Range Of Calcium Concentrations Research Articles

Myocardial cross-bridge kinetics in transition to failure in Dahl salt-sensitive rats.

The role of altered cross-bridge kinetics during the transition from cardiac hypertrophy to failure is poorly defined. We examined this in Dahl salt-sensitive (DS) rats, which develop hypertrophy and failure when fed a high-salt diet (HS). DS rats fed a low-salt diet were controls. Serial echocardiography disclosed compensated hypertrophy at 6 wk of HS, followed by progressive dilatation and impaired function. Mechanical properties of skinned left ventricular papillary muscle strips were analyzed at 6 wk of HS and then during failure (12 wk HS) by applying small amplitude (0.125%) length perturbations over a range of calcium concentrations. No differences in isometric tension-calcium relations or cross-bridge cycling kinetics or mechanical function were found at 6 wk. In contrast, 12 wk HS strips exhibited increased calcium sensitivity of isometric tension, decreased frequency of minimal dynamic stiffness, and a decreased range of frequencies over which cross bridges produce work and power. Thus the transition from hypertrophy to heart failure in DS rats is characterized by major changes in cross-bridge cycling kinetics and mechanical performance.

High-throughput assessment of calcium sensitivity in skinned cardiac myocytes.

Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.

Hippocampal LTP induction is related to dynamic range of postsynaptic calcium concentration

It is widely believed that a rise in postsynaptic calcium concentration is a necessary step in the induction of long-term potentiation. For physiologically plausible short burst trains of pulses, the intracellular calcium dynamics has not only mean level but also variance (or dynamic range). Therefore, it is unclear whether mean level or variance of intracellular calcium dynamic is related to the induction of long-term potentiation. Here we investigate mean and standard deviation of simulated postsynaptic calcium dynamics following the patterned stimulation, which are consisted of bursts (4 pulses at 100 Hz ) repeated at 2.0, 1.0, 0.2, or 0.1 s intervals (i.e. burst frequencies of 0.5, 1.0, 5.0 or 10.0 Hz , respectively). Our results show that variance (or dynamic range) rather than mean level of postsynaptic [Ca 2+] i matches experimental results well.

Intrinsic Coagulation Pathway: An Activation Threshold

The activation threshold for the intrinsic pathway of coagulation was experimentally determined in stirred recalcified plasma from the dependence of plasma clotting time after activation by celite. Concentration of factor XIa was used as a measure of activation. At free calcium concentrations below 0.45 mM, plasma clotting time depended nonlinearly on the factor XIa concentration: with decreasing concentration of factor XIa (or celite), the clotting time dramatically increased until no coagulation was observed at concentrations of factor XIa below the threshold. As the free calcium concentration increased, the threshold concentration of factor XIa sharply decreased, from 30 pM at 0.35 mM free calcium to less than 3 pM at 0.45 mM. In the range of free calcium concentrations from 0.45 mM to physiologic ones, plasma coagulated even in the absence of celite in plastic cuvettes. This fact and extremely low threshold concentrations of factor XI (on the order of 0.5 pM) preclude determining the factor XI threshold at physiologic free calcium. As factor XIa is localized to the activating surfaces, observing the local surface concentrations of factor XIa and the dynamics of fibrin formation in systems without stirring may solve the problem.

Effect of calcium, Bay K 8644, and reduced perfusion on basic indices of myocardial function in isolated hearts from rats after prolonged exposure to ethanol.

We previously reported findings consistent with a marked upregulation in functional L-type voltage-operated calcium channels (L-VOCCs) in hearts obtained from rats exposed over the long term to ethanol. These experiments were undertaken to establish whether detrimental effects on cardiac function were associated with excess calcium entry into the myocardium in these hearts. Isolated hearts from adult male Sprague-Dawley rats received intoxicating concentrations of ethanol for 6-10 days by inhalation, were perfused with Krebs-Henseleit buffer by a modified Langendorff technique, and several functional parameters were assessed continuously. In some experiments, the calcium concentration in the perfusate was first reduced from the physiologic range (1.2 mM) to 0.15 mM and then increased in a step-wise fashion to 4 mM. In other experiments, hearts were exposed to buffer containing concentrations of the L-VOCC activator, (+/-)Bay K 8644, increasing from 10(-9) to 10(-6) M. These perfusion protocols were repeated in hearts from treated animals subject to reduced coronary flow because of induction of partial left ventricular ischemia. There were some close similarities in the effects of these different stimuli. When the calcium concentration in the perfusate exceeded a physiologic level, there were signs of decreased function relative to controls in the hearts from ethanol-exposed rats. Thus R-wave amplitude and systolic pressure were lower, diastolic pressure also was reduced, but heart rate was elevated above that of controls. Similarly the presence of (+/-)Bay K 8644 in the perfusate caused a decrease in systolic and diastolic pressure and an increase in heart rate in hearts from ethanol-exposed rats. When cardiac perfusion was reduced in vitro by inflation of a balloon in the left ventricle, some of the effects of excess calcium and (+/-)Bay K 8644 were reproduced in control hearts. However, imposition of this "ischemic" stress did not appear to exacerbate the effects of prior exposure to ethanol. In general, in control hearts, indices of contractility were increased across the range of calcium concentration or by perfusing with (+/-)Bay K 8644. Hearts from ethanol-exposed rats, however, showed no further increase in these parameters once physiologic levels of calcium were exceeded, or showed inhibition of contractility in the presence of (+/-)Bay K 8644. The results are consistent with calcium entry through L-VOCCs in hearts from ethanol-exposed animals having detrimental effects on cardiac function once physiologic levels are exceeded. However, it is possible that these channels also may be involved in maintenance of cardiac function at hypocalcemic levels.

Calcium imaging with chemiluminescence.

This review updates the imaging of free cytosolic calcium with the chemiluminescent aequorins. Basic principles of chemiluminescence are discussed and the biochemistry of aequorins is briefly described. The review provides practical tips on handling and microinjecting aequorins and describes available ultra low light imaging systems. It is argued that aequorin-based calcium imaging is the method of choice for exploratory studies, since it is extremely sensitive, can detect a broad range of calcium concentrations, and allows for continuous recording during long periods of time. However, fluorescent methods are needed to attain high spatial resolution.

Calcium imaging with chemiluminescence

This review updates the imaging of free cytosolic calcium with the chemiluminescent aequorins. Basic principles of chemiluminescence are discussed and the biochemistry of aequorins is briefly described. The review provides practical tips on handling and microinjecting aequorins and describes available ultra low light imaging systems. It is argued that aequorin-based calcium imaging is the method of choice for exploratory studies, since it is extremely sensitive, can detect a broad range of calcium concentrations, and allows for continuous recording during long periods of time. However, fluorescent methods are needed to attain high spatial resolution. Microsc. Res. Tech. 46:390–397, 1999. © 1999 Wiley-Liss, Inc.

Precise Determination of Element/Calcium Ratios in Calcareous Samples Using Sector Field Inductively Coupled Plasma Mass Spectrometry

A new method was developed for rapid and precise simultaneous determination of Mg/Ca, Sr/Ca, Mn/Ca, Cd/Ca, Ba/Ca and U/Ca ratios in foraminiferal shells using sector field inductively coupled plasma mass spectrometry (ICPMS). Element/calcium ratios were determined directly from intensity ratios using external, matrix-matched standard to correct for instrumental mass discrimination. Because of large differences in the abundance of chemical constituents of the foraminiferal shell, major elemental ratios were determined in analog mode (using (24)Mg, (43)Ca, (44)Ca, (55)Mn, and (88)Sr) whereas trace elemental ratios were determined in pulse-counting mode (using (111)Cd, (138)Ba, (238)U, and the low-abundance (46)Ca isotope). Matrix-induced variations in mass discrimination over a calcium concentration range of 2.0-24.5 mM were observed only for Mg/Ca and Cd/Ca ratios. However, these effects are negligible if the samples and standard calcium concentration are within a factor of 2-3. Multiratio method reproducibility was better than previously reported for other ICPMS methods yielding precision (1σ) of Sr/Ca = 0.45%; Mg/Ca = 0.45%, Mn/Ca = 0.8%, Cd/Ca = 1.7%, Ba/Ca = 0.7%, and U/Ca = 1.4% for foraminifera samples as small as 25 μg. Using this approach for a single-ratio analysis, Sr/Ca ratios were determined with precision of 0.06% (1σ) on carbonate samples as small as a single foraminifera shell (<10 μg). The new method is more sensitive, more precise, and simpler to use than previously available ICPMS techniques. It provides an efficient tool for simultaneous determination of several elemental ratios of paleoceanographic interest in a single foraminiferal sample, thereby reducing overall sample size requirement and analysis time.

Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins.

The direct effects of expressing hypertrophic cardiomyopathy-associated (HCM-associated) mutant troponin T (TnT) proteins on the force generation of single adult cardiac myocytes have not been established. Replication-defective recombinant adenovirus vectors were generated for gene transfer of HCM-associated I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac myocytes in primary culture. We tested the hypothesis that the mutant TnT proteins would be expressed and incorporated into the cardiac sarcomere and would behave as dominant-negative proteins to directly alter calcium-activated force generation at the level of the single cardiac myocyte. Interestingly, under identical experimental conditions, the ectopic expression of the mutant TnTs was significantly less ( approximately 8% of total) than that obtained with expression of wild-type TnT ( approximately 35%) in the myocytes. Confocal imaging of immunolabeled TnT showed a regular periodic pattern of localization of ectopic mutant TnT that was not different than that in normal controls, suggesting that mutant TnT incorporation had no deleterious effects on sarcomeric architecture. Direct measurements of isometric force production in single cardiac myocytes demonstrated marked desensitization of submaximal calcium-activated tension, with unchanged maximum tension generation in mutant TnT-expressing myocytes compared with control myocytes. Collectively, these results demonstrate an impaired expression of the mutant protein and a disabling of cardiac contraction in the submaximal range of myoplasmic calcium concentrations. Our functional results suggest that development of new pharmacological, chemical, or genetic approaches to sensitize the thin-filament regulatory protein system could ameliorate force deficits associated with expression of I79N and R92Q in adult cardiac myocytes.

Does water calcium content influence the distinctness of daily growth increments in the otoliths of larval whitefish (Coregonus lavaretus L.)?

In larval and juvenile whitefish (Coregonus lavaretus L.) from Lake Constance, Germany, the otolith increments are deposited daily, whereas daily deposition could not be confirmed in larval whitefish from Lake Pyhäselkä, Finland. The calcium concentration in Lake Constance is high (around 1.3 m m), while calcium deficiency is typical for Finnish lakes (around 0.15 m m). Therefore, the hypothesis that the distinctness of daily otolith increments in whitefish is related to water calcium content was tested by rearing three groups of Lake Constance whitefish in water of 0.2, 1.3 and 4.7 m m Ca. The eggs were incubated in lake water (1.3 m m Ca), and the larvae were acclimated to the experimental calcium concentrations on the day of hatching. After 39 days of ad libitum-feeding with Artemia nauplii, the three groups did not differ significantly in total length, wet and dry weight, and otolith length and width. The daily increments were easily recognizable, and contrast between dark (D)- and light (L)-zones was the same in the fish of all test groups. For the experimental set-up of this study, and particularly the range of calcium concentrations tested, the hypothesis that water calcium content influences the distinctness of daily otolith increments was rejected.

Gating of cx46 gap junction hemichannels by calcium and voltage.

Connexin 46 (cx46), when expressed in Xenopus oocytes, not only forms typical gap junction channels between paired cells but also forms open gap junction hemichannels in the plasma membrane of single cells. The gap junction hemichannels share properties with complete gap junction channels in terms of permeability and gating. Here we characterize the gate that closes hemichannels in response to increased calcium concentration with whole-cell and single-channel records. The channels close within a narrow range of extracellular calcium concentrations (1-2 mM) which includes the calcium concentration prevailing in the primary site of cx46 expression, the lens. The effect of calcium on the channels is determined by voltage. A cysteine mutant of cx46, cx46L35C, was used to determine the localization of the gate. Experimental evidence suggests that position 35 is pore lining. The localization protocol tests the accessibility of position 35 for thiol reagents applied extra- or intracellularly to the channel closed by calcium. Channel closure by calcium excluded the thiol reagent from the outside but not from the inside. Consequently, the gate results in a regional closure of the pore and it is located extracellular to the position 35 of cx46. The present data also suggest that the cx46 gap junction hemichannel may exert a physiological function in the lens. Considering the association of calcium with cataract formation, it is feasible that misregulation of cx46 gap junction hemichannels could be a cause for cataract.

Computing SI and CCPP using spreadsheet programs

Spreadsheets can automate any chemical equilibrium calculation that can be performed by hand.Lotus 1‐2‐3® worksheets for calculating the calcite saturation index (SI) and calcium carbonate precipitation potential of a water sample are described. A simplified worksheet illustrates the principles of the method, and a more complex worksheet suitable for modeling most potable water sources is briefly described. Both worksheets include ion pairing and adjustments of equilibrium constants for temperature and ionic strength. For several test cases with a wide range of temperature, alkalinity, pH, and calcium concentration, the worksheet SI values differed &lt;1 percent from those computed by WATEQX. For water quality modeling, spreadsheets have several advantages compared with large equilibrium codes: flexible input and output, ease of maintaining the thermodynamic database, and relatively small memory and mass‐storage requirements. Modeling of archived chemical data is possible through the combination of database management and computational capabilities of many commercially available programs. The worksheets can be obtained from the authors.

Quantitative Kinetic Model for Photoassembly of the Photosynthetic Water Oxidase from Its Inorganic Constituents: Requirements for Manganese and Calcium in the Kinetically Resolved Steps,

The process of photoactivation, the assembly of a functional water-oxidizing complex (WOC) from the apoproteins of photosystem II of higher plants and inorganic cofactors (Mn2+, Ca2+, and Cl-), was known from earlier works to be a two-step kinetic process, requiring two light-induced processes separated by a slower dark period. However, these steps had not been directly resolved in any kinetic experiment, until development of an ultrasensitive polarographic O2 electrode and synthesis of an improved chelator for cofactor removal allowed direct kinetic resolution of the first pre-steady state intermediate [Ananyev, G. M. & Dismukes, G. C. (1996a) Biochemistry 35, 4102-4109]. Herein, the dependence of the rates of each of the first two light steps and the dark step of photoactivation was directly determined in spinach PSII membranes over a range of calcium and manganese concentrations at least 10-fold lower than those possible using commercial O2 electrodes. The following results were obtained. (1) One Mn2+ ion binds and is photooxidized to Mn3+ at a high-affinity site, forming the first light-induced intermediate, IM1. Formation of IM1 is coupled to the dissociation of a bound Ca2+ ion either located in the Mn site or coupled to it. (2) The inhibition constant for Ca2+ dissociation from this site is equal to 1.5 mM. (3) The dissociation constant of Mn2+ at this high-affinity site is equal to 8 microM at the optimum calcium concentration for O2-evolving activity of 8 mM, in agreement with the high-affinity site for electron donation to PSII. (4) Prior to the next photolytic step, one Ca2+ ion must bind at its effector site so that stable photooxidation of a second Mn2+ ion can occur, forming the second light-induced intermediate, IM2. This dark process is the rate-determining step. (5) The Michaelis constant for recovery of O2 evolution by Ca2+ binding at this effector site (Km) is equal to 1.4 mM, a value that is the same as that measured for the calcium requirement for O2 evolution in intact PSII. (6) The low quantum yield for the formation of IM2 from IM1 increases linearly with the duration of the dark period up to the longest period we could examine (10 s). Accordingly, the rate limitation in the second photolytic step originates from a slow calcium-induced dark rearrangement of the first intermediate, IM1, which we propose to be a protein conformational change that allows stable binding of the next Mn2+ ion. We further propose that the single Ca2+ ion which is required for assembly of the Mn4 cluster is equivalent to the Ca2+ ion which functions at the "gatekeeper" site in intact O2-evolving centers, where it plays a role in limiting substrate access to the Mn4 cluster [Sivaraja, M., et al. (1989) Biochemistry 28, 9459-9464; Tso, J., et al., (1991) Biochemistry 30, 4734-4739]. A molecular model for photoactivation is proposed and discussed.

Decreased myocyte tension development and calcium responsiveness in rat right ventricular pressure overload.

The contractile dysfunction observed in end-stage myocardial hypertrophy has at its base an abnormality in myocyte function. However, whether depressed contractile function is related to an alteration in contractile protein function is presently unknown. Contractile force, tension, and calcium responsiveness were measured in single-skinned myocytes isolated from rats with right ventricular hypertrophy (RVH) and control rats. RVH was induced by pulmonary artery constriction for 36 weeks and was associated with significant myocyte hypertrophy. Myocytes were attached to micropipettes that extended from a force transducer and motor. Isometric force was measured over a wide range of calcium concentrations at two sarcomere lengths (SLs). Maximal force was increased in the RVH group: 1.20 +/- 0.10 versus 1.62 +/- 0.13 mg at SL = 2.0 microns and 1.33 +/- 0.10 versus 1.84 +/- 0.15 mg at SL = 2.3 microns (P < .05). Maximal tension, however, was reduced in the RVH group: 24.3 +/- 1.91 versus 37.5 +/- 2.92 mN/mm2 at SL = 2.0 microns and 27.4 +/- 1.78 versus 41.8 +/- 3.19 mN/mm2 at SL = 2.3 microns (P < .01). The concentration of calcium ions required for half-maximal activation was increased in the RVH group: 2.64 +/- 0.13 versus 3.47 +/- 0.22 mumol/L at SL = 2.0 microns and 2.23 +/- 0.15 versus 2.86 +/- 0.18 mumol/L at SL = 2.3 microns (P < .01). The slope of the force-calcium relationship (Hill coefficient) was decreased in the RVH group at SL = 2.0 microns (4.3 +/- 0.4 versus 3.1 +/- 0.2, P = .04) but not at SL = 2.3 microns (3.8 +/- 0.2 versus 3.6 +/- 0.2, P = NS). These results suggest that the depressed cardiac function of end-stage myocardial hypertrophy may be due, in part, to altered contractile protein function.

Competitive Binding of Protons, Calcium, Cadmium, and Zinc to Isolated Cell Walls of a Gram-Positive Soil Bacterium

Metal ion binding to the bacterial cell wall is the first step in the interactions of a metal with a bacterium. Cadmium and zinc ion binding to isolated cell walls of Rhodococcus erythropolis A177 has been studied for a wide range of proton and calcium concentrations. The release of calcium ions and protons during metal ion binding is an indication of the competitive nature of the binding. Calculations, based on the metal binding data and the pH-dependent charging behavior, reveal that the cell wall becomes positively charged at high coverage with bivalent ions. A cooperative effect of the presence of calcium on metal ion binding was observed. Apparently, the cell wall structure is altered in the presence of bivalent ions. Since calcium is a dominant bivalent cation in most natural systems, one may regard the calcium as a “structure determining ion”. On the basis of a qualitative interpretation of the data, the NICA model was selected for a quantitative description of the data.

The Cytoplasmic Streaming Response of Tomato Root Hairs to Auxin; the Role of Calcium

The effects of IAA on the cytoplasmic streaming response of tomato root hairs were observed at different external calcium concentrations. The sensitivity of tomato root hairs to IAA was increased by increasing the calcium content of the bathing medium; lower concentrations of IAA were needed to stimulate cytoplasmic streaming at high [Ca2+]ext. The transplasma membrane electrical potential difference was rapidly depolarised by IAA across a wide range of auxin and calcium concentrations; subsequent hyperpolarisation was only seen in the presence of high [Ca2+]ext. Measurements of cytoplasmic calcium levels, using the dye fluo-3, revealed the dynamic nature of the cytoplasm. As in earlier experiments, in which indo-1 and fura-2 were used, IAA appeared to have only small effects on [Ca2+]cyt. Clear, auxin-induced changes in [Ca2+]cyt were recorded only under non-physiological conditions.

Specificity of polyunsaturated fatty acid release from rat brain synaptosomes.

Release of specific polyunsaturated fatty acids for cell membranes may have a significant implication in biological function, considering the involvement of various fatty acids in cell signal transduction. In the present study, release of polyunsaturated fatty acids from rat brain synaptosomes by endogenous synaptosomal lipase activity was examined in comparison to that by cobra venom phospholipase A2 (Naja naja naja). Cobra venom phospholipase A2 (Naja naja naja) preferentially hydrolyzed docosahexaenoic acid (22:6n-3) from both synaptosomes and lipid mixtures containing similar classes of lipids commonly found in the brain. Arachidonic acid (20:4n-6) and oleic acid (18:1n-9) were also hydrolyzed; however, monoene species was hydrolyzed slower than were polyenoic species in synaptosomes. Phosphatidylethanolamine was the most preferred phospholipid class for release of 22:6n-3 fatty acid from both lipid mixtures and synaptosomes. In contrast to hydrolysis by cobra venom phospholipase A2, endogenous synaptosomal lipase activity preferentially hydrolyzed 20:4n-6 from rat brain synaptosomes, despite the high abundance of 22:6n-3 in synaptosomal membranes. Preferential release of 20:4n-6 was observed over a wide range of pH values and calcium concentrations. Synaptosomal 22:6 species appeared to be resistant to hydrolysis even after stimulation with various agents such as phorbolmyristate, suggesting that physiological importance of 22:6n-3 in neuronal membranes may not be as the release fatty acid.

The solubility of calcium and phosphate in two specialty amino acid solutions.

The purpose of this study was to determine precipitation limits and construct curves for calcium and phosphorus in parenteral nutrition solutions compounded with specialty amino acid solutions. The effect of temperature, concentration of amino acids, and pH on the solubility of calcium and phosphate was studied for two specialized amino acid solutions: NephrAmine and HepatAmine. Ten amino acid solutions were manufactured with NephrAmine and 12 with HepatAmine at amino acid concentrations of 0.8%, 1.5%, and 2%. The final dextrose concentration was 10%. Some of the solutions were tested with the additive cysteine hydrochloride, 40 mg cysteine/g of protein, and some were buffered with sodium bicarbonate or hydrochloric acid to model the pH of a combined fat emulsion and total parenteral nutrient (TPN) solution. Calcium gluconate and potassium phosphate were added to test samples of each TPN solution. A range of calcium concentrations of 2.5 to 40 mEq/L and phosphate concentrations of 2.5 to 40 mmol/L were tested. After storage at room temperature (25 degrees C) for 18 hours, solutions were inspected for precipitation. At the end of the 18-hour period, the solutions were held in a water bath at 37 degrees C for 30 minutes and inspected for precipitation and microcrystallization. Solubility curves were plotted to represent Ca-PO4 solubility limits before visual or microscopic precipitation. These curves depend on the variables of time, temperature, concentration, and pH. These data and graphs will help the clinical pharmacist estimate Ca-PO4 solubility limits and prevent precipitation of TPN solutions formulated with NephrAmine or Hepatamine.

Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range.

1. Axonal transection triggers a cascade of pathological processes that frequently lead to the degeneration of the injured neuron. It is generally believed that the degenerative process is triggered by an overwhelming influx of calcium through the cut end of the axon. 2. Theoretical considerations and indirect observations suggest that axotomy is followed by an increase in the free intracellular calcium concentration ([Ca2+]i) to the millimolar level. In contrast, only relatively modest and transient elevation in [Ca2+]i to the micromolar level was revealed by recent fura-2 studies. 3. In the current study we used the low-affinity Ca2+ indicator mag-fura-2 to reexamine the spatiotemporal distribution pattern of Ca2+ after axotomy and to map the free intracellular Mg2+ concentration gradients. 4. We report that axotomy elevates [Ca2+]i well beyond the "physiological" range of calcium concentrations, to levels > 1 mM near the tip of the cut axon and to hundreds of micromolars along the axon further away from the cut end. Nevertheless, [Ca2+]i recovers to the control levels within 2-3 min after the resealing of the cut end. 5. A comparison of the behavior of fura-2 and mag-fura-2 in the cytosol of the axotomized neurons reveals that the determination of [Ca2+]i by fura-2 largely underestimates the actual intracellular Ca2+ concentrations. 6. Experiments in which one branch of a bifurcated axon was transected revealed that the elevation in [Ca2+]i is confined to the transected axonal branch and does not spread beyond the bifurcation point. 7. After axotomy, the intracellular Mg2+ concentration equilibrates rapidly with the external concentration and then recovers at a rate somewhat slower than that of [Ca2+]i. 8. To the best of our knowledge, this study is the first direct demonstration that axotomy elevates [Ca2+]i to the millimolar range and that neurons are able to recover from these extreme calcium concentrations.

5330911 Surfaces having desirable cell adhesive effects

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992–22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (μM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 μM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.