External Ca Concentration Research Articles

Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

Hydroxyapatite (HAP) formation in acetate-driven partial denitrification process: Enhancing sludge granulation and phosphorus removal

Partial denitrification/anammox (PD/A) processes have emerged as a promising technology for efficient nitrogen removal from wastewater. However, these processes fail to remove phosphorus (P), a key pollutant that contributes to water eutrophication. To address this issue, the potential of inducing hydroxyapatite (HAP) precipitation in PD processes to achieve simultaneous P removal was investigated for the first time. Specifically, three SBRs (R1-R3) for PD were operated with adding varying concentrations of external Ca (30, 60, and 120 mg/L, respectively). Results demonstrated significant P reduction in all three SBRs, particularly in R3 with high Ca, which achieved an 80 % removal efficiency. Notably, sludge granulation was observed during operation, with the granule size in R3 with high Ca reaching 906.1 μm during the stable period, exceeding those in R2 (788.7 μm) and R1 (707.1 μm). This led to good settle ability of the PD sludge, as demonstrated by the lowest SVI5 (20 mL/g MLSS). Moreover, the decrease in the MLVSS/MLSS ratio suggested that the inorganic content accumulated, as observed by confocal laser scanning microscopy in the interior of the granules. Elemental composition analysis suggested that PD granules contained high P and Ca, while the X-ray diffraction (XRD) results confirmed the formation of HAP. Overall, this study demonstrated that PD-HAP coupled granular sludge process has potential as a robust and efficient method for nitrite production, as well as effective P removal and recovery, thereby advancing the application of anammox processes in wastewater treatment.

GABABR silencing of nerve terminals.

Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Presynaptic G-protein coupled receptors (GPCRs) are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Among the mechanisms by which GPCRs impact neurotransmission is by inhibiting voltage-gated calcium (Ca2+) influx in the active zone. Here, using quantitative analysis of both single bouton Ca2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca2+ influx and the concentration of external Ca2+ ([Ca2+]e). We find that this unexpected relationship is leveraged by GPCR signaling when operating at the nominal physiological set point for [Ca2+]e, 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or-none fashion at the single synapse level when operating at the physiological set point.

A differential proteomic study reveals the downregulation of several plasma membrane Ca2+-binding proteins in the sperm of Atlantic salmon (Salmo salar) following cold storage

Sperm motility is a key event in fertilization, which is regulated by different factors. Among the factors that most affect sperm motility in fish are the external concentrations of Ca2+, the influence of which is highly regulated by various plasma membrane Ca2+-binding proteins. Concentrations of this cation have also been shown to be one of the most important factors affecting motility in cold-stored sperm cells. Taking into account the aforementioned aspects, we carried out a differential proteomic study on Salmo salar sperm stored at 4 °C with the aim of evaluating the protein profile on day 0 and day 14. The results of our study showed that 401 proteins were significantly downregulated (p < 0.05) on day 14, where four of them are key in the sperm motility of Salmo salar. The results of this study will allow a better understanding of the sperm activation mechanisms of Salmo salar, which will be of great importance in the design of future cold storage strategies for sperm preservation.

CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp.

Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants.

The role of Ca2+ and Na+ membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility

The role of environmental ion composition and osmolality in Ca(2+) signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris-HCl pH 8.5. For investigation of spermatozoa reaction to external Ca(2+) concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na(+) concentration in conditions of low external Ca(2+), 100 µM amiloride was added to the EGTA-containing solutions as a Na(+) transport blocker. Low motility was observed in sucrose (Na(+) free) solutions containing 2 mM EGTA but not in Na(+) solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na(+) free) solutions containing 2 mM EGTA. We conclude that Na(+) transport in Ca(2+)-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na(+) and Ca(2+) transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.

Effects of Ca2+ on activity restoration of the damaged anammox consortium

Intracellular free Ca(2+) are canonically well known as significant "second messenger" in cells and regarded as critical regulators of bacterial metabolism. We investigated the influence of Ca(2+) dosage on the restoration of anammox consortium, in which nearly 80% cells were dead or badly damaged. Chemical analysis and flow cytometry (FCM) demonstrated that Ca(2+) dosage was of primary importance and the restoration process was apparently faster with increasing Ca(2+) as its concentration was ranged 0.02-0.5mM in feeding. Using FCM and Fura red fluorescence labeling for analysis of intracellular free Ca(2+), we found a strong correlation between external Ca(2+) concentration in feeding and the levels of steady-state intracellular free Ca(2+), the abundance of which was considered as the intrinsic causes for favoring anammox consortium restoration. This study provides new insight into the ions effects on rapid restoration of damaged anammox consortium, targeting efficient nitrogen removal from wastewater with anammox process.

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

To investigate the reverse mode function of Na(+)/Ca(2+) exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA. CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca(2+) level ([Ca(2+)]i) was measured using Ca(2+) imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na(+)-free medium. Ca(2+) paradox was induced by Ca(2+)-free EBSS medium, followed by Ca(2+)-containing solution (1.8 or 3.8 mmol/L CaCl2). The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca(2+)]i, which was followed by a Ca(2+) level plateau at higher external Ca(2+) concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca(2+)]i at higher external Ca(2+) concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L). Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca(2+)]i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.

Potentiation of mGlu7 receptor-mediated glutamate release at nerve terminals containing N and P/Q type Ca2+ channels

Calcium channels that mediate glutamate release (N-type and P/Q-type) are expressed in distinct populations of cerebrocortical nerve terminals in adult mice. mGlu7 receptors are exclusively expressed in nerve terminals containing N-type Ca2+ channels, which are less tightly coupled to glutamate release than P/Q-type Ca2+ channels. We recently reported that in addition to inhibit, mGlu7 receptors can also potentiate glutamate release via phosphatidyl inositol (4,5)-bisphosphate hydrolysis and activation of the non-kinase diacylglycerol binding protein Munc13-1, a protein that primes synaptic vesicles for exocytosis. Here, we assessed whether mGlu7 receptor-mediated potentiation of glutamate release is restricted to nerve terminals expressing N-type Ca2+ channels to compensate for their weak coupling to release. In the hippocampus, mGlu7 receptors are expressed both in nerve terminals containing N-type Ca2+ channels and in nerve terminals containing P/Q-type Ca2+ channels. When analyzed, we observed potentiation of mGlu7 receptor mediated release in wild type hippocampal nerve terminals at physiological (1.3 mM) and low (0.1 mM) concentrations of external Ca2+. By contrast, in nerve terminals from mice lacking the α1B subunit of N-type channels (Cav2.2), in which evoked release is mediated by P/Q-type channels only, no release potentiation was observed at 1.3 mM Ca2+. We conclude that release potentiation at 1.3 mM [Ca2+]e occurs in nerve terminals expressing N-type channels, whereas that which occurs at low 0.1 mM [Ca2+]e represents the release from nerve terminals containing P/Q-type Ca2+ channels. Although, mGlu7 receptor mediated potentiation is independent of Ca2+ channel activity, as it was induced by the Ca2+ ionophore ionomycin, release potentiation is influenced by the Ca2+ channel type and/or the associated release machinery.

Abstract 17427: A Unique “Cellular Fibrillation” Status in Mouse Ventricular Myocytes

Different intracellular calcium (Ca i ) wave patterns have been observed in cardiac myocytes. However, their mechanisms and corresponding electrophysiological properties remain unclear. In the present study, we have generated various patterns of Ca i waves in isolated mouse ventricular myocytes under highly-activated Ca i cycling states, such as high external Ca concentration or in the present of -adrenergic stimulation (i.e. isoproterenol and Bay K8644). Our results revealed at least three distinct types of Ca wave dynamics: I) slow propagating planar waves; II) Repetitive single spiral waves; III) Multiple spiral/micro waves, in which average Ca i level remained high. All waves were completely blocked by tetracaine (1 mM), suggesting RyR leaking is the original source for the waves. Simultaneous recording of membrane potential under current-clamp mode revealed that these complex Ca i wave dynamics have several functional consequences. The cell exhibited individual or oscillatory EADs and/or DADs under the conditions with type I or type II Ca i waves. Interestingly, a unique “cellular fibrillation” status was associated with type III Ca i waves. Under this condition, the cell maintained a high Ca i level and a depolarized membrane potential (~ -20 mV) and thus lost its excitability and contractility. We also found that in the same cell, the Ca waves can be converted from one type to another by changing the Ca handling conditions. The multiple spiral/micro waves (type III) were transformed into slow-propagating, planar waves (type I) by a Ca/Na-free Tyrode's solution or by Gadolinium (Gd 3+ ), a nonselective TRPC channel blocker; but not by nifedipine (L-type Ca channel blocker), SEA0400 (Na-Ca exchange blocker) or TTX (Na channel blocker). Hyperpolarizing the cell membrane by external injection of an outward current also transformed type III to type I Ca waves, suggesting the facilitating role of membrane current in the generation of the cellular fibrillation status. Termination of the cellular fibrillation by blocking TRPC channel activity may provide new opportunities for the treatment of clinical ventricular fibrillation.

Effects of external calcium on the biotransformation of ginsenoside Rb1 to ginsenoside Rd by Paecilomyces bainier 229-7

Calcium is a known signalling molecule in eukaryotic cells and plays a central role in the regulation of many cellular processes. In the following study, we report on the effect of external calcium treatments on the biotransformation of ginsenoside Rb1 to ginsenoside Rd by Paecilomyces bainier 229-7. We observed that the intracellular calcium content of P. bainier 229-7 mycelia was increased in response to exposure to high external Ca(2+) concentrations. Both ginsenoside Rd biotransformation and β-glucosidase activity were both found to be dependent on the external calcium concentration. At an optimal Ca(2+) concentration of 45 mM, maximal ginsenoside Rd bioconversion rate of 92.44% was observed and maximal β-glucosidase activity of 0.1778 U was reached in a 72-h biotransformation. The Ca(2+) channel blocker Verapamil blocked the trans-membrane influx of calcium and decreased ginsenoside Rd biotransformatiom. In addition, β-glucosidase activity and ginsenoside Rd content decreased by 36.0 and 29.2% respectively after a 72-h incubation in the presence of 0.05 mM Calmodulin (CaM) antagonist Perphenazine. These results suggest that both Ca(2+) channels and CaM are involved in ginsenoside Rd biotransformation via regulation of β-glucosidase activity. This is the first report regarding the effects of calcium signal transduction on biotransformation and enzyme activity in fungi.

Selectivity in biomineralization of barium and strontium

The desmid green alga Closterium moniliferum belongs to a small number of organisms that form barite (BaSO(4)) or celestite (SrSO(4)) biominerals. The ability to sequester Sr in the presence of an excess of Ca is of considerable interest for the remediation of (90)Sr from the environment and nuclear waste. While most cells dynamically regulate the concentration of the second messenger Ca(2+) in the cytosol and various organelles, transport proteins rarely discriminate strongly between Ca, Sr, and Ba. Herein, we investigate how these ions are trafficked in C. moniliferum and how precipitation of (Ba,Sr)SO(4) crystals occurs in the terminal vacuoles. Towards this goal, we simultaneously visualize intracellular dynamics of multiple elements using X-ray fluorescence microscopy (XFM) of cryo-fixed/freeze-dried samples. We correlate the resulting elemental maps with ultrastructural information gleaned from freeze-fracture cryo-SEM of frozen-hydrated cells and use micro X-ray absorption near edge structure (micro-XANES) to determine sulfur speciation. We find that the kinetics of Sr uptake and efflux depend on external Ca concentrations, and Sr, Ba, and Ca show similar intracellular localization. A highly ion-selective cross-membrane transport step is not evident. Based on elevated levels of sulfate detected in the terminal vacuoles, we propose a "sulfate trap" model, where the presence of dissolved barium leads to preferential precipitation of (Ba,Sr)SO(4) due to its low solubility relative to SrSO(4) and CaSO(4). Engineering the sulfate concentration in the vacuole may thus be the most direct way to increase the Sr sequestered per cell, an important consideration in using desmids for phytoremediation of (90)Sr.

Hippocampal mossy fiber long‐term depression in Grm2/3 double knockout mice

Group II metabotropic glutamate receptors (mGluR2, encoded by Grm2, and mGluR3, encoded by Grm3) are inhibitory autoreceptors that negatively modulate the adenylate cyclase signaling cascade. Within the hippocampus, mGluR2 is believed to play a key role in the induction of long-term depression (LTD) at mossy fiber-CA3 synapses. Here, we used Grm2/3 double knockout (dko) mice to investigate to what extent group II mGluRs are necessary for mossy fiber LTD. Surprisingly, we found that these mice displayed prominent mossy fiber LTD. However, the induction of this form of LTD was sensitive to the external Ca(2+) concentration. Mossy fiber LTD in Grm2/3 dko mice was indistinguishable from that in wild-type mice at 4 mM Ca(2+) , but largely absent at 2 mM external Ca(2+) . Mossy fiber LTD in Grm2/3 dko mice was not blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist D-AP5, confirming that the observed response did not reflect NMDA receptor-dependent LTD in contaminating associational-commissural fibers, and enabling us to use the NMDA receptor-mediated EPSC to monitor mossy fiber LTD. Using whole-cell recordings, we demonstrated that LTD of the NMDA receptor-mediated EPSC in Grm2/3 dko mice was not affected by intracellular application of BAPTA and CsF to block postsynaptic Ca(2+) and G-protein-mediated effects. This presynaptic LTD was, however, blocked by the AMPA/kainate receptor antagonist, NBQX. Thus, an activity-dependent, external Ca(2+) concentration-sensitive form of mossy fiber LTD can be induced in Grm2/3 dko mice. Two mGluR antagonists also failed to block mossy fiber LTD under 4 mM conditions in wild-type mice, strengthening the conclusion that group II mGluRs are not obligatory for mossy fiber LTD.

Anoxia-induced elevation of cytosolic Ca2+ concentration depends on different Ca2+ sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.

Evoked Centripetal Ca 2+ Activation in Cardiac Purkinje Cells: CICR or Ca 2+ Diffusion?

In large mammals, cardiac Purkinje cells (Pcells) lack transverse tubules (T-tubules). However, Pcells respond to stimulations by massive Ca-releases from the sarcoplasmic reticulum (SR) similar to those of ventricular myocytes (VMs) with T-tubules. Ca-imaging revealed a wave of elevated Ca-concentration (Cai) propagating from the sarcolemma (SL) toward the centre of Pcells upon stimulation. Ca-diffusion was proposed to explain this centripetal propagation. A thin layer of SR expressing RyR3 was found ∼5µm below the SL while other SR-regions expressed RyR2. Ca-Induced Ca-Release (CICR) from RyR3-SR-region could relay Ca-signal from Ca-entry to RyR2-Ca-release sites in the core, playing equivalent role as T-tubules in VMs. Our objective wasto examine whether centripetal Ca-signaling results exclusively from Ca-diffusion or involved intermediate RyR3-CICR. Methods:Pcells were prepared from Yucatan swine (30-40kg) and incubated with Fluo4-AM. Ca-dynamics was assessed by 2D-confocal microscopy (30-100fps); pH7.3,35oC. 26 cells were field stimulated at various external Ca-concentrations (Cao). Ca-data were compared with those from a model of Ca-propagation across 3 virtual adjacent SR-Ca-release regions. Results: In stimulated Pcells, Ca-increase first happened under SL with amplitude increasing from 0.1 to 0.5µmol/L with Cao from 1 to 4mmol/L. A Ca-wave then propagated uniformly from the SL toward cell center at 100-250µm/s. An overall Ca-transient appeared when Ca-wave reached the core region. When the model included intermediate CICR-region, predicted data were consistent with Pcells observations: minimal amplitude (minAmp) of peripheral Ca-increase mediating a central Ca-transient similar to that of Pcells, was 130nmol/L; this generated Ca-propagation with velocity (propVel) of 160µm/s; propVel was independent on Cao. In contrast, minAmp and propVel were 220nmol/L and 40µm/s respectively, and propVel increased with Cao when only diffusion was considered. Conclusion: Our data were consistent with an intermediate CICR-mechanism which “boosts” Ca-propagation between SL and core of Pcells.

Chlorophyll fluorescence images demonstrate variable pathways in the effects of plasma membrane excitation on electron flow in chloroplasts of Chara cells

Chlorophyll fluorescence Imaging and Microscopy PAM fluorometry were applied to study spatial dynamics of photosystem II quantum yield (ΔF/F'(m)) and non-photochemical quenching (NPQ) in resting and electrically stimulated Chara corallina cells in the absence and presence of the hydrophilic electron acceptor methyl viologen (MV) in the external medium. Electrical excitation of the plasma membrane temporarily enhanced the heterogeneity of photosynthetic patterns under physiological conditions (in the absence of MV), but irreversibly eliminated these patterns in the presence of MV. These findings suggest that the action potential (AP) of the excitable plant cell affects the spatial patterns of photosynthesis and chlorophyll fluorescence through different pathways operated in the absence and presence of MV. Based on the extent of NPQ as an indicator of MV-dependent electron flow, it is supposed that MV cannot permeate into the chloroplasts of photosynthetically active "acid cell regions" but gains an immediate access to the stroma of these chloroplasts after triggering of an AP. The AP-triggered MV-dependent non-photochemical quenching in the chloroplasts of acidic cell regions was routinely observed at 0.1 mM Ca(2+) in the medium but not at elevated (2 mM) external Ca(2+) concentration. The results are interpreted in terms of competition between two permeant divalent ion species, Ca(2+) and MV(2+), for their passage through the voltage-gated calcium channels of the plasma membrane. It is proposed that the herbicidal activity of MV in characean cells, here serving as model object, can be manipulated by triggering AP and varying Ca(2+) concentration in the environmental medium.

Calcium Nutrition of Peanut Grown in Solution Culture. I. Genetic Variation in Ca Requirements for Vegetative Growth

ABSTRACT Calcium (Ca) deficiency is an important problem in peanut (Arachis hypogaea L.) production, with lines from the sub-species A. hypogaea hypogaea (Virginia type) being considered more susceptible to Ca deficiency than those of A. hypogaea fastigata (Spanish and Valencia types). Fifteen peanut lines comprising five Virginia bunch, one Virginia runner, six Spanish, two Valencia types, and one Valencia × Spanish cross were grown for 30 d in continuously flowing solution culture at constant external Ca concentrations ranging from 9 to 2500 μM. Two lines required <9 μM Ca for near-maximum growth, whereas 5 lines required 15–20 μM, six lines required 25–50 μM, and two lines required >75 μM Ca for maximum growth. The Ca concentration in the youngest fully expanded leaves required for maximum growth also differed considerably among the lines, ranging from <5.7 g kg−1 in ‘SH-2’ to 17.3 g kg−1 in ‘Red Spanish’). The differences in solution and tissue Ca requirement for growth did not coincide with the broad botanical groupings despite that Virginia type peanut lines had consistently lower Ca concentrations in youngest fully expanded leaves than Spanish or Valencia lines at all solution Ca concentrations. Leaf magnesium (Mg), potassium (K), phosphorus (P), iron (Fe), and manganese (Mn) concentrations in the leaves decreased as the solution Ca concentration increased and it seemed probable that growth reductions observed at above-optimum Ca concentrations were due to induced deficiencies of P, Mg, or possibly Fe.

Dihydropyridine‐ and voltage‐sensitive Ca2+ entry in human parathyroid cells

Patch-clamp and fluorescence measurements of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) were performed to directly detect extracellular Ca(2+) entry into cultured parathyroid cells from patients with secondary hyperparathyroidism. Cells loaded with fluo-3 AM or fluo-4 AM showed a transient increase in fluorescence (Ca(2+) transient) following 10 s exposure to 150 mm K(+) solution in the presence of millimolar concentrations of external Ca(2+). The Ca(2+) transient was completely inactivated after 30-40 s exposure to the high-K(+) solution, was reduced by dihydropyridine antagonists and was enhanced by FPL-64176, an L-type Ca(2+) channel agonist. The electrophysiological and pharmacological properties of the whole-cell Ca(2+) and Ba(2+) currents were similar to those of L-type Ca(2+) channels. The Ca(2+) transients induced by 10 s exposure to 3.0 mm extracellular Ca(2+) concentration ([Ca(2+)](o)) were inhibited by dihydropyridine antagonists and were partly inactivated following 30-40 s exposure to the high-K(+) solution. These results demonstrate, for the first time, that human parathyroid cells express L-type-like Ca(2+) channels that are possibly involved in the [Ca(2+)](o)-induced change in [Ca(2+)](i). This Ca(2+) entry system might provide a compensatory pathway for the negative feedback regulation of parathyroid hormone secretion, especially in hyperplastic conditions in which the Ca(2+)-sensing receptor is poorly expressed.

Involvement of actin filaments in rhizoid morphogenesis of Spirogyra

The role of actin filaments in rhizoid morphogenesis was studied in Spirogyra. When the algal filaments were severed, new terminal cells started tip growth and finally formed rhizoids. Actin inhibitors, latrunculin B and cytochalasin D, reversibly inhibited the process. A mesh-like structure of actin filaments (AFs) was formed at the tip region. Gd(3+) inhibited tip growth and decreased AFs in the tip region. Either a decrease in turgor pressure or lowering of the external Ca(2+) concentration also induced similar results. It was suggested that the mesh-like AF structure is indispensable for the elongation of rhizoids. A possible organization mechanism of the mesh-like AF structure was discussed.

Inhibitory Control Over Ca2+ Sparks via Mechanosensitive Channels Is Disrupted in Dystrophin Deficient Muscle but Restored by Mini-Dystrophin Expression

BackgroundIn dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca2+ release events (ECRE) in high Ca2+ external environments. Such ‘uncontrolled’ Ca2+ sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition.Methodology/Principal FindingsWe recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca2+ concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 µM), spider peptide GsMTx-4 (5 µM) and Gd3+ (20 µM) that block unspecific, specific cationic and Ca2+ selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation.Conclusions/SignificanceDystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca2+ homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.