Apparent Ca Research Articles

Xenopus oocyte plasma membrane sheets for FRET analysis

Plasma membrane sheets from Xenopus oocytes have been isolated for use in fluorescence resonance energy transfer (FRET) measurements. This system has the following advantages: 1) fluorescent recordings from a large surface area to maximize the signal-to-noise ratio, 2) reduction in background fluorescence from proteins retained in intracellular compartments, and 3) access to the cytoplasmic surface of the plasma membrane for rapid solution changes. To demonstrate the utility of this approach, we have examined a previously published FRET-based Ca(2+) sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca(2+) additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca(2+) affinity of 1.65 microM was determined. Thus preparation of Xenopus oocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above.

In vitro investigation of the geometric contraction behavior of chemo-mechanical P-protein aggregates (forisomes)

We investigated the contracting behavior of forisomes from Vicia faba by carrying out precise measurements of their changing geometric parameters in vitro in the absence and in the presence of dissolved oxygen. Furthermore, we investigated the fine structure of forisomes by scanning electron microscopy. For the first time, single forisomes were titrated with Ca 2+, protons, and hydroxide ions recording the complete progression of their contractions. An apparent Ca 2+-binding constant of (22 ± 3) μM was calculated from two complete titration curves. The forisomes also contracted in the presence of Ba 2+ and Sr 2+ ions, but the amplitudes of contraction were smaller under the same measuring conditions. The time taken to change from the longitudinally expanded into the longitudinally contracted state was up to 2 s shorter in 10 mM Ca 2+ in comparison to 0.2 mM Ca 2+. However, the contraction time was prolonged by decreasing the Ca 2+ concentration. In the absence of dissolved oxygen, the transition between the two final states of the forisomes was almost reversible and the amplitude of contraction remained almost constant during the first 25 contraction cycles. In the presence of dissolved oxygen the forisomes denaturated after a few cycles and lost their ability to contract, just after only a few cycles with 10 min in the contracted state. Denaturation of the forisomes occurred appreciably in the contracted state. We propose a cycle process to explain the thermodynamic basis of the Ca 2+-induced contraction and its reversal by EDTA. Reducing the pH-value from 7.3 to 4.0 caused the forisomes to shorten by approximately 15%, while increasing the pH to 11.0 caused them to shorten by 28 to 30%. In both cases, the increases of the forisomes volume were greater than during the Ca 2+ induced contraction. The pH values of 4.7 ± 0.3, and 10.2 ± 0.2 marked the inflection points of the acid base titration of different forisomes.

Spatially explicit, nano-mechanical models of the muscle half-sarcomere: Implications for biomechanical tuning in atrophy and fatigue

Astronaut biomechanical performance depends on a wide variety of factors. Results from computational modelling suggest that muscle function—a key component of performance—could be modulated by compliance of the contractile filaments in muscle, especially when force is low such as transient Ca 2 + activation in a twitch, reduced activation in muscle fatigue encountered during EVA, or perhaps atrophy during prolonged space flight. We used Monte-Carlo models to investigate the hypotheses that myofilament compliance influences muscle function during a twitch, and also modulates the effects of cooperative interactions between contractile proteins on force generation. Peak twitch force and the kinetics of force decay were both decreased, while tension cost was increased, when myofilament compliance was increased relative to physiological values. Both the apparent Ca 2 + sensitivity and cooperativity of activation of steady-state isometric force were altered by myofilament compliance even when there were no explicit interactions included between binding sites. The effects of cooperative interactions between adjacent regulatory units were found to be greater than either the effect of myofilament compliance on apparent cooperativity of activation or that due to myosin cross-bridge-induced cooperativity. These results indicate that muscle function may be “tuned” at the molecular level, particularly under conditions of reduced Ca 2 + activation.

Inhibitory Gating Modulation of Small Conductance Ca2+-Activated K+Channels by the Synthetic Compound (R)-N-(Benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) Reduces Afterhyperpolarizing Current in Hippocampal CA1 Neurons

SK channels are small conductance Ca(2+)-activated K(+) channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca(2+) sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In whole-cell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit (125)I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca(2+) sensitivity by shifting the activation curve for Ca(2+) to the right, only slightly affecting the maximal Ca(2+)-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca(2+)-dependently (K(d) = 0.42, 0.60, and 0.73 microM, respectively, at 0.5 microM Ca(2+)), whereas the compound did not affect the Ca(2+)-activated K(+) channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 microM.

Protein and Mineral Digestibility of Three Pelleted Equine Feeds and Subsequent Nitrogen and Phosphorus Waste Excretion

AbstractThis experiment was designed to examine the digestibilities of 3 commercially available equine feeds when fed with Bermudagrass hay, and the N and P waste associated with these types of concentrate feeds. The feeds used were a 12% protein pelleted feed produced by 3 different manufacturers. Nine mature Quarter Horse geldings were used in a 3 × 3 Latin square design. The apparent digestibilities for Ca were 39.02%, 25.47%, and 39.00% for feeds A, B, and C , respectively, with apparent Ca digestibilities of feeds A and C significantly higher (P < .05) than feed B. Apparent digestibility of P was greater (P < .009) for feed A (28.65%, compared with feed B (15.63%) and C (16.08%). There were no differences for apparent CP digestibilities (feed A = 71.19%, feed B = 69.39%, and feed C = 68.37%). The apparent digestibilities for K and Mg were similar between feeds. The P and total kjeldahl N (TKN) in manure was similar among treatment groups. The mean urinary excretion of Ca, P, and TKN was similar among feeds. For feeds A, B, and C the TKN results were 1.08%, 1.13 % and 1.10%, respectively. The mean urinary excretion of K and Mg was greater for feed B compared with feeds A and C (P < .05). Data from this study suggest that similar types of equine pelleted feeds may vary slightly in apparent digestibility of nutrients but appear to produce similar amounts of waste products of environmental concern.

The role of panendoscopy in the staging of high risk head &amp; neck squamous cell carcinoma prior to therapy: Is it of value?

5569 Background: Panendoscopy (triple endoscopy) has historically been advocated to adequately stage patients w/ head &amp; neck squamous cancer, especially in the setting of neoadjuvant therapy. This role is controversial by in view of comparable information provided by current imaging techniques Objective: The primary objective was to analyze the results of panendoscopy in a series of patients enrolled in a neoadjuvant chemo radiation protocol. Methods: We reviewed the panendoscopy results of 69 pts w/ stage III/IV head &amp; neck squamous cell carcinoma (H&amp;N ca) treated w/ neoadjuvant chemo radiation at a single institution from 1996–2005. All pts had detailed laryngoscopy/microlaryngoscopy w/ strategic biopsies, esophago-duodeno-scopy (selected biopsies for Barretts) &amp; placement of percutaneous gastrostomy (PEG) &amp; bronchoscopy washings for cytology. Pts were initially treated w/chemo radiation protocols (H&amp;N 53,67); subsequent protocols utilized induction chemotherapy/chemo radiation (H&amp;N 79,86). Results: Two of 69 pts had malignant cells in bronchial cytology in absence of any lung findings (both were NED at 5 yrs). A radiologically occult primary bronchial cancer was found in an additional pt excluding pt from the protocol. Two pts were diagnosed w/ Barrett’s esophagus; another pt also had a synchronous primary cancer of the cervical esophagus. Nine of 69 pts underwent panendoscopy for cervical metastases from an apparent unknown primary ca (at initial diagnosis). The primary cancer was demonstrated in 6 pts at staging panendoscopy and in another patient at follow up panendoscopy. Conclusion: Panendoscopy is essential in staging high risk H &amp; N ca pts prior to initiation of therapy. A detailed laryngoscopy (micro-laryngoscopy) w/ strategic biopsies is required for adequate staging of all cancers of oropharynx &amp; larynx. Esophago-gastro-duodenoscopy had a low but definable yield, but is also necessary for PEG placement to ensure adequate nutrition during neoadjuvant therapy. Bronchoscopy has a limited role in the absence of radiologic chest findings. No significant financial relationships to disclose.

Deletions in the A L region of the h4xb plasma membrane Ca 2+ pump : High apparent affinity for Ca 2+ of a deletion mutant resembling the alternative spliced form h4zb

Mutants of the plasma membrane Ca 2+ pump (human isoform 4xb) with deletions in the linker between domain A and transmembrane segment M3 (A L region) were constructed and expressed in Chinese hamster ovary cells. The total or partial removal of the amino acid segment 300–349 did not change the maximal Ca 2+ transport activity, but mutants with deletions involving residues 300–338 exhibited a higher apparent affinity for Ca 2+ than the wild type h4xb enzyme. Deletion of the putative acidic lipid interacting sequence (residues 339–349) had no observable functional consequences. The removal of either residues 300–314 or 313–338 resulted in a similar increase in the apparent Ca 2+ affinity of the pump although the increase was somewhat lower than that obtained by the deletion 300–349 suggesting that both deletions affected the same structural determinant. The results show that alterations in the region of the alternative splicing site A change the sensitivity to Ca 2+ of the human isoform 4 of the PMCA.

Dietary Fibers from Mushroom Sclerotia. 4. In Vivo Mineral Absorption Using Ovariectomized Rat Model

The effect of three novel dietary fibers (DFs) prepared from mushroom sclerotia, namely, Pleurotus tuber-regium, Polyporus rhinocerus, and Wolfiporia cocos, on calcium and magnesium absorption was evaluated in ovariectomized (OVX) rats fed with sclerotial DF based and low Ca (0.3%) diets for 14 days. The animals in the W. cocos DF diet group possessed significantly (p < 0.05) higher levels of cecal total short-chain fatty acids (204 mumol/g of cecal content) and had an acidic pH (5.88) in their cecum when compared with those of the cellulose control group. Such an acidic environment was found to promote the ionization of the unabsorbed Ca and Mg in their cecum, which in turn significantly (p < 0.05) increased the concentrations of cecal soluble Ca (2.56-fold) and Mg (1.22-fold). Besides, the apparent Ca and Mg absorptions of the W. cocos DF group were also significantly (p < 0.05) enhanced (Ca, 16.5%; Mg, 15.3%) together with significantly (p < 0.05) higher serum Ca (3.61 mmol/L) and Mg (1.07 mmol/L) levels when compared with those of the cellulose control group. These data suggest that ingestion of W. cocos DF could improve the overall Ca and Mg absorptions of the OVX rats fed a low Ca diet. The potential use of sclerotial DFs as a functional food ingredient for enhancing mineral absorption is also discussed.

Augmenting neurotransmitter release by enhancing the apparent Ca 2+ affinity of synaptotagmin 1

Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.

Quantitative Calcium Measurements in Subcellular Compartments of Plasmodium falciparum-infected Erythrocytes

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.

Inhibition of plasma membrane Ca 2+-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca 2+-occluded state

The bidentate complex of ATP with Cr 3+, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca 2+-ATPase and the Na +,K +-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca 2+ and Na +, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca 2+-ATPase. The complex inhibited with similar efficiency the Ca 2+-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly ( T 1/2 = 30 min at 37 °C) with a K i = 28 ± 9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg 2+ but unaltered when Ca 2+ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca 2+ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La 3+ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca 2+ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca 2+ promoted by the plasma membrane Ca 2+-ATPase goes through an enzymatic phospho-intermediate that maintains Ca 2+ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.

Transient and sustained changes in myofilament sensitivity to Ca2+ contribute to the inotropic effects of sevoflurane in rat ventricle

The volatile anaesthetics isoflurane and sevoflurane induce both negative and positive inotropic effects in ventricular myocytes, the mechanisms of which are not fully understood. Previous data suggest that changes in myofilament Ca(2+) sensitivity contribute to their sustained negative inotropic effects. In this study, the role of changes in myofilament Ca(2+) sensitivity in both positive and negative inotropic effects of these agents was examined in intact ventricular myocytes. Contractility and cytosolic Ca(2+) (fura-2) were recorded optically in ventricular myocytes stimulated electrically (1 Hz) at 30 degrees C. Myofilament Ca(2+) sensitivity was assessed from plots of cell length against fura-2 fluorescence ratio (Fr) from individual twitches at various points before, during and after a 1 or 4 min exposure to 0.6 mM anaesthetic. Isoflurane reduced mean (sd) myofilament Ca(2+) sensitivity from 10.3 (1.9) to 5.9 (1.6) microm Fr(-1) (P<0.001) throughout a 1 min exposure, which returned to control on removal. In contrast, on initial exposure to sevoflurane, Ca(2+) sensitivity was reduced from 10.8 (1.3) to 4.3 (0.9) microm Fr(-1) (P<0.001) but this recovered partially towards control over 3 min. On removal, sensitivity was increased above control (to 17.7 (2.2) microm Fr(-1); P<0.001) before preanaesthetic levels were restored. These data show that both isoflurane and sevoflurane reduce apparent myofilament Ca(2+) sensitivity at steady state. However, sevoflurane (but not isoflurane) induced transient changes in apparent myofilament Ca(2+) sensitivity, which would contribute to its inotropic profile.

Effects of Dietary Phosphorus Intake on Bone Mineralization and Calcium Absorption in Adult Female Rats

We investigated the effects of dietary phosphorus (P) intake on the bone mineralization and calcium (Ca) absorption in adult female rats. Fifteen 16-wk-old female Wistar rats were divided into three groups, and respectively fed a low-P diet containing 0.15% P (LP), a control diet containing 0.5% P (C), and a high-P diet containing 1.5% P (HP) for 42 d. The apparent Ca absorption was significantly increased with decreasing dietary P level. The serum parathyroid hormone concentration was significantly lower in the LP group than in the C and HP groups. The serum osteocalcin concentration and urinary excretion of deoxypyridinoline were significantly higher in the HP groups than in the LP and C groups. The bone mineral density of the fifth lumbar vertebra was significantly increased with decreasing dietary P level. These results indicate that the low-P diet increased Ca absorption, this being effective for bone mineralization in adult female rats.

A Spatially Explicit Nanomechanical Model of the Half-Sarcomere: Myofilament Compliance Affects Ca2+-Activation

The force exerted by skeletal muscle is modulated by compliance of tissues to which it is connected. Force of the muscle sarcomere is modulated by compliance of the myofilaments. We tested the hypothesis that myofilament compliance influences Ca2+ regulation of muscle by constructing a computational model of the muscle half sarcomere that includes compliance of the filaments as a variable. The biomechanical model consists of three half-filaments of myosin and 13 thin filaments. Initial spacing of motor domains of myosin on thick filaments and myosin-binding sites on thin filaments was taken to be that measured experimentally in unstrained filaments. Monte-Carlo simulations were used to determine transitions around a three-state cycle for each cross-bridge and between two-states for each thin filament regulatory unit. This multifilament model exhibited less "tuning" of maximum force than an earlier two-filament model. Significantly, both the apparent Ca(2+)-sensitivity and cooperativity of activation of steady-state isometric force were modulated by myofilament compliance. Activation-dependence of the kinetics of tension development was also modulated by filament compliance. Tuning in the full myofilament lattice appears to be more significant at submaximal levels of thin filament activation.

Diet composition and age determine the effects of inulin–type fructans on intestinal calcium absorption in rat

Although several studies in animals and humans have established that inulintype fructans (inulin, oligofructose, fructooligosaccharides) enhance intestinal Ca absorption, there are also reports that failed to demonstrate any effects of added fructans on Ca absorption. We investigated in a rat model what variables determine inulin actions on mineral absorption with special focus on the basic diet. To determine apparent intestinal mineral absorption, whole body retention and mineral accumulation in bone, we performed feeding experiments with a balance technique by which mineral intake, faecal and urinary output are determined. In a first study we compared the effects of an inulin oligofructose mixture (0 and 10%,respectively) when added either to a standard diet or a semi-synthetic diet. Rats fed the semi-synthetic diet were younger (adolescent) than rats on standard diet (young adult). We observed that the apparent Ca absorption ratio was significantly increased by inulin and oligofructose only when provided in the semisynthetic diet and not in the standard diet that intrinsically already contained some fructans from wheat. In our second study with a semi-synthetic diet fed to growing (adolescent) rats, inulin and oligofructose increased not only Ca but also Mg and Zn absorption, whole body mineral retention and femur mineral content. Inulin-type fructans at dietary levels of 10 % (w/w) do increase mineral absorption, retention and accumulation in bone in the case of Ca, Mg and Zn, but only when the basic diet for the control group contains no intrinsic fructans and when the mineral demand is particularly high as during growth.

Influence of dietary calcium and phytase on phytate phosphorus hydrolysis in broiler chickens

The effect of Ca and phytase on phytate phosphorus (PP) hydrolysis was studied in vitro and in vivo. In vitro, PP hydrolysis by a 3-phytase and a 6-phytase was studied at pH 2.5 and 6.5 with Ca added at levels equivalent to 0, 0.1, 0.2, 0.4, 0.7, or 0.9% of the diet. Irrespective of enzyme, Ca at a level as low as 0.1% reduced (P < 0.05) PP hydrolysis at pH 6.5. To test these effects in vivo, 22-d-old male broilers were fed 1 of 6 diets (10 replicate pens of 4 birds per diet) for 30 h. The experimental design was a 3 × 2 factorial arrangement of 3 phytase treatments (0, 500 U of phytase A/kg of diet, and 500 U of phytase B/kg of diet) and 2 added Ca levels (0 and 0.5% from CaCO3) to a corn-soy basal diet. Adding Ca to the diet resulted in a reduction (P < 0.05) in ileal PP disappearance from 69.2 to 25.4% when the 0 and 0.5% added Ca diets were fed, respectively, and in apparent ileal Ca and P absorption (46.3 to 33.6% and 67.9 to 29.4% when 0 and 0.5% Ca were added, respectively). Inclusion of a 3-phytase improved (P < 0.05) ileal PP disappearance from 25.4 to 58.9% in diets containing 0 and 0.5% added Ca, but the improvement was less pronounced with a 6-phytase. Apparent ileal Ca absorption was improved (P < 0.05) when Ca, phytase, or both were added to the diet.

The apparent digestibility of phytate phosphorus and the influence of supplemental phytase in horses.

Availability of phytate-bound P as influenced by supplemental phytase was studied in eight horses consuming four diets in a 4 x 4 Latin square design experiment. The treatments were a control (containing a low P level, 18.4 g/d) and three high-P diets. These diets contained P as monocalcium phosphate (MCP; 43.7 g/d), myoinositol hexakisphosphate in the form of wheat and rice bran (MIHP; 41.8 g/d), or MIHP with microbial phytase (MIHPP; 42.5 g/d). The proportions of phytate-bound P were 3, 1, 55, and 56% for the control, MCP, MIHP, and MIHPP, respectively. The MIHPP diet was supplemented with 300 phytase units (FTU)/kg (as-fed basis). Feces and urine were collected quantitatively and analyzed for P, Ca, and Mg. Urinary P excretion was lower (P < 0.05) with the control diet (0 g of P/d) than with the MCP diet (1.0 g of P/d). The low urinary P excretion (0.3 g of P/d) for the MIHP diet suggested low P availability compared with the MCP diet, but apparent digestibility of P expressed as a percentage of intake did not differ (P = 0.065) between these diets. Apparent Ca digestibility was lower (P < 0.05) for the MIHP diet than for the MCP diet (26.4 vs. 42.4%). This difference may have been caused by the origin of the Ca in these diets. Phytase supplementation increased apparent Ca digestibility from 26.4 to 31.5% (P < 0.05). Magnesium was not influenced by the level of phytate in the diet. Our data indicate that phytase supplementation had more influence on Ca digestibility than on P digestibility and suggest that phytase supplementation may be beneficial for improving Ca digestibility for horses receiving a phytate-rich diet.

Effects of sodium sulphate, sodium chloride and manganese sulphate on kikuyu (Pennisetum clandestinum) growth and ion uptake

Irrigation with saline water is increasingly practised yet an understanding of plant response to different salt types remains largely elusive. Our study investigated the effects of irrigation with simulated effluent containing three salts on the growth and composition of a common South African pasture grass. We treated pots of kikuyu (Pennisetum clandestinum Hochst, ex Chiov.) in a controlled environment with solutions of sodium sulphate (Na2SO4) or chloride (NaCI) at saturated soil solution electrical conductivity (EC) of 0, 5,10 and 20 dS m-1, combined with 0, 900 or 1800 mg kg-1 Mn as MnSO4. Kikuyu yields in the Na2SO4 treatments were significantly lower than in corresponding NaCI treatments. This could be explained by increased ion imbalance and osmotic stress as a result of higher ionic strength and a greater concentration of neutral ion pairs in the Na2SO4 system at similar EC. An apparent Na2SO4-induced Ca deficiency was attributed to suppressed Ca2+ activity through ion pairing at high ionic strengths. Under the conditions of this experiment ionic strength appeared to be more effective than EC as a measure of salinity stress when comparing the effects of SO4 and CI on growth of kikuyu.

Carbon monoxide activates KCa channels in newborn arteriole smooth muscle cells by increasing apparent Ca2+ sensitivity of alpha-subunits.

Carbon monoxide (CO) is a gaseous vasodilator produced by many cell types, including endothelial and smooth muscle cells. The goal of the present study was to investigate signaling mechanisms responsible for CO activation of large-conductance Ca(2+)-activated K(+) (K(Ca)) channels in newborn porcine cerebral arteriole smooth muscle cells. In intact cells at 0 mV, CO (3 microM) or CO released from dimanganese decacarbonyl (10 microM), a novel light-activated CO donor, increased K(Ca) channel activity 4.9- or 3.5-fold, respectively. K(Ca) channel activation by CO was not blocked by 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (25 microM), a soluble guanylyl cyclase inhibitor. In inside-out patches at 0 mV, CO shifted the Ca(2+) concentration-response curve for K(Ca) channels leftward and decreased the apparent dissociation constant for Ca(2+) from 31 to 24 microM. Western blotting data suggested that the low Ca(2+) sensitivity of newborn K(Ca) channels may be due to a reduced beta-subunit-to-alpha-subunit ratio. CO activation of K(Ca) channels was Ca(2+) dependent. CO increased open probability 3.7-fold with 10 microM free Ca(2+) at the cytosolic membrane surface but only 1.1-fold with 300 nM Ca(2+). CO left shifted the current-voltage relationship of cslo-alpha currents expressed in HEK-293 cells, increasing currents 2.2-fold at +50 mV. In summary, data suggest that in newborn arteriole smooth muscle cells, CO activates low-affinity K(Ca) channels via a direct effect on the alpha-subunit that increases apparent Ca(2+) sensitivity. The optimal tuning by CO of the micromolar Ca(2+) sensitivity of K(Ca) channels will lead to preferential activation by signaling modalities, such as Ca(2+) sparks, which elevate the subsarcolemmal Ca(2+) concentration within this range.

Distinct Potentiation of L-Type Currents and Secretion by cAMP in Rat Chromaffin Cells

We have investigated the potentiating action of cAMP on L-currents of rat chromaffin cells and the corresponding increase of Ca 2+-evoked secretory responses with the aim of separating the action of cAMP on Ca 2+ entry through L-channels and the downstream effects of cAMP/protein kinase A (PKA) on exocytosis. In ω-toxin-treated rat chromaffin cells, exposure to the permeable cAMP analog 8-(4-chlorophenylthio)-adenosine 3′,5′-monophosphate (pCPT-cAMP; 1 mM, 30 min) caused a moderate increase of Ca 2+ charge carried through L-channels (19% in 10 mM Ca 2+ at +10 mV) and a drastic potentiation of secretion (∼100%), measured as membrane capacitance increments (Δ C). The apparent Ca 2+ dependency of exocytosis increased with pCPT-cAMP and was accompanied by 83% enhancement of the readily releasable pool of vesicles with no significant change of the probability of release, as evaluated with paired-pulse stimulation protocols. pCPT-cAMP effects could be mimicked by stimulation of β 1-adrenoreceptors and reversed by the PKA inhibitor H89, suggesting strict PKA dependence. For short pulses to +10 mV (100 ms), potentiation of exocytosis by pCPT-cAMP was proportional to the quantity of charge entering the cell and occurred independently of whether L, N, or P/Q channels were blocked, suggesting that cAMP acts as a constant amplification factor for secretion regardless of the channel type carrying Ca 2+. Analysis of statistical variations among depolarization-induced capacitance increments indicates that pCPT-cAMP acts downstream of Ca 2+ entry by almost doubling the mean size of unitary exocytic events, most likely as a consequence of an increased granule-to-granule rather than a granule-to-membrane fusion.