Sphingosylphosphorylcholine-induced vasoconstriction of pulmonary artery: activation of non-store-operated Ca2+ entry

OBJECTIVE: Sphingosylphosphorylcholine (SPC) is an important lipid mediator that has been implicated in vascular disease. As it has not been studied in the pulmonary circulation, we examined its mechanisms of action in rat small intrapulmonary arteries (IPA). METHODS: IPA were mounted on a myograph for recording tension and intracellular Ca2+ concentration ([Ca2+]i). Ca2+ sensitisation was examined in alpha-toxin permeabilized IPA, and by Western blot analysis of MYPT1 phosphorylation. RESULTS: SPC induced a slow but powerful vasoconstriction in IPA associated with an elevation in [Ca2+]i, with an EC50 for vasoconstriction of 12+/-2 microM. Removal of extracellular Ca2+ increased the EC50 to 76+/-33 microM (p<0.01) and abolished the rise in [Ca2+]i. Endothelial denudation or inhibition of NO synthase with L-NAME enhanced vasoconstriction. Treatment with pertussis toxin or the PLC inhibitor U731223 had no effect on SPC-induced vasoconstriction. The Rho kinase inhibitor Y27632 reduced SPC-induced vasoconstriction by approximately 70% and abolished both SPC-induced Ca2+ sensitisation in permeabilized IPA and the associated increase in MYPT1 phosphorylation; Ca2+ sensitisation was substantially inhibited by GDPbetaS. La3+ and 2-APB, at concentrations previously shown to block capacitative Ca2+ entry in IPA, suppressed SPC-induced vasoconstriction to the same extent as removal of extracellular Ca2+; residual tension was abolished by Y27632. Diltiazem was relatively ineffective. 2-APB also abolished the SPC-induced rise in [Ca2+]i. However, treatment with thapsigargin to empty intracellular stores had no effect on the elevation of [Ca2+]i induced by SPC. CONCLUSION: We present evidence that SPC is a powerful vasoconstrictor of IPA and the novel finding that SPC-induced vasoconstriction in IPA is dependent on activation of a Ca2+ entry pathway with a similar sensitivity to La3+ and 2-APB as capacitative Ca2+ entry, although its activation is not dependent on emptying of PLC/IP3 or thapsigargin-sensitive intracellular stores.     

ER-resident STIM1/2 couples Ca2+ entry by NMDA receptors to pannexin-1 activation

Pannexin-1 (Panx1) is a large-pore ion and solute permeable channel highly expressed in the nervous system, where it subserves diverse processes, including neurite outgrowth, dendritic spine formation, and N-methyl D-aspartate (NMDA) receptor (NMDAR)-dependent plasticity. Moreover, Panx1 dysregulation contributes to neurological disorders, including neuropathic pain, epilepsy, and excitotoxicity. Despite progress in understanding physiological and pathological functions of Panx1, the mechanisms that regulate its activity, including its ion and solute permeability, remain poorly understood. In this study, we identify endoplasmic reticulum (ER)-resident stromal interaction molecules (STIM1/2), which are Ca²⁺ sensors that communicate events within the ER to plasma membrane channels, as binding and signaling partners of Panx1. We demonstrate that Panx1 is activated to its large-pore configuration in response to stimuli that recruit STIM1/2 and map the interaction interface to a hydrophobic region within the N terminus of Panx1. We further characterize a Panx1 N terminus–recognizing antibody as a function-blocking tool able to prevent large-pore Panx1 activation by STIM1/2. Using either the function-blocking antibody or re-expression of Panx1 deletion mutants in Panx1 knockout (KO) neurons, we show that STIM recruitment couples Ca²⁺ entry via NMDARs to Panx1 activation, thereby identifying a model of NMDAR-STIM-Panx1 signaling in neurons. Our study highlights a previously unrecognized and important role of the Panx1 N terminus in regulating channel activation and membrane localization. Considering past work demonstrating an intimate functional relation between NMDARs and Panx1, our study opens avenues for understanding activation modality and context-specific functions of Panx1, including functions linked to diverse STIM-regulated cellular responses.

Glutamatergic signaling plays a critical role in diverse processes linked to learning and memory formation. Ca²⁺ signals generated by the N-methyl D-aspartate (NMDA) subtype of glutamate receptors (NMDARs) are indispensable for several forms of synaptic plasticity, including long-term potentiation (LTP), a prototypic form of plasticity linked to memory formation (1–3). NMDAR-initiated Ca²⁺ signals (e.g., time course, amplitude, and spatial spread) are shaped by secondary events, including those engendered via the endoplasmic reticulum (ER) (4, 5). Ca²⁺ entry via NMDARs can promote Ca²⁺-induced Ca²⁺ release from ER stores by stimulating ryanodine (RyRs) (6–8) and/or IP3 receptors (IP3Rs) (9). In turn, NMDAR-initiated Ca²⁺ store depletion recruits ER-resident and Ca²⁺-sensing STIM proteins (10) to negatively regulate L-type voltage-gated Ca²⁺ channels (VGCCs) (13). This establishes the notion that Ca²⁺ entry via NMDARs can stimulate ER- and STIM-dependent cascades that regulate secondary routes of Ca²⁺ entry, thereby sculpting intracellular Ca²⁺ dynamics and in turn the cellular functions influenced by them. As part of a broader search to identify candidate Ca²⁺ channels able to respond to ER signaling dynamics, we found that Pannexin-1 (Panx1) can be activated through ER-based signaling following sarcoendoplasmic reticulum calcium adenosine triphosphatase (ATPase) (SERCA) pump inhibition by thapsigargin. This led us to consider the role of STIM1/2 as a candidate Panx1 activation mechanism.Panx1 is a large-pore nonselective ion and solute permeable channel with prominent central nervous system (CNS) expression (14, 15). Panx1 activation has been linked to pathophysiological disorders, such as excitotoxicity, stroke, migraine, chronic pain, and epilepsy (16–18). However, Panx1 also mediates physiological processes in the CNS, including contributions to neural development (19, 20), spine formation (21, 22), and NMDAR-dependent synaptic plasticity (23, 24). In this context, there remains an important gap in understanding the mechanisms by which Panx1 can mediate such disparate physiological and pathological functions. Intriguingly, evidence suggests that Panx1 ion versus solute permeability may be mediated by distinct channel pore configurations (i.e., small anion vs. large solute permeable) recruited via distinct activation modalities (25). Thus, identifying novel activation mechanisms is fundamental to understanding context- and modality-specific channel function.Here, we uncover a mechanism by which Panx1 is activated in response to ER-initiated signaling, which we demonstrate is dependent on Panx1 interaction with ER-resident STIM1/2. STIM1/2 recruitment and activation stimulates large-pore Panx1 opening, evident on the basis of increased permeability to Ca²⁺ and the large inorganic ion N-methyl-D-glucamine (NMDG). We map the STIM1/2 binding interface to a hydrophobic region in the N terminus of Panx1, a region not previously linked to channel gating. Our detailed structure-function analysis reveals that the Panx1 N-terminal region is necessary for its STIM1/2 responsiveness, but not for its responsiveness to hypotonic stress, demonstrating that this region mediates modality-specific regulation of Panx1 function. Using reverse genetics, ectopic rescue with Panx1 N-terminal deletion mutants, as well as a function inhibiting antibody targeting the critical N-terminal region of Panx1 identified by us, we demonstrate that NMDARs activate Panx1 in hippocampal neurons in a manner contingent upon ER-initiated signaling and reliant upon STIM proteins. Collectively, our data reveal the molecular mechanism by which STIM1/2 activates Panx1 and establishes a previously unrecognized essential role of its N-terminal region in regulating the transition of Panx1 to its large-pore solute permeable state. Our work will benefit studies aimed at understanding diverse functions of Panx1, including those linked to NMDAR-dependent signaling, stimulated in a modality- and context-specific manner by STIM proteins.     

Characterization of thyroid hormone effects on Na channel synthesis in cultured skeletal myotubes: role of Ca2+

Thyroid hormones (TH) cause an increase in spontaneous electrical activity of cultured rat skeletal myotubes. This activity is associated with tetrodotoxin (TTX)-sensitive Na channels. In addition, the initial effect of TH on Na-K pump synthesis has been shown to be TTX dependent. Accordingly, we have studied effects of TH on expression of TTX-sensitive Na channels in cultured skeletal muscle. Expression of Na channels was determined by measurements of the binding of [3H]saxitoxin (STX). The frequency and rate of rise of spontaneously occurring action potentials, which are related to the density of TTX-sensitive Na channels, were also determined. TH caused dose-dependent increases in Na channels as well as in action potential frequency and rate of rise. The increases were detectable as early as 12 h after treatment with TH was begun, and levels reached a maximum plateau after 36-48 h. The effects of TH were blocked by inhibitors of protein synthesis. Scatchard analysis showed the channels in TH-treated myotubes to have lower affinity for STX than those in control cells. The effect of TH to up-regulate Na channels was reduced by growth of the cells in elevated external calcium. In contrast, treatment with TTX or verapamil, which lower cytosolic Ca2+, resulted in a marked increase in the effect of TH over that in control myotubes. Thus, TH appears to regulate Na channels in cultured myotubes by two opposing mechanisms; 1) direct stimulation of Na channel synthesis, and 2) indirect decrease in synthesis mediated by an increase in cytosolic Ca2+. The results indicate that TH may play an important role in developmental expression of Na channels in excitable tissue.     

Agonist-induced Ca2+ entry determined by inositol 1,4,5-trisphosphate recognition

It has been considered that Ca2+ release is the causal trigger for Ca2+ entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (IP3R), the lack of Ca2+ entry in response to receptor activation is attributed to the absence of Ca2+ release. We reveal in this article that IP3R recognition of IP3 determines agonist-induced Ca2+ entry (ACE), independent of its Ca2+ release activity. In DT40 IP3R(-/-) cells, endogenous ACE can be rescued with type 1 IP3R mutants (both a DeltaC-terminal truncation mutant and a D2550A pore mutant), which are defective in Ca2+ release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an IP3R-dependent manner without Ca2+ store release. Conversely, ACE cannot be rescued with mutant IP3Rs lacking IP3 binding (both the Delta90-110 and R265Q IP3-binding site mutants). We conclude that an IP3-dependent conformational change in the IP3R, not endoplasmic reticulum Ca2+ pool release, triggers ACE.     

Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor

Examination of store-operated Ca(2+) entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP(3)) receptor acts as a sarcoplasmic reticulum [Ca(2+)] sensor and mediates SOC by physical coupling without playing a key role in Ca(2+) release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IP(3) receptors in skeletal muscle, the role of which with respect to Ca(2+) homeostasis was ill defined until now.     

Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca(2+) signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca(2+) channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca(2+) permeation properties suggest an important function in Ca(2+) uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca(2+)-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.     

Ca2+ entry in squid axons during voltage-clamp pulses is mainly Na+/Ca2+ exchange.

Intact squid axons were injected with aequorin and bathed in 3 mM Ca seawater (a concentration close to that of squid blood). Sodium and potassium currents were pharmacologically blocked and repetitive voltage-clamp pulses of a duration of 1.5 ms were applied (to simulate the duration of an action potential) at amplitudes of +30 to +90 mV and at frequencies of 100/s. In a very fresh axon (low internal Na concentration) no detectable change in aequorin glow resulted from this treatment, whether the axons were in Na-containing or in Na-free seawater. In axons subjected to modest Na loading, repetitive voltage-clamp pulsing did not result in an increased aequorin glow when the pulses were delivered in Na seawater, whereas in Na-free seawater there was an easily measurable increase in aequorin light emission during repetitive pulsing. The increase in aequorin photons emitted per voltage-clamp pulse was e-fold for 22 mV of depolarization, and the process showed no signs of saturating at pulse amplitudes of +180 mV (i.e., at a membrane potential close to ECa). The aequorin light emission per voltage-clamp pulse increased linearly with pulse duration (at constant amplitude).     

Visualization of Ca2+ entry through single stretch-activated cation channels

Stretch-activated channels (SACs) have been found in smooth muscle and are thought to be involved in myogenic responses. Although SACs have been shown to be Ca(2+) permeable when Ca(2+) is the only charge carrier, it has not been clearly demonstrated that significant Ca(2+) passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca(2+) fluorescence transient (SCCaFT) arising from Ca(2+) entry through a single SAC opening, we provide direct evidence that significant Ca(2+) can indeed pass through SACs and increase the local [Ca(2+)]. Results were obtained under conditions where the only source of Ca(2+) was the physiological salt solution in the patch pipette containing 2 mM Ca(2+). Single smooth muscle cells were loaded with fluo-3 acetoxymethyl ester, and the fluorescence was recorded by using a wide-field digital imaging microscope while SAC currents were simultaneously recorded from cell-attached patches. Fluorescence increases at the cell-attached patch were clearly visualized before the simultaneous global Ca(2+) increase that occurred because of Ca(2+) influx through voltage-gated Ca(2+) channels when the membrane was depolarized by inward SAC current. From measurements of total fluorescence ("signal mass") we determined that about 18% of the SAC current is carried by Ca(2+) at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca(2+) current at the resting potential. Such Ca(2+) currents passing through SACs are sufficient to activate large-conductance Ca(2+)-activated K(+) channels and, as shown previously, to trigger Ca(2+) release from intracellular stores.     

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) inducing Ca²⁺ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca²⁺ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca²⁺ release, leading to a significant increase in the activity of Ca²⁺ sparks and contraction in airway SMCs. The increased Ca²⁺ sparks are independent of VDCCs and the associated extracellular Ca²⁺ influx. This format of local Ca²⁺ release results from a direct activation of G protein-coupled, M₃ muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP₃), inducing initial Ca²⁺ release through IP₃ receptors and then further Ca²⁺ release via RyR2 due to a local Ca²⁺-induced Ca²⁺ release process. These findings demonstrate an important mechanism for Ca²⁺ signaling and attendant physiological function in SMCs.     

Ca(2+) sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle

Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular-sarcoplasmic reticulum junctions, and the unit signal is the Ca(2+) spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RyR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca(2+) sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RyR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca(2+)-induced Ca(2+) release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RyR3 isoform. Because RyR3 does not contact voltage sensors, their opening was probably activated by Ca(2+), secondarily to Ca(2+) release through junctional RyR1. Physiologically voltage-controlled Ca(2+) sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca(2+), and a slave parajunctional RyR3 cohort.     

Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca(2+) is increased in dystrophic muscle, leading to Ca(2+) accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca(2+) in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca(2+)-activated K(+) channels as subsarcolemmal Ca(2+) probe, we tested the possibility of a Ca(2+) accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca(2+)-activated K(+) channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca(2+)]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca(2+)-activated K(+) channel activation could correspond to a approximately 3-fold increase in the subsarcolemmal Ca(2+) concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca(2+) overload at the subsarcolemmal level.     

Phosphorylation and dephosphorylation of dihydropyridine-sensitive voltage-dependent Ca2+ channel in skeletal muscle membranes by cAMP- and Ca2+-dependent processes.

The phosphorylation and dephosphorylation of the dihydropyridine-sensitive Ca2+ channel was studied in transverse-tubule membranes isolated from rabbit skeletal muscle. Exposure of these membranes to either the cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase resulted in a rapid phosphorylation of a protein with properties similar to the major component of the skeletal muscle Ca2+ channel. The molecular mass of the phosphoprotein was 140 or 160 kDa, depending on the electrophoretic conditions. The stoichiometry of the phosphorylation was calculated to be 0.4-1.0 mol of phosphate per mol of protein. Neither the rate nor the extent of phosphorylation was affected by dihydropyridines. Limited proteolytic digestion of the protein that had been phosphorylated by either or both protein kinases yielded a single phosphopeptide of approximately equal to 5.4 kDa. The Ca2+-dependent phosphatase calcineurin dephosphorylated the membrane-bound Ca2+ channel that had been previously phosphorylated by either protein kinase. The results suggest that the major component of the dihydropyridine-sensitive Ca2+ channel from skeletal muscle can be effectively phosphorylated and dephosphorylated in its native state by cAMP- and Ca2+-dependent processes.     

Rhythmic ryanodine receptor Ca2+ releases during diastolic depolarization of sinoatrial pacemaker cells do not require membrane depolarization

Localized, subsarcolemmal Ca2+ release (LCR) via ryanodine receptors (RyRs) during diastolic depolarization of sinoatrial nodal cells augments the terminal depolarization rate. We determined whether LCRs in rabbit sinoatrial nodal cells require the concurrent membrane depolarization, or are intrinsically rhythmic, and whether rhythmicity is linked to the spontaneous cycle length. Confocal linescan images revealed persistent LCRs both in saponin-permeabilized cells and in spontaneously beating cells acutely voltage-clamped at the maximum diastolic potential. During the initial stage of voltage clamp, the LCR spatiotemporal characteristics did not differ from those in spontaneously beating cells, or in permeabilized cells bathed in 150 nmol/L Ca2+. The period of persistent rhythmic LCRs during voltage clamp was slightly less than the spontaneous cycle length before voltage clamp. In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length; and regardless of the cycle length, LCRs occurred predominantly at a constant time, ie, 80% to 90% of the cycle length. Numerical model simulations incorporating LCRs reproduce the experimental results. We conclude that diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization, and that LCR periodicity is closely linked to the spontaneous cycle length. Thus, the biological clock of sinoatrial nodal pacemaker cells, like that of many other rhythmic functions occurring throughout nature, involves an intracellular Ca2+ rhythm.     

Repetitive increases in cytosolic Ca2+ of guard cells by abscisic acid activation of nonselective Ca2+ permeable channels.

Many signal-transduction processes in higher plant cells have been suggested to be triggered by signal-induced opening of Ca2+ channels in the plasma membrane. However, direct evidence for activation of plasma-membrane Ca2+ channels by physiological signals in higher plants has not yet been obtained. In this context, several lines of evidence suggest that Ca2+ flux into the cytosol of guard cells is a major factor in the induction of stomatal closing by abscisic acid (ABA). ABA closes stomatal pores, thereby reducing transpirational loss of water by plants under drought conditions. To directly investigate initial events in ABA-induced signal transduction in guard cells, we devised an experimental approach that allows simultaneous photometric measurements of cytosolic Ca2+ and patch-clamp recordings of ion currents across the plasma membrane of single Vicia faba guard cells. Using this approach, we found that the resting cytosolic Ca2+ concentration was 0.19 +/- 0.09 microM (n = 19). In responsive guard cells, external exposure to ABA produced transient repetitive increases in the cytosolic free Ca2+ concentration. These Ca2+ transients were accompanied by concomitantly occurring increases in an inward-directed ion current. Depolarization of the membrane terminated both repetitive elevations in cytosolic Ca2+ and inward-directed ion currents, suggesting that ABA-mediated Ca2+ transients were produced by passive influx of Ca2+ from the extracellular space through Ca2(+)-permeable channels. Detailed voltage-clamp measurements revealed that ABA-activated ion currents could be reversed by depolarizations more positive than -10 mV. Interestingly, reversal potentials of ABA-induced currents show that these currents are not highly Ca2(+)-selective, thereby permitting permeation of both Ca2+ and K+. These results provide direct evidence for ABA activation of Ca2(+)-permeable ion channels in the plasma membrane of guard cells. ABA-activated ion channels allow repetitive elevations in the cytosolic Ca2+ concentration, which, in turn, can modulate cellular responses promoting stomatal closure.     

Promoting effects of IL-13 on Ca2+ release and store-operated Ca2+ entry in airway smooth muscle cells

Th2 cytokine interleukin (IL)-13 plays a central role in the pathogenesis of allergic asthma. IL-13 exhibits a direct effect on airway smooth muscle cells (ASMCs) to cause airway hyperresponsiveness. IL-13 has been demonstrated to regulate Ca²⁺ signaling in ASMCs, but the underlying mechanisms are not fully understood. Store-operated Ca²⁺ entry (SOCE) plays an important role in regulating Ca²⁺ signaling and cellular responses of ASMCs, whether IL-13 affects SOCE in ASMCs has not been reported. In this study, by using confocal Ca²⁺ fluorescence imaging, we found that IL-13 (10 ng/ml) treatment increased basal intracellular Ca²⁺ ([Ca²⁺]_i) level, Ca²⁺ release and SOCE induced by SERCA inhibitor thapsigargin in rat bronchial smooth muscle cells. The glucocorticoid dexamethasone and the short-acting β2 adrenergic agonist (β2 agonist) salbutamol suppressed IL-13-augumented basal [Ca²⁺]_i, Ca²⁺ release and SOCE, whereas the long-acting β2 agonist salmeterol had no effect on altered Ca²⁺ signaling in IL-13-treated ASMCs. Membrane-permeable cAMP analog dibutyryl-cAMP (db-cAMP) similarly decreased Ca²⁺ release and SOCE induced by thapsigargin in IL-13-treated ASMCs, confirmed a role of cAMP/PKA signaling pathway in the regulation of SOCE. IL-13 promoted the proliferation of ASMCs stimulated by serum; this effect was inhibited by nonspecific Ca²⁺ channel blockers SKF-96365 and NiCl₂, by salmeterol, but not by salbutamol and dexamethasone. IL-13 treatment did not change the expression of SOC channel-associated molecules STIM1, Orai1 and TRPC1 at mRNA level. Our findings identified a promoting effect of IL-13 on Ca²⁺ release and SOCE in ASMCs, which partially contributes to its effect on the proliferation of ASMCs; the differences of glucocorticoids and β2 agonists in inhibiting Ca²⁺ signal and proliferation potentiated by IL-13 suggest that these therapies of asthma may have distinct effect on the relief of airway contraction and remodeling in bronchial asthma.     

Cytosolic Ca2+-dependent neutral proteinases from rabbit liver: activation of the proenzymes by Ca2+ and substrate.

Two neutral Ca2+-dependent proteinases, differing in molecular size, have been isolated from rabbit liver. Both are recovered as inactive proenzymes that can be converted to the active forms by high (0.1-1.0 mM) concentrations of Ca2+ in the absence of substrate or, in the presence of a protein substrate, by low (1-5 microM) concentrations of Ca2+. The activated proteinases required only 1-5 microM Ca2+ for maximal activity. Substrates hydrolyzed were denatured globin, globin, casein, and to a lesser extent, several extracellular proteins; no digestion was observed with several intracellular cytosolic enzymes tested. Only those proteins that served as substrates were capable of promoting conversion of the proenzymes to the active low-Ca2+-requiring proteinases.     

Ca2+ influx initiates death of hepatocytes injured by activation of complement

To clarify the role of cytosolic Ca²⁺ in hepatocellular death, we exposed cultured hepatocytes to human serum and a monoclonal antibody directed against rat liver plasma membranes to produce complement-mediated cell injury. The change in cytosolic Ca²⁺ concentration was measured by fura2 and fluo3 fluorescence. With the addition of monoclonal antibody, an increase in cytosolic Ca²⁺ was observed, followed by cell death. Both the increase in intracellular Ca²⁺ and cell death were prevented by intracellular Ca²⁺ chelation or removal of extracellular Ca²⁺. We conclude that an increase in cytosolic Ca²⁺ plays a major role in hepatocellular injury induced by exposure of the cell membrane to monoclonal antibody.     

Elastase-induced suppression of endothelin-mediated Ca2+ entry mechanisms of vascular contraction

Abdominal aortic aneurysm (AAA) is associated with increased endothelin (ET-1), both systemically and locally in the aorta. Also, elastase activity is increased in human AAA, and elastase perfusion of the aorta induces aneurysm formation in animal models of AAA. However, whether elastase directly affects the ET-1-induced mechanisms of aortic smooth muscle contraction is unclear. Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats and treated with elastase (5 U/mL). To avoid degradation of the extracellular matrix proteins by elastase, experiments were performed in the presence of elastin (10 mg/mL). In normal Krebs solution (2.5 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused contraction of aortic strips that was inhibited by elastase (5 U/mL). The elastase-induced inhibition of ET-1 contraction was slow in onset (4.6+/-0.4 minutes), time-dependent, complete in 34+/-3 minutes, and reversible. In Ca2+-free Krebs solution, caffeine (25 mmol/L) caused a small contraction that was not inhibited by elastase, suggesting that elastase does not inhibit Ca2+ release from the intracellular stores. Membrane depolarization by 96 mmol/L KCl, which stimulates Ca2+ entry from the extracellular space, caused a contraction that was inhibited by elastase in a concentration-dependent, time-dependent, and reversible fashion. The reversible inhibitory effects of elastase, particularly in the presence of elastin, suggest that they are not due to dissolution of the extracellular matrix or smooth muscle contractile proteins. Elastase also inhibited ET-1 and KCl-induced 45Ca2+ influx. Thus, elastase directly inhibits ET-1-induced Ca2+ entry mechanisms of vascular smooth muscle contraction, which may explain the role of elastase and ET-1 during the development of AAA.