation of the fertilization membrane, although it prevents cleavage after fertilization. The implication is that intracellular calmodulin is not the receptor of Ca⁺⁺ in the Ca⁺⁺-dependent exocytosis of cortical granules induced by fertilization, by ionophore, or by the micro-injection of calcium buffers.     

The involvement of a G-protein in phytochrome-regulated, Ca2+-dependent swelling of etiolated wheat protoplasts

The red light (R)-induced swelling of mesophyll protoplasts, isolated from dark-grown wheat ( Triticum aestivum L. cv. Arminda) leaves, was inhibited by guanosine-5'-0-(2-thiodiphosphate) (GDP-β-S). In darkness or after control irradiation with far-red light (FR), guanosine-5'-O-(3-thiotriphosphate) (GTP-γ-S) induced swelling to the same extent as after R. Both GDP-β-S and GTP-γ-S were introduced into the cytoplasm by means of electroporation. The possibility of R-induced activation of the phosphatidylinositol pathway of transmembrane signalling was investigated. Neomycin, Li⁺ and l-(5-isoquinolinesulfonyl)-2-methylpiperazine (H₇) inhibited the R-induced swelling. Phorbol 12-myristate 13-acetate (PMA) induced swelling after control irradiation with FR. Neomycin and Li⁺ also inhibited GTP-γ-S-induced swelling. These results suggest that a GTP-binding protein is involved in the phytochrome-regulated swelling response. Addition of N⁶, 2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (DB-cAMP) induced swelling to the same extent as R-irradiation. The calmodulin antagonist N-(6-aminohexyl)5-chloro-l-naphthalenesulfonamide (W₇) induced swelling after FR, while R-induced swelling was not affected. The less active analogue N-(6-aminohexyl)-l-naphthalenesulfonamide (W₅) induced no swelling after FR. It is speculated that the protoplast volume is correlated with the cytoplasmic concentration of free Ca²⁺.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca²⁺. The effect of extracellular Ca²⁺ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca²⁺. Verapamil significantly reduced the action of extracellular Ca²⁺ or Ba²⁺ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No ⁴⁵Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca²⁺ from the external medium is not required.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

Lysophosphatidic Acid Induces a Sustained Elevation of Neuronal Intracellular Calcium

Abstract: Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA-induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca²⁺]_i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca²⁺]_i. Moreover, consistent with a receptor-mediated process, there was no further increase in [Ca²⁺]_i after a second addition of LPA. The LPA-induced increase in [Ca²⁺]_i required extracellular calcium. However, studies with Cd²⁺, Ni²⁺, and nifedipine and nystatin-perforated patch clamp analyses did not indicate involvement of voltage-gated calcium channels in the LPA-induced response. In contrast, glutamate appears to have a significant role in the LPA-induced increase in [Ca²⁺]_i, because this increase was inhibited by NMDA receptor antagonists and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg²⁺ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis

The involvement of calcium and different calmodulin isoforms (Ca²⁺-CaM) in heat shock (HS) signal transduction in Arabidopsis ( Arabidopsis thaliana ) was investigated. Using transgenic Arabidopsis plants which have the AtHsp18.2 promoter/GUS fusion gene, it was found that the level of β -glucuronidase (GUS) activity was up-regulated by the addition of CaCl₂ and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl₃ and verapamil, or the CaM antagonists N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), chlorpromazine (CPZ) and trifluoperazine (TFP). CaCl₂ not only increased the GUS activity after HS, but also up-regulated the GUS activity under non-HS conditions. These results provide additional support for the involvement of the Ca²⁺-CaM signalling system in HSP gene expression. The expression of nine CaM genes (AtCaM1–9) from Arabidopsis was differentially regulated by HS at 37 °C. The expression of AtCaM3 and AtCaM7 genes increased during HS. The temporal expression of the AtCaM3, AtCaM7 and hsp18.2 genes demonstrated that up-regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or hsp18.2 .     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

Acid Release during Activation of Barnea candida (Mollusca, Pelecypoda) Oocytes

Barnea caridida oocytes release acid (1.35 pmole H⁺/oocyte) upon fertilization. After artificial activation by an excess of KCl, germinal vesicle breakdown (GVBD) occurs normally and a quite similar, but not identical, acid release is recorded (1.10 pmole H⁺/oocyte). KCl activation of Barnea oocytes is completely inhibited in 100 mM sodium-acetate sea water at pH 6.5 and fertilization does not result in activation when the oocytes are transferred after one minute into 100 mM sodium-acetate sea water at pH 6.3. When D–600, a calcium transmembrane fluxes inhibitor, is added 20 seconds after fertilization, GVBD is inhibited but a normal acid release is recorded. The presence of at least 10 mM sodium ions in the external medium is required for 100% activation of these oocytes by an excess of KCl. These results suggest that while an intracellular pH increase may be a requisite for GVBD, this can not be a sufficient condition to trigger it unless a calcium influx is allowed to occur. Moreover, the acid release does not result from a Ca⁺⁺-H⁺ exchange transport but appears more likely to be due to a Na⁺-H^* exchange as it has been demontrated in sea urchin eggs.