Targeted single-cell electroporation loading of Ca2+ indicators in the mature hemicochlea preparation.

Ca2+ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca2+ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca2+ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca2+ indicator. However, bulk loading takes long time, is variable and non-selective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca2+ imaging by increasing the signal-to-noise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca2+ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca2+ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca2+ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca2+ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cell-specific indicator loading method allowing subcellular functional Ca2+ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca2+ homeostasis in these cells.

Permeation of topically applied Magnesium ions through human skin is facilitated by hair follicles.

Magnesium is an important micronutrient essential for various biological processes and its deficiency has been linked to several inflammatory disorders in humans. Topical magnesium delivery is one of the oldest forms of therapy for skin diseases, for example Dead Sea therapy and Epsom salt baths. Some anecdotal evidence and a few published reports have attributed amelioration of inflammatory skin conditions to the topical application of magnesium. On the other hand, transport of magnesium ions across the protective barrier of skin, the stratum corneum, is contentious. Our primary aim in this study was to estimate the extent of magnesium ion permeation through human skin and the role of hair follicles in facilitating the permeation. Upon topical application of magnesium solution, we found that magnesium penetrates through human stratum corneum and it depends on concentration and time of exposure. We also found that hair follicles make a significant contribution to magnesium penetration.

Characterization of lysophosphatidylcholine-induced changes of intracellular calcium in Drosophila S2 cells.

AIMS: Lysophosphatidylcholine (LPC), a bioactive lipid, regulates a wide array of biological processes. LPC could be deacylated to form glycerophosphocholine by neuropathy target esterase (NTE)/Swiss cheese protein (SWS). Although NTE/SWS is important in maintaining Ca(2+) homeostasis, the role of LPC in regulating the intracellular calcium concentration ([Ca(2+)]i) in Drosophila remains poorly understood. We aimed to study the mechanism of LPC-induced [Ca(2+)]i changes in Drosophila S2 cells. MAIN METHODS: The [Ca(2+)]i of Drosophila S2 cells was measured by fluorescence spectrophotometry after loading the cells with the calcium-sensitive fluorescent probe Fura-2/AM. KEY FINDINGS: Our results demonstrated that LPC could cause a rapid, dose-dependent increase in the [Ca(2+)]i in the presence of external calcium ([Ca(2+)]e). The LPC-induced [Ca(2+)]i increase was reduced by 60.7% in the absence of [Ca(2+)]e. Furthermore, the Ca(2+) influx was inhibited by 37.3% after the cells were preincubated with an L-type Ca(2+) channel blocker. In the Ca(2+)-free medium, the LPC-induced [Ca(2+)]i increase was completely blocked using an inositol triphosphate receptor (IP3R) inhibitor. However, a ryanodine receptor (RyR) inhibitor had no effect on the LPC-induced [Ca(2+)]i increase. SIGNIFICANCE: The LPC-induced [Ca(2+)]i increase in S2 cells was dependent on both the release of Ca(2+) stored in the endoplasmic reticulum and [Ca(2+)]e influx. Both L-type Ca(2+) channels and IP3R might be involved in this process. The LPC-induced [Ca(2+)]i increase in S2 cells characterized in this study may shed light on the study of NTE/SWS protein function in general because the enzyme is responsible for the deacylation of LPC.

Enhanced loading of Fura-2/AM calcium indicator dye in adult rodent brain slices via a microfluidic oxygenator.

A microfluidic oxygenator is used to deliver constant oxygen to rodent brain slices, enabling the loading of the cell-permeant calcium indicator Fura-2/AM into cells of adult brain slices. When compared to traditional methods, our microfluidic oxygenator improves loading efficiency, measured by the number of loaded cells per unit area, for all tested age groups. Loading in slices from 1-year-old mice was achieved, which has not been possible with current bulk loading methods. This technique significantly expands the age range for which calcium studies are possible without cellular injection. This technique will facilitate opportunities for the study of calcium signaling of aging and long term stress related diseases. Moreover, it should be applicable to other membrane-permeant physiological indicator varieties.

Calcium signaling in intact dorsal root ganglia: new observations and the effect of injury.

BACKGROUND: Ca is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca recording techniques use high K or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. METHODS: Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. RESULTS: Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Abeta-type neurons. Unitary Ca transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca transients in C-type neurons, but increased transients in Abeta-type neurons. CONCLUSIONS: Refined observation of Ca signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state.

Fast dual-excitation ratiometry with light-emitting diodes and high-speed liquid crystal shutters.

Dual-excitation ratiometric dyes permit quantitative measurements of Ca2+ concentrations ([Ca2+]s), by minimizing the effects of several artifacts that are unrelated to changes in [Ca2+]. These dyes are excited at two different wavelengths, and the resultant fluorescence intensities are measured sequentially. Therefore, it is difficult to follow fast [Ca2+] dynamics or [Ca2+] changes in highly motile cell samples. To overcome this problem, we have developed a new dual-excitation ratiometry system that employs two high-power light-emitting diodes (LEDs), two high-speed liquid crystal shutters, and a CCD camera. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity. We demonstrate the effectiveness of this system by monitoring changes in [Ca2+] in cardiac muscle cells loaded with Fura-2.

Isoflurane applied during ischemia enhances intracellular calcium accumulation in ventricular myocytes in part by reactive oxygen species.

BACKGROUND: Isoflurane applied before myocardial ischemia has a beneficial preconditioning effect which involves generation of reactive oxygen species (ROS); ROS, however, have been implicated in critical cytosolic calcium ([Ca2+]i) overload during ischemia. We therefore investigated isoflurane's effects on intracellular Ca2+ handling in ischemic ventricular myocytes and the association with ROS. METHODS: Simulated ischemia was induced in electrically stimulated rat ventricular myocytes for 30 min (ischemia). Isoflurane-treated cells were additionally exposed to 1MAC of isoflurane (ischemia + iso). To determine the contribution of ROS to Ca2+ homeostasis during ischemia in both groups, the intracellular ROS scavenger, N-mercaptopropionylglycine (MPG), was added to the superfusion buffer. The fluorescent ratiometric Ca2+ dye fura-2 was employed to determine [Ca2+]i. RESULTS: Resting and peak [Ca2+]i increased in the ischemia and the ischemia + iso group. However, Ca2+ accumulation was most prominent in isoflurane-treated cardiomyocytes (P < 0.05) and could be mitigated by MPG in both groups (P < 0.001). Isoflurane also decreased the rate constant of the Ca2+ transient decline but did not further diminish the amplitude of the transient during ischemia. CONCLUSION: Isoflurane when applied during ischemia appears to worsen [Ca2+]i overload, which is caused by impeding Ca2+ clearance. As MPG mitigated the increase in [Ca2+]i, isoflurane seems to enhance ROS-mediated effects on intracellular Ca2+ handling in cellular ischemia.

AM-loading of fluorescent Ca2+ indicators into intact single fibers of frog muscle.

The AM loading of a number of different fluorescent Ca2+ indicators was compared in intact single fibers of frog muscle. Among the 13 indicators studied, loading rates (the average increase in the fiber concentration of indicator per first 60 min of loading) varied approximately 100-fold, from approximately 3 microM/h to >300 microM/h (16 degrees C). Loading rates were strongly dependent on the molecular weight of the AM compounds, with the rate increasing steeply as molecular weight decreased below approximately 850. Properties of delta F/F (the Ca2(+)-related fluorescence signal observed with fiber stimulation) were also measured in AM-loaded fibers and compared with those previously reported for fibers microinjected with indicator. In general, the time course of delta F/F was very similar with AM-loading and microinjection; however, the amplitude of delta F/F was usually smaller with AM-loading. There was a strong correlation between the rate of indicator loading and the value of the parameter f (the ratio of the amplitude of delta F/F in AM-loaded versus microinjected fibers). For indicators with small loading rates (<10 microM/h, N = 5), f values were generally small (< or =0.4, N = 4); whereas with large loading rates (>100 microM/h, N = 4), f values were large (> or =0.8, N = 4). This suggests that, with any AM indicator, a small concentration may associate nonspecifically with the fiber (either the indicator is incompletely de-esterified or, if completely de-esterified, not located in the myoplasmic compartment). If the loaded concentration is small, the nonspecific indicator will present a significant source of error in the estimation of [Ca2+]i.

Intracellular free Ca2+ and basal Mn2+ influx in cultured aortic smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats.

Numerous studies investigating the possible role of altered Ca2+ homeostasis in hypertension have compared resting and agonist-stimulated intracellular free Ca2+ ([Ca2+]i) in cultured aortic smooth muscle cells from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. However, such studies have not given consistent results. Differences in the method used to load cells with the Ca(2+)-sensitive indicator fura-2 have been investigated here as a possible source of variability between studies. We also describe the adaptation of a fluorescence technique for the assessment of basal Ca2+ permeability in SHR and WKY through the measurement of Mn2+ influx. The results are consistent with the hypothesis that basal Ca2+ influx is elevated in cultured aortic smooth muscle cells from SHR compared to those from WKY. However, this was not reflected as a significant difference between the two strains in basal or angiotensin II (200 nmol/L)-stimulated [Ca2+]i. Furthermore, this result was not dependent on the protocol used to load cells with fura-2. Hence, measurement of bulk [Ca2+]i does not appear to be the most sensitive parameter for altered Ca2+ homeostasis in SHR. Other compartments of the cell may better reflect altered Ca2+ fluxes in hypertension and are discussed in this work.

Parathyroid tissue in normocalcemic and hypercalcemic primary hyperparathyroidism recruited by health screening.

Parathyroid tissue from 57 women (mean age 65.5 years) with sporadic primary hyperparathyroidism (HPT) was analyzed mainly to clarify its characteristics versus tissue from those with normocalcemia. Patients were recruited by population-based health screening of menopausal women. Analysis of three or four total serum calcium values showed normocalcemia in 16 patients (mean 2.53 mmol/L); 20 and 21 of the women were consistently (mean 2.82 mmol/L) or intermittently (mean 2.59 mmol/L) hypercalcemic, respectively. Parathyroid operation demonstrated a single adenoma in 81% of the individuals, and these lesions were most prevalent and commonly dominated by oxyphil parathyroid cells in the persistently hypercalcemic patients. Chief cell hyperplasia (two or three abnormal glands) of the nodular type was found more often in the normocalcemic patients. Total glandular weight was the smallest (mean 270 mg) among the normocalcemic women and contributed to delicate decisions with regard to the extent of resection. Immunostaining of cryosections with a monoclonal antibody recognizing a putative Ca2+ sensor demonstrated variably heterogeneous down-regulation of the recognized glycoprotein in the pathologic parathyroid glands from all the individuals. Dose-response relations for PTH release and the cytoplasmic Ca2+ concentration ([Ca2+]i) were determined in Ca2+ 0.5-3.0 mmol/L by examining dispersed cells with radioimmunoassay and microfluorometry after fura-2 loading, respectively. ED50 for PTH release and [Ca2+]i and the [Ca2+]i concentrations at Ca2+ 3.0 mmol/L were the least deranged in cells from pathologic glands of the normocalcemic patients. The findings substantiate that the abnormal parathyroid tissue of normocalcemic HPT principally is characterized by the same, albeit less extensive, morphologic and functional derangements, which consistently have been demonstrated in patients with HPT accompanied by hypercalcemia and detected clinically.

[Ca2+]i recordings from neural cells in acutely isolated cerebellar slices employing differential loading of the membrane-permeant form of the calcium indicator fura-2.

This paper contains a description of the procedure for monitoring the cytoplasmic free calcium concentration ([Ca2+]i) from intact neurones and glial cells in acutely isolated cerebellar slices. The loading of cells with the calcium indicator fura-2 was achieved by slice incubation in Tyrode solution containing 5 "mu"M fura-2 acetoxymethylester (fura-2/AM) and 0.02% (w/v) pluronic-F127 under a controlled (temperature, 35 degrees C; humidity, 98%; and gas, 5% O2 +95% CO2) environment. In such conditions, different cellular elements of the cerebellum (namely granule neurones, Bergmann glial cells and Purkinje neurones) acquired fura-2 at different rates. Ten minutes of slice incubation gave adequate staining of granule neurones only, 20 min of incubation allowed calcium-dependent changes of fluorescence signal measurements in Bergmann glial cells, whereas loading of Purkinje neurones required 40 min of slice exposure to fura-2/AM. In order to assure dye deesterification, slices were kept in continuously gassed bicarbonate-buffered solution for not less than 1 h thereafter. The fluorescence signals (excited at 360 and 380 nm) were collected from either a 25- "mu"m or 40- "mu"m area limited by fixed diaphragm inserted in front of the photomultiplier tube; an individual cell was positioned in approximately the centre of the fluorescence measurement area. These signals were comprised of [Ca2+]i-related changes in fura-2 fluorescence recorded from a cell of interest and background fluorescence. The latter resulted from the summation of slice autofluorescence, signals from the fura-2 acquired by neighbouring tissue and signals from fura-2 compartmentalized by intracellular organelles. After the end of fluorescence recordings, the cell was internally dialysed with dye-free intracellular solution in order to determine the actual levels of background fluorescence. In parallel, electrophysiological properties were determined, allowing identification of cell type and viability. The background fluorescence values were then used to correct fluorescence recordings by subtraction from original traces. Corrected records were used for [Ca2+]i calculation.

Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2.

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2's absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.

Subcellular localization of calcium release in isolated rat myocardial cells.

Independent determinations of Ca2+ by two indicators showed that subcellular Ca2+ activity (intracellular free calcium concentration) was heterogeneous in rat myocardial cells. Arsenazo III (Az III), a membrane-impermeant absorbance indicator for Ca2+, was loaded into cardiac muscle via liposomes and calcium quantitated at two wavelengths through a focal point diaphragm in 5-100 microns 2 regions by a photometer. Fura-2, a high-affinity fluorescent calcium indicator, was loaded into cells as the ester form, and the light intensity was measured by digital-imaging microscopy using a photon-counting camera. Calcium activity at each picture element was quantitated by division of fluorescence excited by two ultraviolet wavelengths, and corrected for concentration differences in fura-2 by a third, Ca(2+)-insensitive wavelength. Both methods revealed hot spots of Ca2+ release and uptake that could be enlarged, added to, or altered. Addition of 1-10 nM norepinephrine caused up to a 500% increase in Ca2+ in localized regions, although whole cell average Ca2+ increased by only 0-80%, suggesting the importance of localized intracellular Ca2+ release for contraction.