On the endothelial cell ISOC

Ca²⁺ store depletion activates both Ca²⁺ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca²⁺ selective current, I_SOC. Indeed, I_SOC is a relatively small inward Ca²⁺ current that exhibits an approximate +40 mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I_SOC, although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I_SOC requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca²⁺ store depletion and channel activation. Thus, at present findings indicate a TRPC4–protein 4.1 physical linkage regulates I_SOC activation following Ca²⁺ store depletion.     

Interleukin-13 enhanced Ca2+ oscillations in airway smooth muscle cells

Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca²⁺ signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca²⁺ oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs.LTD4-induced Ca²⁺ oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca²⁺ entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca²⁺ oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP₃R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca²⁺ oscillations. Ca²⁺ oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP₃R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3.We conclude that IL-13 enhances frequency of LTD4-induced Ca²⁺ oscillations in human ASMCs, which may be cooperatively modulated by IP₃R, RyR systems and possibly by SOCE.     

9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

It is well established that intracellular calcium ([Ca²⁺]_i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca²⁺ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC_Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC_Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca²⁺ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca²⁺ oscillations followed by an overall increase in [Ca²⁺]_i. The latter occurs also in HL-1 cells in Ca²⁺-free solution and after depletion of sarcoplasmic reticulum Ca²⁺ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca²⁺ oscillations followed by a compensatory increase in [Ca²⁺]_i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Calcium oscillations in human mesenteric vascular smooth muscle

Phenylephrine (PE)-induced oscillatory fluctuations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) of vascular smooth muscle have been observed in many blood vessels isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically-stimulated [Ca²⁺]_i oscillations in human mesenteric artery smooth muscle. In arterial segments preloaded with Fluo-4 AM and mounted on a myograph on the stage of a confocal microscope, we observed PE-induced oscillations in [Ca²⁺]_i, which initiated and maintained vasoconstriction. These oscillations present some variability, possibly due to compromised health of the tissue. This view is corroborated by our ultrastructural analysis of the cells, in which we found only (5 ± 2)% plasma membrane-sarcoplasmic reticulum apposition, markedly less than measured in healthy tissue from laboratory animals. We also partially characterized the oscillations by using the inhibitory drugs 2-aminoethoxydiphenyl borate (2-APB), cyclopiazonic acid (CPA) and nifedipine. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca²⁺ and inositol 1,4,5-trisphosphate receptor (IP3R) in the maintenance of the Ca²⁺ oscillations observed in human blood vessels.     

TRPC6 and TRPC4 Heteromultimerization Mediates Store Depletion-Activated NCX1 Reversal in Proliferative Vascular Smooth Muscle Cells

Store depletion has been shown to induce Ca²⁺ entry by Na+/Ca+ exchange (NCX) 1 reversal in proliferative vascular smooth muscle cells (VSMCs). The study objective was to investigate the role of transient receptor potential canonical (TRPC) channels in store depletion and NCX1 reversal in proliferative VSMCs. In cultured VSMCs, expressing TRPC1, TRPC4, and TRPC6, the removal of extracellular Na⁺ was followed by a significant increase of cytosolic Ca²⁺ concentration that was inhibited by KBR, a selective NCX1 inhibitor. TRPC1 knockdown significantly suppressed store-operated, channel-mediated Ca²⁺ entry, but TRPC4 knockdown and TRPC6 knockdown had no effect. Separate knockdown of TRPC1, TRPC4, or TRPC6 did not have a significant effect on thapsigargin-initiated Na⁺ increase in the peripheral regions with KBR treatment, but knockdown of both TRPC4 and TRPC6 did. Stromal interaction molecule (STIM)1 knockdown significantly reduced TRPC4 and TRPC6 binding. The results demonstrated that TRPC4–TRPC6 heteromultimerization linked Ca²⁺ store depletion and STIM1 accumulation with NCX reversal in proliferative VSMCs.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca²⁺ channel (Ca_V1.2) regulates Ca²⁺ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca_V1.2 under low resting [Ca²⁺], but is poised to change conformation and position when intracellular [Ca²⁺] rises. CaM binding Ca²⁺, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A₁₅₈₈, and C₁₆₁₄ and the IQ motif studied as overlapping peptides IQ₁₆₄₄ and IQ′₁₆₅₀ as well as their effect on calcium binding. (Ca²⁺)₄-CaM bound to all four peptides very favorably (K_d ≤ 2 nM). Linkage analysis showed that IQ_1644-1670 bound with a K_d ~ 1 pM. In the pre-IQ region, (Ca²⁺)₂-N-domain bound preferentially to A₁₅₈₈, while (Ca²⁺)₂-C-domain preferred C₁₆₁₄. When bound to C₁₆₁₄, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.     

Polarized but Differential Localization and Recruitment of STIM1, Orai1 and TRPC Channels in Secretory Cells

Polarized Ca²⁺ signals in secretory epithelial cells are determined by compartmentalized localization of Ca²⁺ signaling proteins at the apical pole. Recently the ER Ca²⁺ sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store‐dependent Ca²⁺ influx. STIM1 also gates the transient receptor potential‐canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca²⁺ influx and frequency of Ca²⁺ oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E‐cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co‐IP of STIM1/Orai1/IP₃ receptors (IP₃Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2‐aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca²⁺ entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca²⁺ permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca²⁺] oscillations and thus impact critical cellular functions.     

A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

We have investigated the molecular basis of intracellular Ca²⁺ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca²⁺ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca²⁺ levels, agonist-induced Ca²⁺ increases, store-operated Ca²⁺ entry (SOCE), and store-operated currents (I_SOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca²⁺ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca²⁺ release-activated Ca²⁺ currents (I_CRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced I_CRAC plus a nonselective I_SOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca²⁺ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca²⁺ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca²⁺ remodeling and tumor features in colon cancer.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

Synthesis and biological evaluation of F-18 labeled tetrahydroisoquinoline derivatives targeting orexin 1 receptor

Orexin 1 receptor (OX₁R) is thought to be involved in various body functions, including arousal maintenance and emotional control, but the full details of its function remain unknown. OX₁R imaging with positron emission tomography (PET) would be useful in elucidating the orexin system including OX₁R, but no PET probes targeting OX₁R have been reported. We, therefore, designed and synthesized tetrahydroisoquinoline (THIQ) derivatives as novel PET probes targeting OX₁R, and evaluated their utility. In an in vitro competitive binding assay, THIQ-1 and THIQ-2 showed significantly higher binding to OX₁R (IC₅₀ = 30 and 31 nM, respectively) than OX₂R (IC₅₀ = 160 and 332 nM, respectively). These features were also observed in a cell binding assay using [¹⁸F]THIQ-1 and [¹⁸F]THIQ-2, demonstrating their OX₁R-specific binding property in vitro. In a biodistribution study using normal mice, the brain uptake of [¹⁸F]THIQ-1 was higher than that of [¹⁸F]THIQ-2, but further improvement is required for in vivo imaging with PET. Taken together, [¹⁸F]THIQ-1 and [¹⁸F]THIQ-2 have the potential to become useful imaging probes for PET targeting the OX₁R, but require additional structural changes to improve their brain uptake.     

Functional organization of TRPC-Ca channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1–TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca²⁺ influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) hydrolysis, generation of IP₃ and DAG, and IP₃-induced Ca²⁺ release from the intracellular Ca²⁺ store via inositol trisphosphate receptor (IP₃R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca²⁺ entry mechanisms. The former is regulated by the emptying/refilling of internal Ca²⁺ store(s) while the latter depends on PIP₂ hydrolysis (due to changes in PIP₂ per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca²⁺ entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca²⁺]_i signals which are critical for precise control of downstream cellular functions.     

Activation of TRPC4β by Gαi subunit increases Ca selectivity and controls neurite morphogenesis in cultured hippocampal neuron

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca²⁺-permeable channels. TRPC channels are activated by stimulation of Gα_q-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gα_i. We studied the essential role of Gα_i subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gα_i2 subunit. Activation of TRPC4 by Gα_i2 increased Ca²⁺ permeability and Ca²⁺ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca²⁺ influx. Moreover, both TRPC4β and the TRPC4β-Gα_i2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gα_i2^Q205L-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gα_i proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.     

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP₃Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP₃Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca²⁺ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP₃Rs at different cytosolic free Ca²⁺ concentrations ([Ca²⁺]_c) indicates that the association between these proteins is finely regulated by cytosolic Ca²⁺ via association of CaM and displacement of the IP₃Rs at high [Ca²⁺]_c. Thrombin-stimulated association of TRPC6 with CaM or the IP₃Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca²⁺-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP₃Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca²⁺ entry in the presence of low or high, respectively, extracellular Ca²⁺ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca²⁺ entry and aggregation through its interaction with CaM and IP₃Rs.     

Cellular and molecular effects of unoprostone as a BK channel activator

Effects of unoprostone isopropyl (unoprostone), a prostaglandin metabolite analog; latanoprost, a PGF_2α analog; and PGF_2α were examined in HCN-1A cells, a model system for studies of large conductance Ca²⁺ activated K⁺(BK) channel activator-based neuroprotective agents. Unoprostone and latanoprost, both used as anti-glaucoma agents, have been suggested to act through FP receptors and have neuroprotective effects. Ion channel activation, plasma membrane polarization, [Ca²⁺]_i changes and protection against long-term irreversible glutamate-induced [Ca²⁺]_i increases were studied. Unoprostone activated iberiotoxin (IbTX)-sensitive BK channels in HCN-1A cells with an EC₅₀ of 0.6 ± 0.2 nM and had no effect on Cl⁻ currents. Unoprostone caused IbTX-sensitive plasma membrane hyperpolarization that was insensitive to AL8810, an FP receptor antagonist. In contrast, latanoprost and PGF_2α activated a Cl⁻ current sensitive to [Ca²⁺]_i chelation, tamoxifen and AL8810, and caused IbTX-insensitive, AL8810-sensitive membrane depolarization consistent with FP receptor-mediated Ca²⁺ signaling Cl⁻ current activation. Latanoprost and PGF_2α, but not unoprostone, increased [Ca²⁺]_i. Unoprostone, PGF_2α only partially, but not latanoprost protected HCN-1A cells against glutamate-induced Ca²⁺ deregulation. These findings show that unoprostone has a distinctly different mechanism of action from latanoprost and PGF_2α. Whether unoprostone affects the BK channel directly or an unidentified signaling mechanism has not been determined.     

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells

There is controversy as to whether TRP channels participate in mediating store-operated current (I_SOC) and store-operated Ca²⁺ entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca²⁺ store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I_SOC. We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4^−/− or trpc1^−/− knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.     

Crosstalk between cellular morphology and calcium oscillation patterns Insights from a stochastic computer model

Agonist-induced oscillations in the concentration of intracellular free calcium ([Ca²⁺]₁) display a wide variety of temporal and spatial patterns. In non-excitable cells, typical oscillatory patterns are somewhat cell-type specific and range from frequency-encoded, repetitive Ca²⁺ spikes to oscillations that are more sinusoidal in shape. Although the response of a cell population, even to the same stimulus, is often extremely heterogeneous, the response of the same cell to successive exposures can be remarkably similar. We propose that such ‘Ca ²⁺ fingerprints’ can be a consequence of cell-specific morphological properties. The hypothesis is tested by means of a stochastic computer simulation of a two-dimensional model for oscillatory Ca ²⁺ waves which encompasses the basic elements of the two-pool oscillator introduced by Goldbeter et al. (Goldbeter A., Dupont G., Berridge M.J. Minimal model for signal-induced Ca²⁺-oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461–1465). In the framework of our extended spatiotemporal model, single cells can display various oscillation patterns which depend on the agonist dose, Ca²⁺ diffusibility, and several morphological parameters. These are, for example, size and shape of the cell and the cell nucleus, the amount and distribution of Ca²⁺ stores, and the subcellular location of the inositol(1,4,5)-trisphosphate-generating apparatus.     

Two mechanisms of calcium oscillations in adipocytes

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca²⁺ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) and in some cases through ryanodine receptors (RyR). In the present work, oscillations in single mature adipocytes of mice epididymal fat on the ninth day of cultivation are studied. Cells were stimulated by acetylcholine (ACh) or by fetal bovine serum (FBS). ACh at a concentration of 0.1–5 μM evoked a rise in [Ca²⁺]_i to a peak and subsequent oscillations whose peaks and troughs declined along with increasing amplitude while frequency decreased. In most cells oscillations lasted less than 5 min. The new constant or interspike level exceeded the initial one or was equal to it (at 1 μM ACh). The removal of ACh stopped oscillations immediately. An inhibitor of phospholipase C (U73122) or of IP₃R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP₃. At the same time, suppression of responses by ryanodine, which blocks RyR, was observed. Besides, oscillatory responses were abolished by inhibitors of phosphatidylinositol 3-kinase, NO synthase, and cGMP-dependent protein kinase. FBS (1%) initiated oscillations characterized by return of [Ca²⁺]_i after each peak to the baseline level, occurring prior to stimulation, and by maintenance of roughly constant amplitude and frequency (of the order of 1 min⁻¹). Oscillations persisted longer (more than 15 min in 87% of cells) than with ACh. Repeated stimulation of cells by FBS revealed a strongly reduced sensitivity after 1 h of rest, whereas responses to ACh partially restored within 3 min. Investigation of the involvement of IP₃R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP₃R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca²⁺ entry through the plasma membrane was necessary only as a support of oscillations. The results show that in adipocytes different agonists can engage distinct subsystems of Ca²⁺ signaling, each of them generating oscillations with a specific temporal pattern.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.