Simultaneous monitoring of Zn2+ secretion and intracellular Ca2+ from islets and islet cells by fluorescence microscopy

A method for simultaneously imaging Zn2+ secretion and intracellular Ca2+ at beta-cell clusters and single islets of Langerhans was developed. Cells were loaded with the Ca2+ indicator Fura Red, incubated in buffer containing the Zn2+ indicator FluoZin-3, and imaged via laser scanning fluorescence confocal microscopy. FluoZin-3 and Fura Red are excited at 488 nm and emit at 515 and 665 nm, respectively. Zn2+, which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product. Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca2+ and Zn2+ secretion that were correlated with each other and were synchronized among cells. In single islets, spatially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed. Fura Red did not yield detectable Ca2+ signals at islets. For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially illuminating the islets with 340, 380, and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca2+ and secretion to be observed with approximately 2 s temporal resolution. This method should be useful for studying Ca2+ secretion coupling and any application, requiring rapid assays of secretion.     

Spike-triggered dendritic calcium transients depend on synaptic activity in the cricket giant interneurons.

The relationship between electrical activity and spike-induced Ca2+ increases in dendrites was investigated in the identified wind-sensitive giant interneurons in the cricket. We applied a high-speed Ca2+ imaging technique to the giant interneurons, and succeeded in recording the transient Ca2+ increases (Ca2+ transients) induced by a single action potential, which was evoked by presynaptic stimulus to the sensory neurons. The dendritic Ca2+ transients evoked by a pair of action potentials accumulated when spike intervals were shorter than 100 ms. The amplitude of the Ca2+ transients induced by a train of spikes depended on the number of action potentials. When stimulation pulses evoking the same numbers of action potentials were separately applied to the ipsi- or contra-lateral cercal sensory nerves, the dendritic Ca2+ transients induced by these presynaptic stimuli were different in their amplitude. Furthermore, the side of presynaptic stimulation that evoked larger Ca2+ transients depended on the location of the recorded dendritic regions. This result means that the spike-triggered Ca2+ transients in dendrites depend on postsynaptic activity. It is proposed that Ca2+ entry through voltage-dependent Ca2+ channels activated by the action potentials will be enhanced by excitatory synaptic inputs at the dendrites in the cricket giant interneurons.     

Development of depolarization-induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K(+) concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K(+) (1 min) with a transient increase in [Ca(2+)](i) of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K(+), the amplitude of the [Ca(2+)](i) transients averaging 592 nM. At later stages, all cells responded to 25 mM K(+) with [Ca(2+)](i) transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca(2+)](i) transients. The amplitudes of these [Ca(2+)](i) transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca(2+)](i) transients were greatly reduced in Ca(2+)-free, EGTA-buffered saline, and in the presence of the Ca(2+) channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca(2+) influx through voltage-activated Ca(2+) channels into antennal lobe glial cells. The development of the depolarization-induced Ca(2+) transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna.     

Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons

ABSTRACT: BACKGROUND: Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain. RESULTS: Glutamate robustly increased [Ca2+]i in 14.4 +/- 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3). 75% of cells that responded to glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals. CONCLUSIONS: Bulk-loading fura-2AM into spinal cord slices is a successful means for determining Ca2+ responses in adult dorsal horn neurons. Glutamate-evoked Ca2+ signals in adult dorsal horn neurons are mediated predominantly by AMPA channels and are potentiated by peripheral neuropathic injury.     

Facilitatory and inhibitory transmitters modulate calcium influx during action potentials in aplysia sensory neurons

Serotonin (5-HT) produces presynaptic facilitation and FMRFamide produces presynaptic inhibition in Aplysia sensory neurons. These effects may involve the modulation of Ca2+ influx into sensory neuron terminals during action potentials. Here, we have used the Ca2+ indicator dye fura-2 to monitor directly the effects of 5-HT and FMRFamide on internal Ca2+ concentration ([Ca2+]i). 5-HT caused a 50% increase in the transient rise in [Ca2+]i in response to action potentials, whereas FMRFamide decreased the [Ca2+]i transient by 40%. Neither transmitter altered the resting [Ca2+]i, the time course of recovery of the [Ca2+]i transient, or the [Ca2+]i transients produced by intracellular injection of CaCl2 or inositol 1,4,5-trisphosphate. We conclude that the effects of the transmitters on the action potential-induced [Ca2+]i transient are due to changes in Ca2+ influx and not in intracellular Ca2+ homeostasis.     

Pheromone-evoked potentials and oscillations in the antennal lobes of the sphinx moth Manduca sexta

Using intra- and extracellular recording methods, we studied the activity of pheromone-responsive projection neurons in the antennal lobe of the moth Manduca sexta. Intracellularly recorded responses of neurons to antennal stimulation with the pheromone blend characteristically included both inhibitory and excitatory stages of various strengths. To observe the activity of larger groups of neurons, we recorded responses extracellularly in the macroglomerular complex of the antennal lobe. The macroglomerular complex is part of a specialized olfactory subsystem and the site of first-order central processing of sex-pheromonal information. Odors such as the pheromone blend and host-plant (tobacco) volatiles gave rise to evoked potentials that were reproducible upon repeated antennal stimulation. Evoked potentials showed overriding high-frequency oscillations when the antenna was stimulated with the pheromone blend or with either one of the two key pheromone components. The frequency of the oscillations was in the range of 30–50 Hz. Amplitude and frequency of the oscillations varied during the response to pheromonal stimulation. Recording intracellular and extracellular activity simultaneously revealed phase-locking of action potentials to potential oscillations. The results suggest that the activity of neurons of the macroglomerular complex was temporally synchronized, potentially to strengthen the pheromone signal and to improve olfactory perception. Accepted: 19 December 1997     

Role of voltage-dependent Na+ channels for rhythmic Ca2+ signalling in glucose-stimulated mouse pancreatic beta-cells.

The putative role of voltage-dependent Na+ channels for glucose induction of rhythmic Ca2+ signalling was studied in mouse pancreatic beta-cells with the use of the Ca2+ indicator fura-2. A rise in glucose from 3 to 11 mM resulted in slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i). These oscillations, as well as superimposed transients seen during forskolin-induced elevation of cAMP, remained unaffected in the presence of the Na+ channel blocker tetrodotoxin. During exposure to 1-10 microM veratridine, which facilitates the opening of voltage-dependent Na+ channels, the slow oscillations were replaced by repetitive and pronounced [Ca2+]i transients arising from the basal level. The effects of veratridine were reversed by tetrodotoxin. The veratridine-induced [Ca2+]i transients were critically dependent on the influx of Ca2+ and persisted after thapsigargin inhibition of the endoplasmic reticulum Ca2+-ATPase. Both tolbutamide and ketoisocaproate mimicked the action of glucose in promoting [Ca2+]i transients in the presence of veratridine. It is suggested that activation of voltage-dependent Na+ channels is a useful approach for amplifying Ca2+ signals for insulin release.     

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.     

CRH-induced electrical activity and calcium signalling in pituitary corticotrophs

Pituitary corticotroph cells generate repetitive action potentials and associated Ca2+ transients in response to the agonist corticotropin releasing hormone (CRH). There is indirect evidence suggesting that the agonist, by way of complex intracellular mechanisms, modulates the voltage sensitivity of the L-type Ca2+ channels embedded in the plasma membrane. We have previously constructed a Hodgkin-Huxley-type model of this process, which indicated that an increase in the L-type Ca2+ current is sufficient to generate repetitive action potentials (LeBeau et al. (1997). Biophys. J.73, 1263-1275). CRH is also believed to inhibit an inwardly rectifying K+ current. In this paper, we have found that a CRH-induced inhibition of the inwardly rectifying K+ current increases the model action potential firing frequency, [Ca2+]i transients and membrane excitability. This dual modulatory action of CRH on inward rectifier and voltage-gated Ca2+ channels better describes the observed CRH-induced effects. This structural alteration to the model along with parameter changes bring the model firing frequency in line with experimental data. We also show that the model exhibits experimentally observed bursting behaviour, where the depolarization spike is followed by small oscillations in the membrane potential.     

Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines

We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).     

Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts

Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca(2+) signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca(2+) homeostasis and stimulus-induced Ca(2+) signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca(2+)-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca(2+) concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca(2+) concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca(2+) concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca(2+) concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca(2+) concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.     

Mechanisms underlying odorant-induced and spontaneous calcium signals in olfactory receptor neurons of spiny lobsters, Panulirus argus

We determined if a newly developed antennule slice preparation allows studying chemosensory properties of spiny lobster olfactory receptor neurons under in situ conditions with Ca²⁺ imaging. We show that chemical stimuli reach the dendrites of olfactory receptor neurons but not their somata, and that odorant-induced Ca²⁺ signals in the somata are sufficiently stable over time to allow stimulation with a substantial number of odorants. Pharmacological manipulations served to elucidate the source of odorant-induced Ca²⁺ transients and spontaneous Ca²⁺ oscillations in the somata of olfactory receptor neurons. Both Ca²⁺ signals are primarily mediated by an influx of extracellular Ca²⁺ through voltage-activated Ca²⁺ channels that can be blocked by CoCl₂ and the L-type Ca²⁺ channel blocker verapamil. Intracellular Ca²⁺ stores contribute little to odorant-induced Ca²⁺ transients and spontaneous Ca²⁺ oscillations. The odorant-induced Ca²⁺ transients as well as the spontaneous Ca²⁺ oscillations depend on action potentials mediated by Na⁺ channels that are largely TTX-insensitive but blocked by the local anesthetics tetracaine and lidocaine. Collectively, these results corroborate the conclusion that odorant-induced Ca²⁺ transients and spontaneous Ca²⁺ oscillations in the somata of olfactory receptor neurons closely reflect action potential activity associated with odorant-induced phasic-tonic responses and spontaneous bursting, respectively. Therefore, both types of Ca²⁺ signals represent experimentally accessible proxies of spiking.     

Relationship between myoplasmic calcium transients and calcium currents in frog skeletal muscle

Ca2+ currents (ICa) and myoplasmic Ca2+ transients were simultaneously recorded in single muscle fibers from the semitendinosus muscle of Rana pipiens. The vaseline-gap voltage-clamp technique was used. Ca2+ transients were recorded with the metallochromic indicator dye antipyrylazo III. Ca2+ transients consisted of an early fast rising phase followed by a late slower one. The second phase was increased by experimental maneuvers that enlarged ICa, such as augmenting [Ca2+]o (from 2 to 10 mM) or adding (-)-Bay K 8644 (2 microM). When [Ca2+]o was increased, the second phase of the Ca2+ transients and ICa showed an average increase at 0 mV of 2 +/- 0.9 microM (4) and 1.4 +/- 0.3 mA/ml (4), respectively. (-)-Bay K 8644 increased the late phase of the Ca2+ transients and ICa at 0 mV by 0.8 +/- 0.3 microM (3) and 6.7 +/- 2.0 mA/ml (4), respectively. The initial fast rising phase of the Ca2+ transients was not modified. (-)-Bay K 8644 slowed the time constant of decay of the transients by 57 +/- 6 ms. In other experimental conditions, Ca2+ release from the sarcoplasmic reticulum (SR) was impaired with repetitive stimulation in 1 mM [EGTA]i-containing fibers. Under those circumstances, Ca2+ transients directly followed the time integral of ICa. Pulses to 0 mV caused a large Ca2+ transient that became suppressed when large pulses to 100 mV were applied. In fibers with functioning SR, pulses to 100 mV elicited somewhat smaller or similar amplitude Ca2+ transients when compared with those elicited by pulses to 0 mV. The increase in ICa after raising [Ca2+]o or adding (-)-Bay K 8644 cannot directly explain the change in Ca2+ transients in fibers with functioning SR. On the other hand, when Ca2+ release from the SR is impaired Ca2+ transients depend on ICa.     

Calcium buffering and clearance in spider mechanosensory neurons

Spider VS-3 mechanoreceptor neurons have a low-voltage-activated Ca2+ current that raises intracellular calcium concentration [Ca2+] when they are depolarized by agonists of GABAA receptors or fire action potentials. The Ca2+ rise produces negative feedback by modulating the mechanoreceptor current and regulates Ca2+- and voltage-activated K+ currents. However, nothing is known about Ca2+ buffering in VS-3 neurons. Dynamic changes in VS-3 neuron intracellular [Ca2+] were measured using the fluorescent Ca2+ indicator Oregon Green BAPTA-1 (OG488) to understand Ca2+ buffering and clearance. Intracellular OG488 concentration increased slowly over more than 2 h as it diffused through a sharp intracellular microelectrode and spread through the cell. This slow increase was used to measure endogenous Ca2+ buffering and clearance by the added buffer technique, with OG488 acting as both added exogenous buffer and Ca2+ indicator. [Ca2+] was raised for brief periods by regular action potential firing, produced by pulsed electric current injection through the microelectrode. The resulting rise and fall of [Ca2+] were well fitted by the single compartment model of Ca2+ dynamics. With earlier ratiometric [Ca2+] estimates, these data gave an endogenous Ca2+ binding ratio of 684. Strong Ca2+ buffering may assist these neurons to deal with rapid changes in mechanical inputs.     

Activation of Nuclear Calcium Dynamics by Synaptic Stimulation in Cultured Cortical Neurons

L-type voltage-sensitive Ca2+ channels (VSCCs) are enriched on the neuronal soma and trigger gene expression during synaptic activity. To understand better how these channels regulate somatic and nuclear Ca2+ dynamics, we have investigated Ca2+ influx through L-type VSCCs following synaptic stimulation, using the long-wavelength Ca2+ indicator fluo-3 combined with laser scanning confocal microscopy. Single synaptic stimuli resulted in rapid Ca2+ transients in somatic cytoplasmic compartments (<5 ms rise time). Nuclear Ca2+ elevations lagged behind cytoplasmic levels by approximately 60 ms, consistent with a dependence on diffusion from a cytoplasmic source. Pharmacological experiments indicated that L-type VSCCs mediated approximately 50% of the nuclear and somatic (cytoplasmic) Ca2+ elevation in response to strong synaptic stimulation. In contrast, relatively weak excitatory postsynaptic potentials (EPSPs; approximately 15 mV) or single action potentials were much less effective at activating L-type VSCCs. Antagonist experiments indicated that activation of the NMDA-type glutamate receptor leads to a long-lasting somatic depolarization necessary to activate L-type VSCCs effectively during synaptic stimuli. Simulation of action potential and somatic EPSP depolarization using voltage-clamp pulses indicated that nuclear Ca2+ transients mediated by L-type VSCCs were produced by sustained depolarization positive to -25 mV. In the absence of synaptic stimulation, action potential stimulation alone led to elevations in nuclear Ca2+ mediated by predominantly non-L-type VSCCs. Our results suggest that action potentials, in combination with long-lived synaptic depolarizations, facilitate the activation of L-type VSCCs. This activity elevates somatic Ca2+ levels that spread to the nucleus.     

Simultaneous dual-excitation ratiometry using orthogonal linear polarized lights

Dual-excitation ratiometric dyes permit quantitative Ca2+ measurements by minimizing the effects of several artifacts that are unrelated to changes in the concentration of free Ca2+ ([Ca2+]). These dyes are excited alternately at two different wavelengths, and the pair of intensity measurements must be collected sequentially. Therefore, it is difficult to follow very fast Ca2+ dynamics or Ca2+ changes in highly motile cell samples. Here, we present a novel but simple dual-excitation ratiometric method which overcomes this problem. By the use of our home-made illuminator, each sample is illuminated by two orthogonal linear polarized lights of different wavelengths. Fluorescence images are captured by two CCD cameras through two analyzers, whose polarization directions are at right angles. This methodology allows us to perform simultaneous measurements of any dual-excitation ratiometric dye, and we demonstrate its validity by monitoring [Ca2+] changes in rat cardiac muscle cells loaded with Fura Red.     

Cyclic compression of chondrocytes modulates a purinergic calcium signalling pathway in a strain rate- and frequency-dependent manner

Mechanical loading modulates cartilage homeostasis through the control of matrix synthesis and catabolism. However, the mechanotransduction pathways through which chondrocytes detect different loading conditions remain unclear. The present study investigated the influence of cyclic compression on intracellular Ca2+ signalling using the well-characterised chondrocyte-agarose model. Cells labelled with Fluo4 were visualised using confocal microscopy following a period of 10 cycles of compression between 0% and 10% strain. In unstrained agarose constructs, not subjected to cyclic compression, a subpopulation of approximately 45% of chondrocytes exhibited spontaneous global Ca2+ transients with mean transient rise and fall times of 19.4 and 29.4 sec, respectively. Cyclic compression modulated global Ca2+ signalling by increasing the percentage of cells exhibiting Ca2+ transients (population modulation) and/or reducing the rise and fall times of these transients (transient shape modulation). The frequency and strain rate of compression differentially modulated these Ca2+ signalling characteristics providing a potential mechanism through which chondrocytes may distinguish between different loading conditions. Treatment with apyrase, gadolinium and the P2 receptor blockers, suramin and basilen blue, significantly reduced the percentage of cells exhibiting Ca2+ transients following cyclic compression, such that the mechanically induced upregulation of Ca2+ signalling was completely abolished. Thus cyclic compression appears to activate a purinergic pathway involving the release of ATP followed by the activation of P2 receptors causing a combination of extracellular Ca2+ influx and intracellular Ca2+ release. Knowledge of this fundamental cartilage mechanotransduction pathway may lead to improved therapeutic strategies for the treatment of cartilage damage and disease.     

Insulin-secreting INS-1 cells generate a novel type of poorly synchronized Ca2+ transients

Pancreatic beta-cells have an intrinsic oscillatory Ca2+ activity supposed to be synchronized among the islets by cytoplasmic Ca2+ transients elicited by nonadrenergic, noncholinergic (NANC) neurons. To improve the understanding of this process, the cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in two insulin-releasing cell lines using dual wavelength microfluorometry and the indicator fura-2. INS-1 cells but not RINm5F cells were found to generate transients of [Ca2+]i in the presence of the Ca2+ channel blocker methoxyverapamil. These transients differed from those occurring in native beta-cells persisting in the presence of thapsigargin or during prolonged exposure to ATP. Moreover, the [Ca2+]i transients were poorly synchronized whether or not the INS-1 cells had physical contact. If appearing in native beta-cells, the type of [Ca2+]i transients now observed may interfere with the coordination of the beta-cell rhythmicity evoked by NANC neurons.     

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.