Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.     

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H₁Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca ²⁺ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca ²⁺ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca ²⁺- and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca ²⁺ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca ²⁺ response was suppressed by the inhibition of H ₁Rs with pyrilamine, whereas H ₁R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca ²⁺ oscillations were initiated by endogenous Ca ²⁺ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca ²⁺ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca ²⁺ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca ²⁺ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca ²⁺ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Muscarinic Receptor-Mediated Inositol 1,4,5-Trisphosphate Formation in SH-SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP₃) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ～60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca²⁺ with EGTA or inhibition of Ca²⁺ channels with Ni²⁺ showed that the plateau phase was dependent upon Ca²⁺ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca²⁺ from intracellular stores revealed that Ca²⁺ from this source was capable of supporting the peak phase of the InsP₃ response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca²⁺ concentration (20–100 nM) and suggests that under normal conditions, InsP₃ formation is enhanced by increases in cytosolic free Ca²⁺ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li⁺ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP₃ accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca²⁺ concentration amplify InsP₃ formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP₃ accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca²⁺ or protein kinase C activity.     

InsP3R-associated cGMP Kinase Substrate Determines Inositol 1,4,5-Trisphosphate Receptor Susceptibility to Phosphoregulation by Cyclic Nucleotide-dependent Kinases

Ca²⁺ release through inositol 1,4,5-trisphosphate receptors (InsP₃R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca²⁺ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca²⁺ release via InsP₃R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP₃R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP₃R-1 subtype resulted in enhanced Ca²⁺ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP₃R subtype, and phosphorylation of IRAG by PKG attenuated Ca²⁺ release through all InsP₃R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca²⁺ release through InsP₃R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP₃R-2. Phosphorylation of IRAG resulted in reduced Ca²⁺ release through all InsP₃R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways.     

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP₃), which operates as an InsP₃-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP₃-sensitive intracellular Ca²⁺ stores, and a sustained phase of Ca²⁺ influx. These events are accompanied by a redistribution of type II InsP₃ receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP₃ receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP₃ receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP₃ receptor aggregation may be a characteristic cellular response to Ca²⁺-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36^M3R cells. Stimulation of these three cell types leads to a reduction in InsP₃ receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP₃ receptors may contribute to the previously observed changes in affinity for InsP₃ in the presence of elevated Ca²⁺ and/or may establish discrete regions within refilled stores with varying capacity to release Ca²⁺ when a subsequent stimulus results in production of InsP₃.     

Differential functional properties of Ca stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i) are controlled by inositol 1,4,5-trisphosphate (InsP₃) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca²⁺ oscillations are regulated differentially by InsP₃ and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca²⁺ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP₃ production or activation of InsP₃ receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP₃-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca²⁺ free solutions did not prevent the ATP-induced Ca²⁺ release from InsP₃-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP₃- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca²⁺ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.     

Ca<Superscript>2+</Superscript> Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells

We have investigated the dynamics of the free [Ca²⁺] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca²⁺-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca²⁺] ([Ca²⁺]_SG] was around 20–40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca²⁺ pumps with thapsigargin largely blocked Ca²⁺ uptake by the granules in Ca²⁺-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca²⁺ pump inhibitor benzohydroquinone induced a rapid release of Ca²⁺ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca²⁺ pumps is essential to maintain the steady-state [Ca²⁺]_SG. Both inositol 1,4,5-trisphosphate (InsP₃) and caffeine produced a rapid Ca²⁺ release from the granules, suggesting the presence of InsP₃ and ryanodine receptors in the granules. The response to high-K⁺ depolarization was different in both cell types, a decrease in [Ca²⁺]_SG in PC12 cells and an increase in [Ca²⁺]_SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca²⁺]_SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca²⁺ through SERCA-type Ca²⁺ pumps and can release it through InsP₃ and ryanodine receptors, supporting the hypothesis that secretory granule Ca²⁺ may be released during cell stimulation and contribute to secretion.     

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The presence and physiological role of Ca²⁺-induced Ca²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca²⁺] ([Ca²⁺]_c). Using targeted aequorin, we have directly monitored [Ca²⁺] changes inside the ER ([Ca²⁺]_ER) in bovine adrenal chromaffin cells. Ca²⁺ entry induced by cell depolarization triggered a transient Ca²⁺ release from the ER that was highly dependent on [Ca²⁺]_ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca²⁺ release was quantal in nature due to modulation by [Ca²⁺]_ER. Whereas caffeine released essentially all the Ca²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP₃)- producing agonists released only 60–80%. Both InsP₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca²⁺]_c measurements showed that the wave of [Ca²⁺]_c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca²⁺ pool that can release Ca²⁺ both via InsP₃ receptors or CICR.     

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP₃)-mediated increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Local Ca²⁺ release from an InsP₃ receptor (InsP₃R) cluster present on the sarcoplasmic reticulum is termed a Ca²⁺ puff. Ca²⁺ released via InsP₃R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca²⁺ rise. In smooth muscle, mitochondrial Ca²⁺ uptake maintains global InsP₃-mediated Ca²⁺ release by preventing a negative feedback effect of high [Ca²⁺] on InsP₃R. Mitochondria may regulate InsP₃-mediated Ca²⁺ signals by operating between or within InsP₃R clusters. In the former mitochondria could regulate only global Ca²⁺ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP₃-mediated Ca²⁺ release by operating within (local) or between (global) InsP₃R clusters has been addressed. Ca²⁺ puffs evoked by localized photolysis of InsP₃ in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F₀, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨ_M to prevent mitochondrial Ca²⁺ uptake and attenuated Ca²⁺ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca²⁺ puffs by ∼62%. The “fast” Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP₃-mediated Ca²⁺ release suggesting that mitochondrial influence is via their Ca²⁺ uptake facility. These results indicate Ca²⁺ uptake occurs quickly enough to influence InsP₃R communication at the intra-cluster level and that mitochondria regulate both local and global InsP₃-mediated Ca²⁺ signals.     

Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

Spatially Defined InsP3-Mediated Signaling in Embryonic Stem Cell-Derived Cardiomyocytes

The functional role of inositol 1,4,5-trisphosphate (InsP₃) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP₃ receptors can translate Ca²⁺ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP₃-mediated Ca²⁺ release. [InsP₃]_i was monitored with the FRET-based InsP₃-biosensor FIRE-1 (Fluorescent InsP₃ Responsive Element) and heterogeneity in sub-cellular [InsP₃]_i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP₃ 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca²⁺ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca²⁺]_i and spontaneous activity was modulated by [InsP₃]_i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP₃ by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP₃ signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca²⁺ released from InsP₃Rs is more effective than Ca²⁺ released from RyRs to enhance I_NCX. The results support the hypothesis that in ESdCs InsP₃Rs form a functional signaling domain with NCX that translates Ca²⁺ release efficiently into a depolarization of the membrane potential.     

Imaging of intracellular Ca waves induced by muscarinic receptor stimulation in rat parotid acinar cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca²⁺, the increase in [Ca²⁺]_i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca²⁺ wave toward the basolateral region. The [Ca²⁺]_i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca²⁺ was eliminated from the extracellular medium, the Ca²⁺ wave was a result of Ca²⁺ release from intracellular stores. The magnitude and velocity of the Ca²⁺ wave decreased with decreasing concentration of CCh, and the increase in [Ca²⁺]_i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca²⁺ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca²⁺ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca²⁺ pump, caused a slow and homogeneous increase in [Ca²⁺]_i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca²⁺]_i suggesting that the inositol-1,4,5-trisphosphate (InsP₃) and ThG-sensitive Ca²⁺ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP₃ is essential to trigger the Ca²⁺ wave and that the development of the Ca²⁺ wave may be attributed to regional differences in InsP₃ sensitivity of Ca²⁺ stores. The agonist-induced Ca²⁺ wave is probably a general phenomenon in exocrine acinar cells.     

Attenuated endothelium-mediated relaxation by acteoside in rat aorta: Role of endothelial [Ca2+]i and nitric oxide/cyclic GMP pathway

Acteoside and other phenylethanoid glycoside are contained in many plants that are widely used in traditional Chinese herbal medicine. Acteoside possesses multiple biological actions. Its effect on the vascular system is, however, incompletely understood. This study was aimed to investigate the role of endothelial [Ca²⁺]_i, nitric oxide (NO), and cyclic GMP in acteoside-induced inhibition of endothelial NO-mediated relaxation in rat aorta. Acteoside reduced endothelial NO-dependent relaxation induced by acetylcholine (Ach) or A23187. Acteoside inhibited Ach-stimulated increase in tissue content of cyclic GMP in endothelium-intact rings. L-NNA abolished the stimulatory effect of Ach. Treatment with acteoside significantly suppressed bradykinin-induced increase in [Ca²⁺]_i of cultured rat aortic endothelial cells. Acute exposure to acteoside (30 μM) did not affect the expression of eNOS mRNA in endothelium-intact rings. In summary, acteoside impairs endothelial NO-mediated aortic relaxation partially through inhibition of agonist-induced endothelial Ca²⁺ mobilization and Ca²⁺-dependent NO production and subsequent suppression of cyclic GMP formation. This novel pharmacological action if occurring in small vessels in vivo, may contribute to the reported anti-inflammatory effect of acteoside against NO-mediated vascular permeability-related acute edema.     

Elevated InsP3R expression underlies enhanced calcium fluxes and spontaneous extra-systolic calcium release events in hypertrophic cardiac myocytes

Cardiac hypertrophy is associated with profound remodelling of Ca²⁺ signalling pathways. During the early, compensated stages of hypertrophy, Ca²⁺ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca²⁺ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca²⁺ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca²⁺ store are primarily responsible for the Ca²⁺ flux that induces myocyte contraction, a role for Ca²⁺ release via the inositol 1,4,5-trisphosphate receptor (InsP₃R) in cardiac physiology has also emerged. Specifically, InsP₃-induced Ca²⁺ signals generated following myocyte stimulation with an InsP₃-generating agonist (e.g. endothelin, ET-1), lead to modulation of Ca²⁺ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca²⁺ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP₃R (InsP₃R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca²⁺ release via InsP₃Rs serves to sensitise neighbouring RyRs causing an augmentation of Ca²⁺ fluxes during ECC as well as an increase in non-triggered Ca²⁺ release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP₃R expression during hypertrophy, the spontaneous Ca²⁺ release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP₃R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP₃R expression may be a general phenomenon that underlies Ca²⁺ changes during hypertrophy.     

Redox-Regulated Heterogeneous Thresholds for Ligand Recruitment among InsP3R Ca-Release Channels

To clarify the molecular mechanisms behind quantal Ca²⁺ release, the graded Ca²⁺ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP₃R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP₃R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP₃R gene or from cells solely expressing one recombinant InsP₃R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca²⁺ signaling, including quantal Ca²⁺ release, by tuning ligand sensitivities of InsP₃R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca²⁺ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.     

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

Background

Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP₃/Ca²⁺ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca²⁺ signaling and InsP₃-induced Ca²⁺ release from the endoplasmic reticulum (ER).

Results

Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca²⁺]_i changes into a sustained increase. Intraluminal Ca²⁺ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP₃-induced Ca²⁺ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP₃ receptor Ca²⁺ channel for InsP₃. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP₃-induced Ca²⁺ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP₃/Ca²⁺ signaling pathway for 5-HT, because IBMX potentiated Ca²⁺-dependent Cl^- transport activated by a threshold 5-HT concentration.

Conclusion

This report shows, for the first time for an insect system, that cAMP can potentiate InsP₃-induced Ca²⁺ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP₃/Ca²⁺ signaling pathways enhances transepithelial electrolyte transport.     

Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes

In hepatocytes, as in other cell types, Ca²⁺ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP₃R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca²⁺ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP₃ receptors (InsP₃R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP₃R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP₃-induced Ca²⁺ release in hepatocytes. This can be explained by the rather low expression level expression of InsP₃R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca²⁺ signaling via an inhibitory effect on InsP₃ synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP₃ synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca²⁺ release by photolysis of caged InsP₃ have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca²⁺-activated Cl⁻ and K⁺ currents. Photolytic release of InsP₃ from caged InsP₃ at 100 Joules caused transient inward (V_H = 60 mV) and outward (V_H = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl⁻ concentration was decreased, and the outward current was suppressed by K⁺ channel blockers, indicating that they were carried by Cl⁻ and K⁺, respectively. Intracellular pre-loading of the InsP₃ receptor antagonist heparin or the Ca²⁺ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca²⁺-dependent Cl⁻ and K⁺ currents underlies the inward and the outward currents. At low flash intensities, InsP₃ caused Ca²⁺ release which normally activated the K⁺ and Cl⁻ currents in a mono-transient manner. At higher intensities, however, InsP₃ induced an additional delayed outward K⁺ current (I_K(delay)). I_K(delay) was independent of the initial K⁺ current, independent of extracellular Ca²⁺, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca²⁺ from caged Ca²⁺ did not mimic the I_K(delay). It is suggested that Ca²⁺ releases from the InsP₃-sensitive pools in an InsP₃ concentration-dependent manner. Low concentrations of InsP₃ induce the transient Ca²⁺-dependent Cl⁻ and K⁺ currents, which reflects the local Ca²⁺ release, whereas high concentrations of InsP₃ induce a delayed Ca²⁺-dependent K⁺ current, which may reflect the Ca²⁺ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc.