Pavel V. Belan

Cytoplasmic free Ca in isolated snail neurons as revealed by fluorescent probe fura-2: Mechanisms of Ca recovery after Ca load and Ca release from intracellular stores

SummaryUsing the fluorescent probe fura-2, we measured the cytoplasmic concentration of free Ca2+ ([Ca]i) and its changes in isolated, nonidentified neurons of the snailHelix pomatia. [Ca]i increased during membrane depolarization due to opening of Ca channels in the surface membrane. When the membrane potential returned to the resting level, [Ca]i recovered monoexponentially, with the time constant ranging from 10 to 30 sec. The rate of recovery remained unchanged after treatments that interferred with the normal functioning of both Ca/Na exchange and Ca-ATPase in the surface membrane or mitochondria. [Ca]i recovery slowed down upon cooling according to Q10=2.3 and after intracellular injection of vanadate. The data obtained suggest that the rate of [Ca]i recovery after membrane depolarization is mainly determined by Ca pump of intracellular stores (presumably by the endoplasmic reticulum). Ca release from these stores could be induced in the presence of millimolar caffeine or theophylline in the external medium when [Ca]i increased up to a certain threshold level. This depolarization-induced Ca load triggered further transient increase in [Ca]i, which was accompanied by membrane hyperpolarization due to the development of Ca-activated potassium conductance. 1mm procaine or tetracaine, but not lidocaine, inhibited this Ca-induced Ca release. In some cases stable oscillations of [Ca]i were observed. They could be induced by producing a steady Ca influx by membrane depolarization.

Specific functioning of Cav3.2 T-type calcium and TRPV1 channels under different types of STZ-diabetic neuropathy.

Streptozotocin (STZ)-induced type 1 diabetes in rats leads to the development of peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia at early stages (4th week) followed by hypoalgesia after 8weeks of diabetes development. Here we found that 6-7 week STZ-diabetic rats developed either thermal hyper- (18%), hypo- (25%) or normalgesic (57%) types of PDN. These developmentally similar diabetic rats were studied in order to analyze mechanisms potentially underlying different thermal nociception. The proportion of IB4-positive capsaicin-sensitive small DRG neurons, strongly involved in thermal nociception, was not altered under different types of PDN implying differential changes at cellular and molecular level. We further focused on properties of T-type calcium and TRPV1 channels, which are known to be involved in Ca(2+) signaling and pathological nociception. Indeed, TRPV1-mediated signaling in these neurons was downregulated under hypo- and normalgesia and upregulated under hyperalgesia. A complex interplay between diabetes-induced changes in functional expression of Cav3.2 T-type calcium channels and depolarizing shift of their steady-state inactivation resulted in upregulation of these channels under hyper- and normalgesia and their downregulation under hypoalgesia. As a result, T-type window current was increased by several times under hyperalgesia partially underlying the increased resting [Ca(2+)]i observed in the hyperalgesic rats. At the same time Cav3.2-dependent Ca(2+) signaling was upregulated in all types of PDN. These findings indicate that alterations in functioning of Cav3.2 T-type and TRPV1 channels, specific for each type of PDN, may underlie the variety of pain syndromes induced by type 1 diabetes.

Distribution of Ca2+ extrusion sites on the mouse pancreatic acinar cell surface

The localizations of Ca2+ extrusion sites in mouse pancreatic acinar cells during elevation of the intracellular free calcium concentration ([Ca2+]i) have been studied. During an agonist stimulated calcium elevation as well as when intracellular calcium is released from a caged compound, Ca2+ is primarily extruded from the apical secretory pole of the cells in spite of different spatial patterns of [Ca2+]i different sources of Ca2+, and the presence or absence of agonist. This is most likely due to a relatively high density of calcium pumps in the secretory granule region, although it could be explained by calcium pumps in this part of the cell having different characteristics from those in the basal membrane. The intensity of Ca2+ extrusion in the apical secretory pole is such that substantial (several millimoles per litre) changes of the free calcium concentration in the lumen of the acinus can occur during agonist stimulation.

Calcium clamp in isolated neurones of the snail Helix pomatia.

1. Intracellular free calcium concentration ([Ca2+]i) in isolated non‐identified Helix pomatia neurones has been clamped at different physiologically significant levels by a feedback system between the fluorescent signal of fura‐2 probe loaded into the cell and ionophoretic injection of Ca2+ ions through a CaCl2‐loaded microelectrode. The membrane potential of the neurone has also been clamped using a conventional two‐microelectrode method. 2. Special measurements have shown that the transport indices of injecting microelectrodes filled with 50 mM CaCl2 are quite variable (0.11 +/‐ 0.06, mean +/‐ S.D.). However, for each electrode the transport indices remained stable during several injection trials into a solution drop having the size of a neurone. The spread of calcium ions from the tip of the microelectrode across the cytosol of the neurone terminated within 2‐4 s. The spatial difference in [Ca2+]i at this time did not exceed 10%. 3. Clamping of [Ca2+]i at a new increased level was accompanied by a transient of the Ca(2+)‐injecting current. To increase [Ca2+]i by 0.1 microM, the amount of calcium ions injected during this stage had to be 36 +/‐ 20 microM Ca2+ per cell volume. Obviously, this transient represents the filling of a fast cytosolic buffer which has to be saturated to reach a new increased level of [Ca2+]i. It was followed by a steady component of Ca(2+)‐injecting current, which was quite low (corresponding to injection of 0.39 +/‐ 0.20 microM s‐1 for a 0.1 microM change of [Ca2+]i). This may represent the functioning of Ca(2+)‐eliminating systems and corresponds to a similar amount of Ca2+ extruded from the cytoplasm. 4. Changes in the injection current also developed when Ca2+ influx through the membrane was triggered by the activation of voltage‐gated calcium channels. The amount of Ca2+ entering the cell during the first seconds of depolarization to‐‐15 mV was equal to 0.59 +/‐ 0.31 microM s‐1 per cell volume. 5. No activation of Ca(2+)‐dependent potassium current was observed during the changes in [Ca2+]i to levels exceeding the basal one by several times. Obviously, to activate this current, a much stronger increase in [Ca2+]i is needed in the immediate vicinity of the corresponding channels.

Extrusion of calcium from a single isolated neuron of the snail Helix pomatia

SummarySimultaneous optical measurements of extra- and intracellular Ca2+ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca2+. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the microchamber containing the cell. The velocity of Ca2+ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2–0.5 μm, the velocity of Ca2+ extrusion from the neuron was approximately 0.3–4.6 μm/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca2+ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 μm/sec per cell volume.

Free calcium transients and oscillations in nerve cells

SummaryChanges in the intracellular level of free calcium induced by different influences in neurones of the snail Helix pomatia have been measured by changes in Fura-2 fluorescence. Thymol in submillimolar concentrations induced the release of stored intracellular calcium. This effect was similar to xantine-induced release. IP3 and Gpp[NH]p injections also released intracellular calcium. The response to cAMP injections was more complicated and included, probably, both the release of stored calcium and its influx through membrane channels. Oscillations of intracellular free calcium are described. It has been suggested that oscillations can occur only in cases where the mechanism of Ca-dependent calcium release is activated.

A NEW TECHNIQUE FOR ASSESSING THE MICROSCOPIC DISTRIBUTION OF CELLULAR CALCIUM EXIT SITES

Abstractu2002This paper contains a description of a new method designed to monitor the distribution of Ca2+ efflux from cells or small cellular aggregates. The idea behind this method is to use a fluorescent Ca2+ indicator bound to dextrans of high molecular weight to slow down Ca2+ diffusion. Due to the decrease in diffusion rate, Ca2+ ions should be held close to the site of their release from the cells for a relatively long time, enough for the confocal microscope to detect such a local increase in Ca2+ concentration. This paper gives a detailed description of the method, illustrated with results of measurements of agonist-dependent and agonist-independent Ca2+ extrusion from pancreatic acinar cells. An appendix provides the mathematical background that should allow selection of the concentration of buffer which is necessary to achieve a particular Ca2+ diffusion coefficient.

Hippocalcin signaling via site-specific translocation in hippocampal neurons

Hippocalcin is a Ca2+-binding protein, which belongs to the family of neuronal Ca2+ sensors. It is highly expressed in the hippocampus but molecular mechanisms underlying its action in this part of the brain have not been investigated in detail. To study whether intrinsic neuronal activity could result in hippocalcin-mediated signal transduction we examined spontaneous and action potential (AP)-dependent changes in fluorescence of yellow fluorescent protein-tagged hippocalcin (HPCA-YFP) in transiently transfected hippocampal cultured neurons. In 6–12 DIV neurons HPCA-YFP spontaneously translocated longitudinally to specific sites within diffusionally confined domains of neuronal processes. The translocations to these sites were expressed as fast, reversible increases in HPCA-YFP fluorescence coincided with a decrease in adjacent sites indicating genuine protein translocation. Physiologically relevant neuronal stimulation with short trains of action potentials also resulted in fast, simultaneous, reversible, and [Ca2+]i-dependent translocations of HPCA-YFP to several sites synchronizing hippocalcin signaling in different parts of neuronal processes. The amount of translocated protein increased with the number of action potentials in a train decoding the number of APs into the amount of translocated protein. We conclude that hippocalcin may signal within diffusionally restricted domains of neuronal processes in which it might play a physiological role in Ca2+-dependent local activation of specific molecular targets.

Post-tetanic depression of GABAergic synaptic transmission in rat hippocampal cell cultures.

The effect of tetanic stimulation (30 Hz, 4 s) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. It was found that tetanic stimulation elicited post-tetanic depression (PTD) of the evoked IPSCs with a duration of more than 50 s in about 60% of the connections tested; post-tetanic potentiation was induced in 25% of the connections. We propose that the opposite effects of tetanization on IPSC amplitude are due to differences in the type of the interneuron that was tetanized. Since PTD in our experiments was usually accompanied by changes in the IPSC coefficient of variation and changes of a paired pulse depression, which are thought to reflect presynaptic mechanisms of modulation, we suggest that part of the PTD is due to a presynaptic mechanism(s).

Endocytic adaptor protein intersectin 1 forms a complex with microtubule stabilizer STOP in neurons.

Intersectin 1 (ITSN1) is a multidomain adaptor protein that functions in clathrin-mediated endocytosis and signal transduction. This protein is highly abundant in neurons and is implicated in Down syndrome, Alzheimers disease and, possibly, other neurodegenerative disorders. Here we used an in vitro binding assay combined with MALDI-TOF mass spectrometry to identify novel binding partners of ITSN1. We found that the neuron-specific isoform of the stable tubule-only polypeptide (STOP) interacts with SH3A domain of ITSN1. STOP and ITSN1 were shown to form a complex in vivo and to partially co-localize in rat primary hippocampal neurons. As STOP is a microtubule-stabilizing protein that is required for several forms of synaptic plasticity in the hippocampus, identification of this interaction raises the possibility of ITSN1 participation in this process.