Somatic Exocytosis of Serotonin Mediated by L-Type Calcium Channels in Cultured Leech Neurones

We studied somatic exocytosis of serotonin and its mediation by L-type calcium (Ca²⁺) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 m m potassium (K⁺), and was quantified by use of the fluorescent dye FM 1–43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1–43 fluorescent spots of 1.28 ± 0.01 μm in diameter, provided that Ca²⁺ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 m m K⁺. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 ± 5 spots per soma, 77.9 ± 13.9 spots per soma were produced at 10 Hz and 91.5 ± 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ . In the presence of the L-type Ca²⁺ channel blocker nimodipine (10 μ m ), a 20 Hz train produced only 22.9 ± 6.4 spots per soma, representing a 75 % reduction compared to control cells (   P < 0.05  ). Subsequent incubation with 10 m m caffeine to induce Ca²⁺ release from intracellular stores increased the number of spots to 73.22 ± 12.5. Blockers of N-, P-, Q- or invertebrate Ca²⁺ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.     

Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium-aggravated myotonia, long QT–3 syndrome, and neuropathic pain. This drug elicits tonic block of Na⁺ channels when cells are stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation-deficient mutant Na⁺ channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a β1 subunit. The 50% inhibitory concentrations (IC₅₀) for the inactivated-state block and the resting-state block of wild-type Na⁺ channels by mexiletine were measured as 67.8 ± 7.0 μ m and 431.2 ± 9.4 μ m , respectively ( n = 5). In contrast, the IC₅₀ for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 μ m ( n = 5), which was within the therapeutic plasma concentration range (2.8–11 μ m ). Estimated on- and off-rates for the open-state block by mexiletine at +30 mV were 10.4 μ m ⁻¹ s⁻¹ and 54.4 s⁻¹, respectively. Use-dependent block by mexiletine was greater in inactivation-deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC₅₀ values for the block of persistent late hNav1.4 currents in chloramine-T-pretreated cells by mexiletine was 7.5 ± 0.8 μ m ( n = 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open-channel block of persistent late Na⁺ currents, which may arise during various pathological conditions.     

The sources and sequestration of Ca2+ contributing to neuroeffector Ca2+ transients in the mouse vas deferens

The detection of focal Ca²⁺ transients (called neuroeffector Ca²⁺ transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca²⁺ in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 µ m ) decreased the amplitude of NCTs by 45 ± 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 ± 10 ms to 800 ± 100 ms). Caffeine (3 m m ) induced spontaneous focal smooth muscle Ca²⁺ transients (sparks). Neither of the T-type Ca²⁺ channel blockers NiCl₂ (50 µ m ) or mibefradil dihydrochloride (10 µ m ) affected the amplitude of excitatory junction potentials (2 ± 5 % and −3 ± 10 %) or NCTs (−20 ± 36 % and 3 ± 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the α₁-adrenoceptor antagonist prazosin (100 n m ), showing that NCTs can initiate local contractions. Slow (5.8 ± 0.4 µm s⁻¹), spontaneous smooth muscle Ca²⁺ waves were occasionally observed. Thus, Ca²⁺ stores initially amplify and then sequester the Ca²⁺ that enters through P2X receptors and there is no amplification by local voltage-gated Ca²⁺ channels.     

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.     

Role of the Transverse-Axial Tubule System in Generating Calcium Sparks and Calcium Transients in Rat Atrial Myocytes

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse-axial tubular system (TATS). To investigate the possible role of the TATS in excitation-contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di-8-ANEPPS) simultaneously with Ca²⁺ transients and/or Ca²⁺ sparks (fluo-4). In confocal transverse linescan images of field-stimulated cells, whole-cell Ca²⁺ transients had two morphologies: 'U-shaped' transients and irregular or 'W-shaped' transients with a varying number of points of origin of the Ca²⁺ transient. About half (54 %, n =289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 ± 2.8 μm) than cells without TATS (11.7 ± 2.0 μm) and were more common in the left atrium ( n = 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca²⁺ sparks and sarcolemmal structures showed that cells without TATS had U-shaped transients that started at the cell periphery, and cells with TATS had W-shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) 'spontaneous' (non-depolarized) Ca²⁺ sparks occurred within 1 μm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 μm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca²⁺ transient and for spontaneous Ca²⁺ sparks.     

Effects of Mg2+ and SR luminal Ca2+ on caffeine-induced Ca2+ release in skeletal muscle from humans susceptible to malignant hyperthermia

Regulation of the ryanodine receptor (RYR) by Mg²⁺ and SR luminal Ca²⁺ was studied in mechanically skinned malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) fibres from human vastus medialis. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca²⁺] were detected using fura-2 fluorescence. At 1 m m cytosolic Mg²⁺, MHS fibres had a higher sensitivity to caffeine (2-40 m m ) than MHN fibres. The inhibitory effect of Mg²⁺ on caffeine-induced Ca²⁺ release was studied by increasing [Mg²⁺] of the solution containing 40 m m caffeine. Increasing [Mg²⁺] from 1 to 3 m m reduced the amplitude of the caffeine-induced Ca²⁺ transient by 77 ± 7.4 % ( n = 8) in MHN fibres. However, the caffeine-induced Ca²⁺ transient decreased by only 24 ± 8.1 % ( n = 9) in MHS fibres. In MHN fibres, reducing the Ca²⁺ loading period from 4 to 1 min (at 1 m m Mg²⁺) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca²⁺ content released in response to 40 m m caffeine by 90.4 ± 6.2 % ( n = 6). However, in MHS fibres the response was reduced by only 31.2 ± 17.4 % ( n = 6) under similar conditions. These results suggest that human malignant hyperthermia (MH) is associated with reduced inhibition of the RYR by (i) cytosolic Mg²⁺ and (ii) SR Ca²⁺ depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.     

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 .     

Histamine promotes excitability in bovine adrenal chromaffin cells by inhibiting an M-current

The current study has investigated the electrophysiological responses evoked by histamine in bovine adrenal chromaffin cells using perforated-patch techniques. Histamine caused a transient hyperpolarization followed by a sustained depolarization of 7.2 ± 1.4 mV associated with an increase in spontaneous action potential frequency. The hyperpolarization was abolished after depleting intracellular Ca²⁺ stores with thapsigargin (100 n m ), and was reduced by 40 % with apamin (100 n m ). Membrane resistance increased by about 60 % during the histamine-induced depolarization suggesting inhibition of a K⁺ channel. An inward current relaxation, typical of an M-current, was observed in response to negative voltage steps from a holding potential of −30 mV. This current reversed at −81.6 ± 1.8 mV and was abolished by the M-channel inhibitor linopirdine (100 μ m ). During application of histamine, the amplitude of M-currents recorded at a time corresponding with the sustained depolarization was reduced by 40 %. No inward current rectification was observed in the range −150 to −70 mV, and glibenclamide (10 μ m ) had no effect on either resting membrane potential or the response to histamine. The results show that an M-current is present in bovine chromaffin cells and that this current is inhibited during sustained application of histamine, resulting in membrane depolarization and increased discharge of action potentials. These results demonstrate for the first time a possible mechanism coupling histamine receptors to activation of voltage-operated Ca²⁺ channels in these cells.     

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.     

Calcium buffering in rodent olfactory bulb granule cells and mitral cells

In the mammalian olfactory bulb, axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between mitral cells (MCs) via reciprocal dendrodendritic synapses. Calcium signals in the GC dendrites and reciprocal spines appear to decay unusually slowly, hence GC calcium handling might contribute to the known asynchronous release at this synapse. By recording fluorescence transients of different Ca²⁺-sensitive dyes at variable concentrations evoked by backpropagating action potentials (APs) and saturating AP trains we extrapolated Ca²⁺ dynamics to conditions of zero added buffer for juvenile rat GC apical dendrites and spines and MC lateral dendrites. Resting [Ca²⁺] was at ∼50 n m in both GC dendrites and spines. The average endogenous GC buffer capacities (κ_E) were within a range of 80–90 in the dendrites and 110–140 in the spines. The extrusion rate (γ) was estimated as 570 s⁻¹ for dendrites and 870 s⁻¹ for spines and the decay time constant as ∼200 ms for both. Single-current-evoked APs resulted in a [Ca²⁺] elevation of ∼250 n m . Calcium handling in juvenile and adult mouse GCs appeared mostly similar. In MC lateral dendrites, we found AP-mediated [Ca²⁺] elevations of ∼130 n m with a similar decay to that in GC dendrites, while κ_E and γ were roughly 4-fold higher. In conclusion, the slow GC Ca²⁺ dynamics are due mostly to sluggish Ca²⁺ extrusion. Under physiological conditions this slow removal may well contribute to delayed release and also feed into other Ca²⁺-dependent mechanisms that foster asynchronous output from the reciprocal spine.     

Segmentation induced by intraluminal fatty acid in isolated guinea-pig duodenum and jejunum

Small intestinal movements depend on the composition of the chyme with mixing predominating at high nutrient levels and propulsion being prevalent at low nutrient levels. The mechanisms coupling nutrients to motility are unknown. We used computer analysis of video recordings of isolated guinea-pig duodenum, jejunum and ileum to examine movements induced by a fatty acid, decanoic acid. Increasing intraluminal pressure past a threshold using control saline consistently evoked propulsive reflexes: lumen-occluding constrictions appeared at the oral end propagating at 20.4 ± 2.4 mm s⁻¹ (mean ± s.d. , jejunum) to the anal end before being repeated until the intraluminal pressure was returned to control. Subthreshold pressure increases sometimes evoked a transient series of constrictions appearing at the oral end and propagating anally at 18.4 ± 4.7 mm s⁻¹ (jejunum). At basal pressures, decanoic acid dose-dependently induced motor activity consisting of 40–60 s episodes of constrictions separated by 40–200 s periods of quiescence and lasting up to 2 h. Five contraction patterns were identified within episodes including localized stationary constrictions; constrictions that propagated slowly (5–8 mm s⁻¹) for short distances orally or anally; and constrictions that propagated orally or anally for the length of the preparation at 14–20 mm s⁻¹. Decanoic acid induced motor activity was reversibly abolished by tetrodotoxin (3 μ m ), hyoscine (1 μ m ) and hexamethonium (100 μ m ), but was insensitive to blockade of P2 purinoceptors by PPADS (60 μ m ). Thus, decanoic acid induces motor activity equivalent to segmentation in guinea-pig small intestine in vitro and this depends on intrinsic neural pathways.     

Effects of Perfusion Rate on Permeability of Frog and Rat Mesenteric Microvessels to Sodium Fluorescein

The permeability, P _S, to sodium fluorescein (Stokes-Einstein radius = 0.45 nm) has been measured in single mesenteric capillaries of pithed frogs and anaesthetised rats as perfusion velocity, U , was varied over a range from 400 up to 2000–10 000 μm s⁻¹. P _S increased linearly with U . In 20 frog capillaries, mean (± S.E.M.) P _S (in μm s⁻¹) = 9.35 (± 1.55) U × 10⁻⁵+ 0.244 (± 0.0291). Similarly, in nine rat venules, mean P _S= 1.62 (± 0.385) U × 10⁻⁴+ 0.375 (± 0.025). The flow-dependent component of permeability could be reversibly abolished in frog capillaries by superfusing with 100 μM noradrenaline and by superfusing rat venules with the nitric oxide synthase inhibitor, N ^G-nitro-L-arginine (20 μM). It was shown that changes in microvascular pressure accompanying changes in U during free perfusion could account for only 15 % of the changes in P _S, i.e. 85 % of the changes in P _S were changes in the permeability coefficient itself. A comparison between the changes in P _S with U and the previously described changes in microvascular permeability to K⁺ with U , suggest that if the flow-dependent component of permeability is modelled as a population of pores of constant size, these have radii of 0.8 nm. Such a pathway would limit flow-dependent permeability to small hydrophilic molecules and have minimal effect on net fluid exchange.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex

Loading slices of rat barrel cortex with 50 μ m BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca²⁺ stores with 20 μ m cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca²⁺ stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca²⁺ is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 μ m ryanodine or 10 m m caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca²⁺ release from ryanodine stores. Blocking IP₃ receptors with 14 μ m 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP₃ stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 μ m ( RS) -3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP₃ receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca²⁺-induced Ca²⁺ release (CICR) from ryanodine stores after the spontaneous opening of IP₃ stores.     

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.     

Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ

Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca²⁺]_i (Ca²⁺ transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca²⁺ transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca²⁺ transients synchronously with each other, but did not often show a close temporal relationship with Ca²⁺ transients in USMCs. Nicardipine (1 μ m ) suppressed Ca²⁺ transients in USMCs but not in ICC-LCs. Ca²⁺ transients in ICC-LCs were abolished by cyclopiazonic acid (10 μ m ), ryanodine (50 μ m ) and caffeine (10 m m ) or by removing extracellular Ca²⁺, and inhibited by 2-aminoethoxydiphenyl borate (50 μ m ) and 3-morpholino-sydnonimine (SIN-1; 10 μ m ), but facilitated by increasing extracellular Ca²⁺ or phenylephrine (1–10 μ m ). These results indicated that Ca²⁺ transients in urethral ICC-LCs in situ rely on both Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx through non-L-type Ca²⁺ channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.     

Effect of taurine on sarcoplasmic reticulum function and force in skinned fast-twitch skeletal muscle fibres of the rat

We examined the effect of taurine on depolarisation-induced force responses and sarcoplasmic reticulum (SR) function in mechanically skinned skeletal muscle fibres from the extensor digitorum longus (EDL) of the rat. Taurine (20 m m ) produced a small but significant ( P < 0.01) decrease in the sensitivity of the contractile apparatus to Ca²⁺ (increase in the [Ca²⁺] corresponding to 50 % of maximum force of about 7 %; n = 10) and in maximum force (92.0 ± 1.0 % of controls) in the skinned fibres. Taurine had no statistically significant effect on the slope of the force-pCa curve. Depolarisation-induced force responses in the skinned fibres were markedly increased in peak value by 20 m m taurine, to 120.8 ± 5.3 % of control measurements ( P = 0.0006, n = 27). Taurine (20 m m ) significantly increased the SR Ca²⁺ accumulation in the skinned fibres by 34.6 ± 9.3 % compared to control conditions (measured by comparing the integral of caffeine contractures in fibres previously loaded with Ca²⁺ in the absence or presence of taurine; P = 0.0014, n = 10). Taurine (20 m m ) also increased both the peak and rate of rise of caffeine-induced force responses in the fibres by 29.2 ± 9.7 % ( P = 0.0298, n = 6) and 27.6 ± 8.9 % ( P = 0.037), respectively, compared with controls. This study shows that taurine is a modulator of contractile function in mammalian skeletal muscle. Taurine may increase the size of depolarisation-induced force responses by augmenting SR Ca²⁺ accumulation and release.     

Role of extracellular Ca2+ in gating of CaV1.2 channels

We examined changes in ionic and gating currents in Ca_V1.2 channels when extracellular Ca²⁺ was reduced from 10 m m to 0.1 μ m . Saturating gating currents decreased by two-thirds ( K _D≈ 40 μ m ) and ionic currents increased 5-fold ( K _D≈ 0.5 μ m ) due to increasing Na⁺ conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca²⁺], which appeared to reflect the rapid activation of channels that were not blocked by Ca²⁺ and a slower reversal of Ca²⁺ blockade of the remaining channels. Removal of Ca²⁺ following inactivation of Ca²⁺ currents showed that Na⁺ currents were not affected by Ca²⁺-dependent inactivation. Ca²⁺-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca²⁺ conductance and Ca²⁺-dependent inactivation depend on extracellular Ca²⁺ and are linked to changes in selectivity.     

Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries

The present study was aimed at investigating whether, besides its pivotal role in Ca²⁺-independent contraction of smooth muscle, Rho-kinase is involved in the mechanisms underlying the Ca²⁺ signal activated by noradrenaline in arteries. In rat aorta and mesenteric artery, the Rho-kinase inhibitor Y-27632 (10 μM) completely relaxed the contraction evoked by noradrenaline (1 μM) and simultaneously inhibited the Ca²⁺ signal by 54 ± 1 % (mesenteric artery) and 71 ± 15 % (aorta), and the cell membrane depolarisation by 56 ± 11 % (mesenteric artery). A similar effect was observed in arteries contracted by AlF₄⁻, while in KCl-contracted arteries, Y-27632 decreased tension without changing cytosolic Ca²⁺. The same effects were observed with another inhibitor of Rho-kinase (HA1077) but not with an inhibitor of protein kinase C (Ro-31-8220). Effects of Y-27632 were not prevented by incubating the artery in 25 mM KCl, with K⁺ channel blockers or with the Ca²⁺ channel blocker nimodipine. Y-27632 did not affect either the increase in the production of inositol phosphates activated by noradrenaline, or the release of Ca²⁺ from non-mitochondrial stores evoked by Ins P ₃ in permeabilised aortic cells, or the Ca²⁺ signals evoked by thapsigargin or caffeine. The capacitative Ca²⁺ entry activated by thapsigargin was not impaired by Y-27632, but the entry of Ba²⁺ activated by noradrenaline in the presence of nimodipine was blocked by 10 μM Y-27632. These results indicate that Rho-kinase is involved in noradrenaline activation of a Ca²⁺ entry distinct from voltage- or store-operated channels in rat arteries.     

Action potential initiation and propagation in hippocampal mossy fibre axons

Dentate gyrus granule cells transmit action potentials (APs) along their unmyelinated mossy fibre axons to the CA3 region. Although the initiation and propagation of APs are fundamental steps during neural computation, little is known about the site of AP initiation and the speed of propagation in mossy fibre axons. To address these questions, we performed simultaneous somatic and axonal whole-cell recordings from granule cells in acute hippocampal slices of adult mice at ∼23°C. Injection of short current pulses or synaptic stimulation evoked axonal and somatic APs with similar amplitudes. By contrast, the time course was significantly different, as axonal APs had a higher maximal rate of rise (464 ± 30 V s⁻¹ in the axon versus 297 ± 12 V s⁻¹ in the soma, mean ± s.e.m. ). Furthermore, analysis of latencies between the axonal and somatic signals showed that APs were initiated in the proximal axon at ∼20–30 μm distance from the soma, and propagated orthodromically with a velocity of 0.24 m s⁻¹. Qualitatively similar results were obtained at a recording temperature of ∼34°C. Modelling of AP propagation in detailed cable models of granule cells suggested that a ∼4 times higher Na⁺ channel density (∼1000 pS μm⁻²) in the axon might account for both the higher rate of rise of axonal APs and the robust AP initiation in the proximal mossy fibre axon. This may be of critical importance to separate dendritic integration of thousands of synaptic inputs from the generation and transmission of a common AP output.