Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Amyloid β Protein Potentiates Ca2+ Influx Through L-Type Voltage-Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage-sensitive Ca²⁺ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L-type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω-conotoxin GVIA (ω-CgTX-GVIA) or ω-agatoxin IVA (ω-Aga-IVA), the blocker for N- or P/Q-type VSCCs, had no effects. Whole-cell patch-clamp studies indicated that the Ca²⁺ current density of β(25–35)-treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca²⁺ uptake, which was sensitive to nimodipine. The 2',7'-dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca²⁺ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca²⁺ influx via the L-type VSCC, thereby inducing neurotoxicity.     

Cytotoxic Effect of β-Amyloid on a Human Differentiated Neuron Is Not Mediated by Cytoplasmic Ca2+ Accumulation

Abstract: The effects of synthetic β-amyloid (Aβ1–42) on cell viability and cellular Ca²⁺ homeostasis have been studied in the human neuron-like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolism. Aβ1–42 dose-dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 µ M , and the strongest effect was produced by 40 µ M . Control NT2N cells produced spontaneous cytosolic Ca²⁺ oscillations under basal conditions. These oscillations were inhibited dose-dependently (0.4–40 µ M ) by Aβ1–42 that was present in the cell culture for 1 or 4 days. Ca²⁺ wave frequency was decreased from 0.21 ± 0.02 to 0.05 ± 0.02/min, amplitude from 88 ± 8 to 13 ± 4 n M , and average Ca²⁺ level from 130 ± 8 to 58 ± 3 n M . The Ca²⁺ responses to 30 m M K⁺ and 100 µ M glutamate were not different between control and Aβ-treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca²⁺ accumulation mediates Aβ-induced NT2N cell death.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

Down-Regulation of β-Adrenergic Receptors by Pindolol in Gsα-Transfected S49 cyc− Murine Lymphoma Cells

The role of the alpha subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (GS alpha) in the down-regulation of beta-adrenergic receptors by pindolol was studied in S49 cyc- cells (normally GS alpha-deficient) transfected to express functional recombinant rat GS alpha. An inducible cell line (S49 GS alpha IND) was derived from S49 cyc- cells transfected with a vector containing the full-length coding sequence of GS alpha under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. GS alpha was not detectable in S49 GS alpha IND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [alpha-32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, GS alpha was present at a level 40-50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [alpha-32P]ADP-ribosylation. Membranes prepared from GS alpha IND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGS alpha +, contained the coding sequence of GS alpha under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc- cells, and the resulting cells, S49 GS alpha CST cells, expressed GS alpha at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 GS alpha CST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down-regulated the density of beta-adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc- or GS alpha IND cells. When GS alpha CST cells were exposed to 50 nM pindolol for 12 h, the density of beta-adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that GS alpha is necessary to restore the ability of pindolol to down-regulate beta-adrenergic receptors in S49 cyc- cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.     

Functional Analysis and Tissue-Specific Expression of Drosophila Na+,K+-ATPase Subunits

Abstract: We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the β subunits of Na⁺,K⁺-ATPase from various other species. In this study we have isolated a new Drosophila Na⁺,K⁺-ATPase α subunit cDNA clone (PSα; GenBank accession no. AF044974) and demonstrate expression of functional Na⁺,K⁺-ATPase activity when PSα mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na⁺,K⁺-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na⁺,K⁺-ATPase expression in various cell and tissue types.     

Bovine haemoglobin βA Zebu, βA43(CD3)Ser Thr: an intermediate globin type between the βA and βD Zambia is present in Indian zebu cattle

Two bovine haemoglobin beta chains, electrophoretically identical with the beta A chain of Herefords, were obtained from Ongole and Banteng, Bos javanicus, cattle. The amino acid residue differences of the two beta chains were compared by electrophoresis, cation-exchange and reverse-phase chromatography, amino acid analyses, and Edman degradation in comparison with beta A chain. The results showed that two beta chains differed from the beta A chain of the Hereford breed by the substitution of serine with threonine at the beta 43 position. No other difference was found between the two chains and beta A. This new beta chain type was termed beta A Zebu, which forms a possible evolutionarily transitional type between the beta A and the rare variant beta D Zambia found previously in African zebu cattle. The beta A Zebu differentiates from the previous beta B by at least four amino acid substitutions involving five codon-base changes.     

β-Amyloid1–40 Increases Expression of β-Amyloid Precursor Protein in Neuronal Hybrid Cells

Abstract: Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β-amyloid peptide (β-AP), a 40–43-amino-acid peptide derived from a larger membrane glycoprotein, β-amyloid precursor protein (β-APP). Previous experiments have demonstrated that β-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β-APP mRNA content is increased 3.5-fold in 24 h after treatment with β-AP_1–40. Accompanying β-AP_1–40-induced cell injury, levels of cell-associated β-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of β-APP and 12- and 4-kDa fragments are also increased. Application of β-APP antisense oligodeoxynucleotide reduces both cytotoxicity and β-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β-APP expression. These results suggest a selective positive feedback mechanism whereby β-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β-APP with the net consequence of further neuronal damage.     

Rapid Communication: Ca2+ Channel Blockers Attenuate β-Amyloid Peptide Toxicity to Cortical Neurons in Culture

Abstract: Deposit of β-amyloid protein (Aβ) in Alzheimer's disease brain may contribute to the associated neurodegeneration. We have studied the neurotoxicity of Aβ in primary cultures of murine cortical neurons, with the aim of identifying pharmacologic ways of attenuating the injury. Exposure of cultures to Aβ (25–35 fragment; 3–25 4mU M ) generally triggers slow, concentration-dependent neurodegeneration (over 24–72 h). With submaximal Aβ- (25–35) exposure (10 μ M ), substantial (>40% within 48 h) degeneration often occurs and is markedly attenuated by the presence of the Ca²⁺ channel blockers nimodipine (1–20 μ M ) and Co²⁺ (100 μ M ) during the Aβ exposure. However, Aβ neurotoxicity is not affected by the presence of glutamate receptor antagonists. We suggest that Ca²⁺ influx through voltage-gated Ca²⁺ channels may contribute to Aβ-induced neuronal injury and that nimodipine and Co²⁺, by attenuating such influx, are able to attenuate Aβ neurotoxicity.     

Lysophosphatidic Acid-Induced Neurite Retraction in PC12 Cells: Control by Phosphoinositide-Ca2+ Signaling and Rho

Abstract: The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP-ribosylation of the small-molecular-weight G protein Rho by the Clostridium botulinum C-3 toxin. LPA induced a transient rise in the level of inositol 1,4,5-trisphosphate, and retraction was blocked by inhibitors of phospholipase β. Repeated application of LPA elicited homologous desensitization of the Ca²⁺ mobilization response. The activation of the phosphoinositide (PIP)-Ca²⁺ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C—chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long-term phorbol ester treatment—all diminished neurite retraction by interfering with LPA-induced Ca²⁺ mobilization, which was required for the withdrawal of neurites. A brief 15-min treatment with 4β-phorbol 12-myristate 13-acetate also blocked retraction and Ca²⁺ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP-Ca²⁺ second messenger system appears necessary for the Rho-mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA-induced morphoregulatory response.     

稀土La3+跨PC12细胞膜行为研究

使用AR-CM-M1C阳离子测定系统,发展Fura-2荧光测定技术,将其应用于测定细胞内游离稀土离子La³⁺,并以此研究了La³⁺跨PC12细胞（大鼠嗜铬细胞瘤细胞）膜的行为.结果表明：在模拟细胞内离子组分,pH=7.05的溶液中,测得La³⁺-Fura-2的表观解离常数为3.27×10^-11 mol·L^-1.对于PC12细胞,静息条件下La³⁺不能跨越细胞膜进入胞内.与钙离子通道相关的KCl和去甲肾上腺素均不能刺激稀土La³⁺过膜.用哇巴因（ouabain）使胞内Na⁺超载后,La³⁺可过膜进入细胞内,且过膜量与胞外La³⁺浓度和胞内Na⁺超载程度有一定的浓度依赖关系,提示La³⁺可以经由Na⁺／La³⁺交换机制过膜而进入细胞内.     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

Amyloid β Toxicity Consists of a Ca2+-Independent Early Phase and a Ca2+-Dependent Late Phase

Abstract: Amyloid β protein (Aβ), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of Aβ. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of Aβ and reaches a plateau in 12 h. This effect of Aβ is not blocked by Ca²⁺ channel blockers or in Ca²⁺-free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of Aβ and are blocked by Ca²⁺ channel blockers such as Co²⁺, nicardipine, and diltiazem. When Aβ was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long-lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that Aβ toxicity consists of two phases—a Ca²⁺-independent early phase and a Ca²⁺-dependent late phase—and that the early phase may be required to induce the late phase.     

Effects of interrupted K+ supply on growth and uptake of K+, Ca2+, Mg2+ and Na+ in spring wheat

Effects of interrupted K⁺ supply on different parameters of growth and mineral cation nutrition were evaluated for spring wheat (Triticum aestivum L. cv. Svenno). K⁺ (2.0 mM) was supplied to the plants during different periods in an otherwise complete nutrient solution. Shoot growth was reduced before root growth after interruption in K⁺ supply. Root structure was greatly affected by the length of the period in K⁺ -free nutrient solution. Root length was minimal, and root branching was maximal within a narrow range of K⁺ status of the roots. This range corresponded to cultivation for the last 1 to 3 days, of 11 in total, in K⁺ -free nutrient solution, or to continuous cultivation in solution containing 0.5 to 2 mM K⁺. In comparison, both higher and lower internal/external K⁺ concentrations had inhibitory effects on root branching. However, the differing root morphology probably had no significant influence on the magnitude of Ca²⁺, Mg²⁺ and Na⁺ uptake. Uptake of Ca²⁺ and especially Mg²⁺ significantly increased after K⁺ interruption, while Na⁺ uptake was constant in the roots and slowly increased in the shoots. The two divalent cations could replace K⁺ in the cells and maintain electroneutrality down to a certain minimal range of K⁺ concentrations. This range was significantly higher in the shoot [110 to 140 μmol (g fresh weight)^?1] than in the root [20 to 30 μmol (g fresh weight)^?1]. It is suggested that the critical K⁺ values are a measure of the minimal amount of K⁺ that must be present for physiological activity in the cells. At the critical levels, K⁺ (⁸⁶Rb) influx and Ca²⁺ and Mg²⁺ concentrations were maximal. Below the critical K⁺ values, growth was reduced, and Ca²⁺ and Mg²⁺ could no longer substitute for K⁺ for electrostatic balance. In a short-term experiment, the ability of Ca²⁺ to compete with K⁺ in maintaining electroneutrality in the cells was studied in wheat seedlings with different K⁺ status. The results indicate that K⁺, which was taken up actively and fastest at the external K⁺ concentration used (2.0 mM), partly determines the size of Ca²⁺ influx.     

Fibroblast growth factor 9 prevents MPP+-induced death of dopaminergic neurons and is involved in melatonin neuroprotection in vivo and in vitro

Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP⁺ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP⁺ intoxication and melatonin protection. We found that MPP⁺ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP⁺-induced cell death of DA neurons. Melatonin co-treatment attenuated MPP⁺-induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro . Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP⁺, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP⁺ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection.     

Effects of Na+, Ca2+, and Acetylcholine on Phosphoinositide- and ATP-Phosphate Turnover in 32P-Labeled Rabbit Iris Smooth Muscle

Parallel studies were carried out in the rabbit iris on (a) the effects of Na+ and/or Ca2+ on the acetylcholine-stimulated 32P labeling of phosphatidic acid (PA) and phosphatidylinositol (PI) and the breakdown of polyphosphoinositides (poly PI), and (b) the effects of these cations on the specific radioactivity of [gamma-32P]ATP. Incorporation of 32P1 into ATP and phosphoinositides is time-dependent, and it is remarkably dependent upon Na+ concentration in the incubation medium. The Na+ effect is reversible. Calcium ion, in the absence of Na+, had no effect on the specific radioactivity of ATP in 32P-labeled iris muscle; however, it moderately stimulated the 32P labeling of PA and PI and the breakdown of poly PI. In contrast, the addition of Na+, in the presence or absence of Ca2+, significantly reduced the specific radioactivity of ATP and 32P labeling of phospholipids in the 32P-labeled iris muscle. Acetylcholine had no measurable effect on the specific radioactivity of ATP. Furthermore, the neurotransmitter stimulated the 32P labeling of PA and PI and the breakdown of poly PI in the 32P-labeled muscle only in the presence of both Na+ and Ca2+. These data provide additional support for the concept that in the rabbit iris receptor-activated Ca2+ fluxes mediate or precede the effects of alpha-adrenergic and cholinergic muscarinic agents on phosphoinositide breakdown into 1,2-diacylglycerol and inositol phosphates and that restoration of the polar head groups to the 1,2-diacylglycerol (i.e., the recovery stage) is probably associated with Na+ outflux, via the Na+ -pump mechanism.     

H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.