Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca²⁺ concentration by uncaging of a caged-Ca²⁺ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.     

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+-induced glucagon exocytosis in pancreas

Hormones such as glucagon are secreted by Ca²⁺-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic β-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca²⁺-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca²⁺ sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca²⁺ sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca²⁺-triggered glucagon release from α-cells than insulin secretion from β-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca²⁺-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca²⁺-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic α-cells lacking synaptotagmin-7 exhibited little Ca²⁺-induced exocytosis, whereas all other physiological and morphological parameters of the α-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca²⁺ sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca²⁺ sensors in neurotransmitter, neuropeptide, and hormone secretion.     

Exocytosis and endocytosis of small vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) in the lateral membranes not facing the glass-cover slip. Upon photolysis of a caged Ca²⁺ compound, TEP imaging with FM1-43 (a polar membrane tracer) detected massive exocytosis of vesicles with a time constant of about 1 s. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that the diameter of vesicles was small (55 nm). Extensive exocytosis of small vesicles (SVs) was shown to be mediated by the transient opening of a fusion pore with a diameter less than about 1.6 nm, and to be followed by direct ('kiss-and-run') endocytosis and translocation of the endocytic vesicles (EVs) deep into the cytoplasm. These processes were unaffected by GTP-γ-S. In contrast, constitutive endocytic vesicles exhibited a diameter of 90 nm, took up molecules with a diameter of > 12 nm, and their formation was blocked by GTP-γ-S. Electron-microscopic investigation with photoconversion of diaminobenzidine using FM1-43 confirmed an abundance of EVs with a diameter of 54 nm in stimulated cells. They rapidly translocated into the cytosol, and fused with endosomal organelles. The number of SV exocytosis events vastly exceeded the number of SVs morphologically docked at the plasma membrane. Simultaneous capacitance and FM1-43 measurements indicated that TEP imaging detected most SV exocytosis, and the fusion pore was closed within 2 s. Thus, we have, for the first time, directly visualized massive exocytosis of small vesicles in a non-synaptic preparation, and have revealed their fusion-pore mediated exocytosis and endocytosis.     

Rapid glucose sensing by protein kinase A for insulin exocytosis in mouse pancreatic islets

The role of protein kinase A (PKA) in insulin exocytosis was investigated with the use of two-photon excitation imaging of mouse islets of Langerhans. Inhibitors of PKA selectively reduced the number of exocytic events during the initial period (< 250 s) of the first phase of glucose-induced exocytosis (GIE), without affecting the second phase, in intact islets or small clusters of islet cells. The PKA inhibitors did not reduce the extent of the glucose-induced increase in [Ca²⁺]_i. The actions of glucose and PKA in Ca²⁺-induced insulin exocytosis (CIE) triggered by photolysis of a caged-Ca²⁺ compound, which resulted in large increases in [Ca²⁺]_i and thereby bypassed the ATP-sensitive K⁺ channel-dependent mechanism of glucose sensing, were therefore studied. A high concentration (20 m m ) of glucose potentiated CIE within 1 min, and this effect was blocked by inhibitors of PKA. This PKA-dependent action of glucose required glucose metabolism, given that increasing the intracellular concentration of cAMP by treatment with forskolin potentiated CIE only at the high glucose concentration. Finally, PKA appeared to reduce the frequency of 'kiss-and-run' exocytic events and to promote full-fusion events during GIE. These data indicate that a PKA-dependent mechanism of glucose sensing, which is operative even at the basal level of PKA activity, plays an important role specifically in the first phase of GIE, and they suggest that the action of PKA is mediated at the level of the fusion reaction.     

Novel aspects of the molecular mechanisms controlling insulin secretion

Pancreatic β-cells secrete insulin by Ca²⁺-dependent exocytosis of secretory granules. β-cell exocytosis involves SNARE (soluble NSF-attachment protein receptor) proteins similar to those controlling neurotransmitter release and depends on the close association of L-type Ca²⁺ channels and granules. In most cases, the secretory granules fuse individually but there is ultrastructural and biophysical evidence of multivesicular exocytosis. Estimates of the secretory rate in β-cells in intact islets indicate a release rate of ∼15 granules per β-cell per second, 100-fold higher than that observed in biochemical assays. Single-vesicle capacitance measurements reveal that the diameter of the fusion pore connecting the granule lumen with the exterior is ∼1.4 nm. This is considerably smaller than the size of insulin and membrane fusion is therefore not obligatorily associated with release of the cargo, a feature that may contribute to the different rates of secretion detected by the biochemical and biophysical measurements. However, small molecules like ATP and GABA, which are stored together with insulin in the granules, are small enough to be released via the narrow fusion pore, which accordingly functions as a molecular sieve. We finally consider the possibility that defective fusion pore expansion accounts for the decrease in insulin secretion observed in pathophysiological states including long-term exposure to lipids.     

Real-time monitoring of cAMP levels in living endothelial cells: thrombin transiently inhibits adenylyl cyclase 6

The crosstalk between Ca²⁺ and cAMP signals plays a significant role for the regulation of the endothelial barrier function. The Ca²⁺-elevating agent thrombin was demonstrated to increase endothelial permeability and to decrease cAMP levels. Since Ca²⁺ and cAMP signals are highly dynamic, we aimed to study the temporal resolution between thrombin-evoked Ca²⁺ signals and subsequent changes of cAMP levels. Here we conduct the first real-time monitoring of thrombin-mediated regulation of cAMP signals in intact human umbilical vein endothelial cells (HUVECs) by utilising the Ca²⁺-sensitive dye Fluo-4 and the fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps. We calibrated in vitro FRET responses of Epac1-camps to [cAMP] in order to estimate changes in intracellular [cAMP] evoked by thrombin treatment of HUVECs. After increasing [cAMP] to 1.2 ± 0.2 μ m by stimulation of HUVECs with isoproterenol (isoprenaline), we observed a transient decrease of cAMP levels by 0.4 ± 0.1 μ m which reached a minimum value 30 s after thrombin application and 15 s after the thrombin-evoked Ca²⁺ peak. This transient decrease in [cAMP] was Ca²⁺-dependent and independent of a G_i-mediated inhibition of adenylyl cyclases (ACs). Instead the knock down of the predominant subtype AC6 in HUVECs provided the first direct evidence that the Ca²⁺-mediated inhibition of AC6 accounts for the thrombin-induced decrease in cAMP levels.     

α-Latrotoxin increases spontaneous and depolarization-evoked exocytosis from pancreatic islet β-cells

α-Latrotoxin (α-LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential-evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of α-LT on single human, mouse and canine β-cells. In isolated and combined measurements, α-LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca²⁺, into the micromolar range, that are dependent on extracellular Ca²⁺; (ii) large conductance non-selective cation channels; and (iii) Ca²⁺-dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin. Furthermore, at picomolar concentrations, α-LT potentiates depolarization-induced exocytosis often without evidence of inducing channel activity or increasing cytosolic Ca²⁺. These results strongly support the hypothesis that α-LT, after binding to specific receptors, has at least two complementary modes of action on excitable cells. (i) α-LT inserts into the plasma membrane to form Ca²⁺ permeable channels and promote Ca²⁺ entry thereby triggering Ca²⁺-dependent exocytosis in unstimulated cells. (ii) At lower concentrations, where its channel forming activity is hardly evident, α-LT augments depolarization-evoked exocytosis probably by second messenger-induced enhancement of the efficiency of the vesicle recruitment or vesicle fusion machinery. We suggest that both modes of action enhance exocytosis from a newly described highly Ca²⁺-sensitive pool of insulin granules activated by global cytosolic Ca²⁺ concentrations in the range of ∼1 μ m .     

Paradoxical SR Ca2+ release in guinea-pig cardiac myocytes after β-adrenergic stimulation revealed by two-photon photolysis of caged Ca2+

In heart muscle the amplification and shaping of Ca²⁺ signals governing contraction are orchestrated by recruiting a variable number of Ca²⁺ sparks. Sparks reflect Ca²⁺ release from the sarcoplasmic reticulum (SR) via Ca²⁺ release channels (ryanodine receptors, RyRs). RyRs are activated by Ca²⁺ influx via L-type Ca²⁺ channels with a specific probability that may depend on regulatory mechanisms (e.g. β-adrenergic stimulation) or diseased states (e.g. heart failure). Changes of RyR phosphorylation may be critical for both regulation and impaired function in disease. Using UV flash photolysis of caged Ca²⁺ and short applications of caffeine in guinea-pig ventricular myocytes, we found that Ca²⁺ release signals on the cellular level were largely governed by global SR content. During β-adrenergic stimulation resting myocytes exhibited smaller SR Ca²⁺ release signals when activated by photolysis (62.3% of control), resulting from reduced SR Ca²⁺ content under these conditions (58.6% of control). In contrast, local signals triggered with diffraction limited two-photon photolysis displayed the opposite behaviour, exhibiting a larger Ca²⁺ release (164% of control) despite reduced global and local SR Ca²⁺ content. This apparent paradox implies changes of RyR open probabilities after β-adrenergic stimulation, enhancing local regenerativity and reliability of Ca²⁺ signalling. Thus, our results underscore the importance of phosphorylation of RyRs (or of a related protein), as a regulatory physiological mechanism that may also provide new therapeutic avenues to recover impaired Ca²⁺ signalling during cardiac disease.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.     

Synaptotagmin regulates mast cell functions

Summary: Synaptotagmin(s) (Syts), are products of a gene family implicated in the control of Ca²⁺-dependent exocytosis. Mast cells, specialized secretory cells that release mediators of inflammatory and allergic reactions in a process of regulated exocytosis, express Syt homologues and SNAREs (Soluble NSF Attachment proteins Receptors), which together with Syt constitute the core complex which mediates exocytotic vesicle docking and fusion. Rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, express the Syt homologues Syt II, Syt III and Syt V. Expression of Syt I, the neuronal Ca²⁺ sensor, in the RBL cells, resulted in its targeting to secretory granules and in prominent potentiation and acceleration of Ca²⁺-dependent exocytosis. Syt II is localized to an amine-free lysosomal compartment, which is also subjected to regulated exocytosis. Lysosomal exocytosis is negatively regulated by Syt II: overexpression of Syt II inhibited Ca²⁺-triggered exocytosis of lysosomes, while suppression of Syt II expression markedly potentiated this release. These findings implicate Syt homologues as key regulators of mast cell function.
We thank Drs. T.C. Sudhof, R.H. Scheller and M. Takahashi for their generous gifts of antibodies and cDNAs.     

Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice

At the nerve terminal, both N- and P/Q-type Ca²⁺ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca²⁺ channel subtypes, we recorded N-type and P/Q-type Ca²⁺ currents directly from calyces of Held nerve terminals in α_1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca²⁺ currents was activity-dependent facilitation, which was absent for N-type Ca²⁺ currents. EPSCs mediated by P/Q-type Ca²⁺ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca²⁺ currents. In addition, the maximal inhibition by the GABA_B receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca²⁺ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.     

Calcium oscillations in interstitial cells of the rabbit urethra

Measurements were made (using fast confocal microscopy) of intracellular Ca²⁺ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca²⁺ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with d - myo -inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release using 100 μ m 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N , N -diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca²⁺, and frequency was directly proportional to the external Ca²⁺ concentration. Frequency of Ca²⁺ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca²⁺ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca²⁺ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca²⁺ concentration and that conversion of the primary oscillation to a propagated Ca²⁺ wave depends upon IP₃-induced Ca²⁺ release.     

Physiological modulation of inactivation in L-type Ca2+ channels: one switch

The relative contributions of voltage- and Ca²⁺-dependent mechanisms of inactivation to the decay of L-type Ca²⁺ channel currents ( I _CaL) is an old story to which recent results have given an unexpected twist. In cardiac myocytes voltage-dependent inactivation (VDI) was thought to be slow and Ca²⁺-dependent inactivation (CDI) resulting from Ca²⁺ influx and Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum provided an automatic negative feedback mechanism to limit Ca²⁺ entry and the contribution of I _CaL to the cardiac action potential. Physiological modulation of I _CaL by β-adrenergic and muscarinic agonists then involved essentially more or less of the same by enhancing or reducing Ca²⁺ channel activity, Ca²⁺ influx, sarcoplasmic reticulum load and thus CDI. Recent results on the other hand place VDI at the centre of the regulation of I _CaL. Under basal conditions it has been found that depolarization increases the probability that an ion channel will show rapid VDI. This is prevented by β-adrenergic stimulation. Evidence also suggests that a channel which shows rapid VDI inactivates before CDI can become effective. Therefore the contributions of VDI and CDI to the decay of I _CaL are determined by the turning on, by depolarization, and the turning off, by phosphorylation, of the mechanism of rapid VDI. The physiological implications of these ideas are that under basal conditions the contribution of I _CaL to the action potential will be determined largely by voltage and by Ca²⁺ following β-adrenergic stimulation.     

Synaptotagmin–Ca2+ triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation

Synaptotagmin I (Syt I), the putative Ca²⁺ sensor in regulated exocytosis, has two Ca²⁺-binding modules, the C2A and C2B domains, and a number of putative effectors to which Syt I binds in a Ca²⁺-dependent fashion. The role of Ca²⁺ binding to these domains remains unclear, as efforts to address questions about Ca²⁺-triggered effector interactions have led to conflicting results. We have studied the effects of Ca²⁺ on fusion pores using amperometry to follow the exocytosis of single vesicles in real time and analyse the kinetics of fusion pore transitions. Elevating [Ca²⁺] in permeabilized cells reduced the fusion pore lifetime, indicating an action of Ca²⁺ during the actual fusion process. Analysing the Ca²⁺ dependence of the fusion pore lifetime, together with the frequency of pore openings and the proportion of openings that close without dilating (kiss-and-run events) enabled us to resolve exocytosis into a sequence of kinetic steps representing functional transitions in the fusion pore. Fusion pore opening and dilation were both accelerated by Ca²⁺, indicating separate Ca²⁺ control over each of these steps. Ca²⁺ ligand mutations in either the C2A or C2B domains of Syt I reduced fusion pore opening, but had opposite actions on the rate of fusion pore closure. These studies resolve two separate and distinct Ca²⁺-triggered steps during regulated exocytosis. The C2A and C2B domains of Syt I have different actions during these steps, and these actions may be linked to their distinctive effector interactions.     

Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na⁺-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca²⁺-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca²⁺-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca²⁺ current ( I _Ca) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K⁺ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca²⁺ influx ( I _K(Ca)). I therefore investigated whether proton-mediated feedback inhibition of I _Ca affected the activation of I _K(Ca). A transient inhibition of I _K(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I _Ca but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca²⁺-dependent currents and regenerative potentials in bipolar-cell terminals.     

Propagation in the transverse tubular system and voltage dependence of calcium release in normal and mdx mouse muscle fibres

Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation–contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca²⁺ release by measuring Ca²⁺ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca²⁺ release elicited by voltage pulses was reduced by ∼67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca²⁺ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca²⁺ may be altered in the mdx mouse.     

Local recovery of Ca2+ release in rat ventricular myocytes

Excitation–contraction coupling in the heart depends on the positive feedback process of Ca²⁺-induced Ca²⁺ release (CICR). While CICR provides for robust triggering of Ca²⁺ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca²⁺ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca²⁺ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca²⁺ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca²⁺ spark sites. Examination of these repetitive Ca²⁺ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by ∼80 ms. We conclude that restitution of Ca²⁺ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca²⁺ to SR regulatory proteins.     

Olfactoretinal centrifugal input modulates zebrafish retinal ganglion cell activity: a possible role for dopamine-mediated Ca2+ signalling pathways

The vertebrate retina receives centrifugal input from the brain. In zebrafish, the major centrifugal input originates in the terminal nerve (TN). TN cell bodies are located in the olfactory bulb and ventral telencephalon. The TN projects axons to the retina where they branch in the inner plexiform layer (IPL) and synapse onto several inner retinal cell types, including dopaminergic interplexiform cells (DA-IPCs). This olfactoretinal centrifugal input plays a role in modulating retinal ganglion cell (RGC) activity, probably via dopamine-mediated Ca²⁺ signalling pathways. Normally, dopamine inhibits RGC firing by decreasing the inward Ca²⁺ current. Olfactory stimulation with amino acids decreases dopamine release in the retina, thereby reducing dopaminergic inhibition of RGCs. This model of olfacto-visual integration was directly tested by recording single-unit RGC activity in response to olfactory stimulation in the presence or absence of dopamine receptor blockers. Stimulation of the olfactory neurones increased RGC activity. However, this effect diminished when the dopamine D1 receptors were pharmacologically blocked. In isolated RGCs, the application of dopamine or a dopamine D1 receptor agonist decreased voltage-activated Ca²⁺ current and lowered Ca²⁺ influx. Together, the data suggest that olfactory input has a modulatory effect on RGC firing, and that this effect is mediated by dopamine D1 receptor-coupled Ca²⁺ signalling pathways.