Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca²⁺-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca²⁺-transporter showed high affinity for Ca²⁺ (K_Ca ²⁺ = 0.04 ± 0.01 M), an apparent requirement for Mg²⁺ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca²⁺ or Mg²⁺ but showed a low affinity for these cations. At low concentrations, Mg²⁺ appeared to inhibit the Ca²⁺-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg²⁺ concentration (0.5–1 mM), a high affinity Ca²⁺-dependent ATPase activity was observed (K_Ca ²⁺ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca²⁺-transport nor high affinity Ca²⁺-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca²⁺ (or Mg⁺)-ATPase activity, the presence of both a high affinity Ca²⁺-pump and high affinity Ca²⁺-dependent ATPase. However, the high affinity Ca²⁺-ATPase activity does not appear to be the biochemical expression of the Ca²⁺-pump.Abbreviations Ca²⁺-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate     

The role of Ca2+ in elicitation of phytoalexin synthesis in cell culture of onion (Allium cepa L.)

Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca²⁺ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca²⁺ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca²⁺ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca²⁺ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187 4-bromo-calcium ionophore - cAMP adenosine 3,5-cyclic monophosphate - [Ca²⁺]_cyt cytoplasmic Ca²⁺ concentration - EGTA ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid - EtOH ethanol - Et₂O diethyl ether - fr.wt fresh weight - HR hypersensitive response - PIPES piperazine N,N-bis-(2-ethanesulfonic acid) - TMB-8 [8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate - Tsl tsibulin     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Striated ciliary root-golgi association in branchial crown epithelial cells ofOwenia. Visualization of Ca2+-binding sites and ATPase activities

Summary Striated Ciliary Roots (SCRs), about 3 m long, are attached to the basal bodies of branchial crown ciliated epithelial cells ofOwenia. These SCRs appear to consist of 5–7-nm diameter filaments organized in a cross-striation pattern with an apparent variable periodicity of 50 to 80 nm. The most exciting observation emerging from this study is the constant and conspicuous close spatial relationship between SCRs and fairly well developed Golgi apparatus. By enhancing contrast and preservation of cell components, the OsFeCN postfixation-staining of material prefixed in glutaraldehyde in the presence of calcium has revealed some fine-structural details within the SCR-Golgi Association. By means of the calcium precipitation method, with antimonate or oxalate in conjunction with X-ray microanalysis, we have identified calcium within SCR dark bands and SCR-associated Golgi bodies. The ability to bind calcium makes the Golgi apparatus a likely candidate for Ca²⁺ regulation of putative contraction of the SCRs and/or ciliary motility. The slight period variability measured in the SCRs and cytochemical localization of Mg²⁺, Ca²⁺-dependent ATPase activities associated with cross striations support the view that theOwenia SCRs may be contractile organelles.The striking and specific close structural association between the Golgi apparatus and the SCR showing Ca²⁺-binding capabilities suggests that some sort of Ca²⁺-mediated functional relationship between these organelles may exist.Abbreviations SCR striated ciliary root - OsFeCN method osmium tetroxide-ferricyanide method - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - ATP adenosine 5-triphosphoric acid - ATPase adenosine triphosphatase - ASW artificial sea-water     

Mitochondria in Ca2+ Signaling and Apoptosis

Cellular Ca²⁺ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa²⁺ ([Ca²⁺]_c) signals. Mitochondria are endowed with multiple Ca²⁺ transport mechanismsby which they take up and release Ca²⁺ across their inner membrane. These transport processesfunction to regulate local and global [Ca²⁺]_c, thereby regulating a number of Ca²⁺-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca²⁺ effluxpathway from mitochondria. In addition, Ca²⁺ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na⁺/Ca²⁺ exchanger. Duringcellular Ca²⁺ overload, mitochondria take up [Ca²⁺]_c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (_m) and cell death. In apoptosis signaling,collapse of ;_m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed.     

Involvement of Ca2 + and flowing endoplasm in recovery of cytoplasmic streaming after K+-induced cessation

Summary When K⁺ of high concentration (50 mM) was applied toNitella cells, the cytoplasmic streaming stopped instantly as in the case of electrical stimulation. Recovery of the streaming after chemical stimulation was much slower than after electrical stimulation. When the endoplasm content was modified by centrifugation, streaming recovery was accelerated in the centrifugal cell fragments rich in endoplasm and deccelerated in those poor in it. The recovery was also accelerated either by permeabilizing the plasmalemma in the presence of EGTA in the external solution or by removing the tonoplast by vacuolar perfusion with the EGTA-containing medium. We concluded that the streaming was recovered due to decrease of the cytoplasmic Ca²⁺ concentration, which seems to be accelerated by sequestering of Ca²⁺ by endoplasmic components. The slow recovery of the streaming after KCl-stimulated cessation is assumed to be caused by continuous influx of Ca^{2 +} during the prolonged membrane depolarization.Abbreviations ATP adenosine 5-triphosphoric acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

Ca2+-induced fragmentation of actin filaments in pollen tubes

Summary To control the intracellular free Ca²⁺ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca²⁺ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca²⁺ concentration of the external medium ([Ca²⁺]) was raised to 5×10^–6 M or higher. At [Ca²⁺] below 1×10^–6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca²⁺] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca²⁺ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.Abbreviations ATP adenosine-5-triphosphoric acid - [Ca²⁺] concentration of free Ca²⁺ - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - Rh-ph rhodamine-conjugated phalloidin     

Possible mechanism of ciliary stimulation by extracellular ATP: Involvement of calcium-dependent potassium channels and exogenous Ca2+

Summary Ciliary motility was examined optically in tissue cultures from frog palate epithelium and frog's esophagus as a function of extracellular concentration of adenosine 5-triphosphate (ATP) and related compounds. The addition of micromolar concentration of ATP caused a strong enhancement of frequency and wave velocity in the direction of the effective stroke. Since adenosine 5-[, imido]-triphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, produces the same effects, ATP hydrolysis is not required. The overall potency is ATP AMP-PNP>ADP adenosine>AMP. It is suggested that both the phosphate and the base moieties are involved in ATP binding.The enhancement of ciliary activity by extracellular ATP is dependent on the presence of extracellular Ca²⁺, which can be replaced by extracellular Mg²⁺. The effect of a number of potent inhibitors of the voltage-gated calcium channels on the stimulation of ciliary activity by ATP were examined. No effect was detected in the concentration range within which these agents are specific. On the other hand, quinidine, a potent inhibitor of K⁺ (calcium-dependent) channels, inhibits the effect of ATP.The following model is suggested: exogenous ATP interacts with a membrane receptor in the presence of Ca²⁺, a cascade of events occurs which mobilizes intracellular calcium, thereby increasing the cytosolic free Ca²⁺ concentration which consequently opens the calcium-activated K⁺ channels, which then leads to a change in membrane potential. The ciliary response to these changes is the enhancement of ciliary activity.This work was supported by a grant from the Fund for Basic Research administered by the Israel Academy of Science and Humanities.     

Calcium and tip growth inNeurospora crassa

Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K⁺) or change dramatically (e.g., Ca²⁺ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca²⁺ influx) may not be required for tip growth. However, intracellular Ca²⁺ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca²⁺ directly by ionophoresis. Elevated Ca²⁺ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca²⁺ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca²⁺ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca²⁺ and dissipation of the tip-high gradient. We conclude that Ca²⁺ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca²⁺ gradient is generated from intracellular Ca²⁺ stores in the ascomyceteN. crassa.Abbreviations BAPTA 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - [Ca²⁺]_i intracellular Ca²⁺ concentration - fluo-3 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1

Under diurnal 16/8-h light-dark cycles, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) at 1 mM completely blocked the appearance of rhythmic N₂-fixing activity in Synechococcus RF-1. Ca²⁺ at 2 mM, when supplied either together with or several hours after the EGTA application, restored the nitrogenase activity, whereas, when Ca²⁺ was supplied several hours later, the peak of nitrogenase activity was shifted from the dark to the light period in which the activity is normally suppressed. Sr²⁺ also reversed the inhibition by EGTA, but only partially. When O₂ in the gas phase above the culture was below 1%, the inhibition of nitrogenase activity by EGTA was reduced to less than 20% of the control value without EGTA. Thus Ca²⁺ appears to be required by the cell to protect its nitrogenase from inactivation by O₂. In media without EGTA, a close correlation between nitrogenase activity and concentrations of Ca²⁺ was also observed.Abbreviation EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA). A free-Ca²⁺ activity of less than 40 M restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca²⁺-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca²⁺ during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca²⁺ addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N,-tetracetic acid - FR far-red light - Mes 2-(N-morpholino)ethanesulphonic, acid - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Phosphorylation-dephosphorylation is involved in Ca2+-controlled cytoplasmic streaming of characean cells

Summary The mechanism of Ca²⁺ regulation of the cytoplasmic streaming in characean cells was studied in relation to protein phosphorylation and dephosphorylation. A tonoplast-free cell model was developed which was sensitive to Ca²⁺. Protein phosphatase-1 and its inhibitor-1 were applied into the tonoplast-free cells. A synthetic inhibitor of protein phosphatase, -naphthylphosphate, was applied either to tonoplast-free cells from inside or to the outside of plasmalemma-permeabilized cells which are known to be very sensitive to Ca²⁺. ATP--S applied to permeabilized cells strongly inhibited the recovery of the streaming which had been stopped by 10 M Ca²⁺. Both inhibitor-l and -naphthylphosphate inhibited the streaming even in the absence of Ca²⁺. On the other hand, protein phosphatase-l recovered the streaming even in the presence of Ca²⁺.The results indicate that characean streaming is regulated by the phosphorylation state of a regulatory and/or motile protein component. Streaming is activated when the component is dephosphorylated and inactivated when the component is phosphorylated. Ca²⁺ is assumed to stimulate both phosphorylation and dephosphorylation of the component. Involvement of Ca²⁺/calmodulin in the streaming recovery was discussed in terms of the stimulation of dephosphorylation.Abbreviations ATP -S, Adenosine-5-O-(3-thiotriphosphate) - -NP -naphthylphosphate - EGTA ethylenglycol-bis-(-aminoethylether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

The role of calcium ions in phytochrome-mediated germination of spores of Onoclea sensibilis L.

Phytochrome is confirmed to be the photoreceptor pigment in the germination response of Onoclea sensibilis L. by demonstrating red-far-red (R-FR) photoreversibility. External Ca²⁺ is required for this response with a threshold at a submicromolar concentration. Ethylene glycol-bis(-amino-ethyl ether)-N,N,N,N-tetraacetic acid, La³⁺ and Co²⁺ reversibly inhibit germination. Lanthanum only inhibits germination when applied before or during irradiation, indicating that the external Ca²⁺ requirement is transient, although in the absence of Ca²⁺ the R-stimulated system remains maximally poised to accept the ion for over 4 h after irradiation. The ability to respond to Ca²⁺ 4.1 h after R-irradiation is not reversed by FR-irradiation, indicating that Ca²⁺ transport has been uncoupled from phytochrome. Barium and Sr²⁺, but not Mg²⁺ can substitute for Ca²⁺. Artificially increasing the concentration of intracellular free Ca²⁺ with the ionophore A 23187 stimulates germination in the dark. The Ca²⁺-calmodulin antagonists, trifluoperizine and chlorpromazine, reversibly inhibit germination. Calcium is required in phytochrome-mediated fern spore germination; it may be acting as a second messenger.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - R fed light     

Functional mitochondria in snake Bothrops alternatus erythrocytes and modulation of HbO2 affinity by mitochondrial ATP

Digitonin was applied to permeabilize the plasma membrane of Bothrops alternatus erythrocytes to study respiration, oxidative phosphorylation and Ca²⁺ transport by mitochondria in situ. These mitochondria oxidized added NAD-linked substrates, succinate and N,N,N, N-tetramethyl-p-phenylenediamine. Respiration was sensitive to rotenone and cyanide but not to antimycin A. This indicates that Bothrops mitochondria possess the respiratory complexes NADH-ubiquinone, succinate-ubiquinone, and ferrocytochrome c-oxygen oxidoreductases, although the lack of sensitivity to antimycin A raises doubt about the composition of the ubiquinol cytochrome c-reductase complex. An ability to build up and sustain a membrane potential was documented by their capacity to accumulate tetraphenylphosphonium and Ca²⁺ through an uncoupler-sensitive mechanism. Addition of ADP caused a transient decrease in the membrane potential, indicating that this is the predominant driving force for ATP synthesis as in most types of mitochondria. Uncoupling of phosphorylation from the oxidative process increased hemoglobin O₂ affinity, which suggests that ATP production by mitochondria may participate in modulation of O₂ transport by hemoglobin.Abbreviations membrane potential - BAE Bothrops alternatus erythrocytes - DNP 2,4-dinitrophenol - DPG 2,3-diphosphoglycerate - EGTA ethyleneglycol tetra-acetic acid - FCCP carbonylcyanide p-trifloromethoxyphenylhydrazone - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TPP⁺ tetraphenylphosphonium - TRIS tris-(hydroxymethyl)aminomethane     

Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with “Ca2+ receptor” activation

We report the effects of tetracycline analogues on cytosolic Ca²⁺ transients resulting from application of ionic nickel (Ni²⁺), a potent surrogate agonist of the osteoclast Ca²⁺ receptor. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca²⁺] response to an application of 5 mM-[Ni²⁺]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca²⁺]. The results suggest a novel action of tetracyclines on the osteoclast Ca²⁺ receptor.