Opposed ciliary bands in the feeding larvae of sabellariid annelids

The larvae of marine annelids capture food using an unusual diversity of suspension-feeding mechanisms. Many of the feeding mechanisms of larval annelids are poorly known despite the abundance and ecological significance of both larvae and adults of some annelid taxa. Here we show that larvae of two species of sabellariid annelids, Sabellaria cementarium and Phragmatopoma californica, bear prototrochal and metatrochal cilia that beat in opposition to each other. For larvae of S. cementarium, we provide evidence that these opposed bands of cilia are used to capture suspended particles. In video recordings, captured particles were overtaken by a prototrochal cilium and then moved with the cilium to the food groove, a band of cilia between the prototroch and metatroch. They were then transported by cilia of the food groove to the mouth. Lengths of the prototrochal cilia, lengths of the prototrochal ciliary band, size range of the particles captured, and estimated rates of clearance increased with larval age and body size. Confirmation of the presence of opposed bands in larvae of sabellariids extends their known occurrence in the annelids to members of 10 families. Opposed bands in these different taxa differ in the arrangements and spacing of prototrochal and metatrochal cilia, and in whether they are used in combination with other feeding mechanisms. Opposed bands appear to be particularly widespread among the larvae of sabellidan annelids (a clade that includes sabellariids, sabellids, and serpulids), even in some species whose larvae do not feed. A parsimony analysis suggests that opposed bands are ancestral in this clade of annelids.     

Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium

Intracellular Ca²⁺ induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca²⁺ signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO₃, which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca²⁺-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca²⁺-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca²⁺-induced ciliary reversal.     

Neural correlates of settlement in veliger larvae of the gastropod,Crepidula fornicata

Settlement behavior of molluscan veliger larvae prior to metamorphosis requires cessation of swimming, accomplished by arrest of prototrochal cilia on the margin of the velum (the larval swimming organ). Ciliary arrest in larvae of gastropods is mediated by an action potential that occurs synchronously across the velum as a consequence of electrical coupling between the prototrochal ciliated cells. We developed a preparation for extracellular recording of such ciliary arrest spikes from intact swimming and crawling veliger larvae of the caenogastropod Crepidula fornicata, using a fine wire electrode. Ciliary arrest spike rates during bouts of substrate crawling were significantly higher than those recorded during preceding swimming periods in larvae that were competent for metamorphosis, but not in precompetent larvae. Spike rates were similar on clean polystyrene substrates, and on substrates that had been coated with a natural cue for metamorphosis (mucus from conspecific adults). We used immunohistochemical methods to localize neuromodulators that might regulate the function of velar cilia. Labeled terminals for serotonin, FMRFamide, and tyrosine hydroxylase (an enzyme for catecholamine synthesis) were located in positions consistent with modulatory effects on the prototrochal ciliated cells. Prototrochal ciliary arrest spike rates and beat frequencies were measured in isolated velar lobes from competent larvae, which were exposed to serotonin, FMRFamide, and dopamine (10^?5 mol L^?1). Serotonin abolished arrest spiking and increased beat frequency; dopamine also increased beat frequency, and FMRFamide depressed it. Competent larvae tested in a small static water column swam to the top of the column when exposed to serotonin, but occupied lower positions than controls when in the presence of dopamine and FMRFamide. The larval nervous system appears to regulate velar functions that are critical for settlement behavior, and is likely to do so by integrating different sensory modalities in an age‐dependent manner.     

Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells,and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

In order to develop a novel method of visualizing possible Ca~(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca~(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca~(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca~(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca~(2+) transients were generated from day 1 onward. That ATP was inducing Ca~(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca~(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.     

Effect of calcium on phototactic orientation of individual cryptomonas cells

The effect of Ca²⁺ ions on the phototactic orientation of Cryptomonas cells was determined by observation of the direction of swimming of individual cells using infrared video-micrography. When 2 mM EGTA was added to the medium, changes in the swimming direction of cells became smaller, consequently the phototactic orientation was inhibited. The EGTA-induced inhibition was almost completely overcome by the addition of 2 mM CaCl₂. Consequently the presence of Ca²⁺ ions appears to be crucial for the orientation that is necessary during phototaxis of Cryptomonas cells.     

β2 adrenoceptor signaling regulates ion transport in 16HBE14o- human airway epithelial cells

We investigated the regulation of Cl⁻ secretion by adrenoceptors in polarized 16HBE14o- human bronchial epithelial cells. Treatment with the nonselective β adrenoceptor agonist isoprenaline stimulated an increase in short-circuit current (I_SC), which was inhibited by the β adrenoceptor blocker propranolol. Treatment with procaterol, an agonist specific for the β₂ adrenoceptor subtype, stimulated a similar increase in I_SC, which was inhibited by the β₂ adrenoceptor antagonist ICI 118551. Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl⁻ channel (CaCC), but not K⁺ channel blockers, were able to inhibit the increase in I_SC. “Trimultaneous” recording of I_SC and intracellular cyclic adenosine monophosphate (cAMP) and Ca²⁺ levels in 16HBE14o- epithelia confirmed that the I_SC induced by isoprenaline or procaterol involved both cAMP and Ca²⁺ signaling. Our results demonstrate that β₂ adrenoceptors regulate Cl⁻ secretion in the human airway epithelium by activating apical CFTRs and CaCCs via cAMP-dependent and intracellular Ca²⁺-dependent mechanisms, respectively.     

Alpha-adrenergic agonist and endothelin-1 induced intracellular Ca response in the presence of a Ca entry blocker in cultured rat ventricular myocytes

Previously we demonstrated that stimulation of cultured neonatal rat ventricular myocytes by either α₁-adrenergic agonist or endothelin-1 resulted in a rapid formation of total inositolphosphates, although the levels of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate did not rise significantly. The aim of this study was to examine whether stimulation by α₁-adrenergic agonist and endothelin-1 could still elicit phosphatidylinositol cycle mediated intracellular Ca²⁺ mobilization in these cells. The intracellular free Ca²⁺ concentration ([Ca ²⁺]i) was measured by single cell imaging dual wavelength fluorescence microscopy in Fura-2-loaded cardiomyocytes. The interference of agonist induced [Ca²⁺]; responses by the beat to beat variation of [Ca²⁺]i was prevented by arresting the cells with the Ca²⁺ entry blocker diltiazem (10 μM). The [Ca²⁺]i response (expressed as % of baseline ratio of fluorescence intensities of Fura-2 at 340 nm and 380 nm excitation wavelength), induced by phenylephrine (10⁻⁴ M) and endothelin-1 (10⁻⁸ M) was small, up to 20% of baseline after 9–20 min. In contrast, Ca²⁺-influx induced by incubation in Na⁺-free buffer caused a steep increase of [Ca²⁺]; up to 150% of baseline after 30 s.Analysis of single cells following stimulation with phenylephrine or endothelin-1 showed heterogeneity with respect to a rise in [Ca²⁺. However, if rapid Ca²⁺-influx was induced by incubation in Na⁺-free buffer, [Ca²⁺]i responses in individual myocytes occurred homogeneously.It is concluded that the α₁-adrenergic agonist and endothelin-1induced [Ca²⁺]i responses are delayed in time, small and quite heterogeneous among cells. The findings are in agreement with earlier observations which revealed no detectable overall increase of the Ca²⁺ releasing inositolphosphates under these conditions and suggest that other second messengers, such as 1,2-diacylglycerol, are involved in the agonist mediated Ca²⁺ signals.     

In Silico Determination of the Effect of Multi-Target Drugs on Calcium Dynamics Signaling Network Underlying Sea Urchin Spermatozoa Motility

The motility of spermatozoa of both Lytechinus pictus and Strongylocentrotus purpuratus sea urchin species is modulated by the egg-derived decapeptide speract via an oscillatory [Ca²⁺]-dependent signaling pathway. Comprehension of this pathway is hence directly related to the understanding of regulated sperm swimming. Niflumic acid (NFA), a nonsteroidal anti-inflammatory drug alters several ion channels. Though unspecific, NFA profoundly affects how sea urchin sperm respond to speract, increasing the [Ca²⁺]_i oscillation period, amplitude, peak and average level values of the responses in immobilized and swimming cells. A previous logical network model we developed for the [Ca²⁺] dynamics of speract signaling cascade in sea urchin sperm allows integrated dissection of individual and multiple actions of NFA. Among the channels affected by NFA are: hyperpolarization-activated and cyclic nucleotide gated Na⁺ channels (HCN), [Ca²⁺]-dependent Cl⁻ channels (CaCC) and [Ca²⁺]-dependent K⁺ channels (CaKC), all present in the sea urchin genome. Here, using our model we investigated the effect of blocking in silico HCN and CaCC channels suggested by experiments. Regarding CaKC channels, arguments can be provided for either their blockage or activation by NFA. Our study yielded two scenarios compliant with experimental observations: i) under CaKC inhibition, this [Ca²⁺]-dependent K⁺ channel should be different from the Slo1 channel and ii) under activation of the CaKC channel, another [Ca²⁺] channel not considered previously in the network is required, such as the pH-dependent CatSper channel. Additionally, our findings predict cause-effect relations resulting from a selective inhibition of those channels. Knowledge of these relations may be of consequence for a variety of electrophysiological studies and have an impact on drug related investigations. Our study contributes to a better grasp of the network dynamics and suggests further experimental work.     

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Specific labelling of the (Ca2+ + Mg2+)-ATPase of Escherichia coli with 8-azido-ATP and 4-chloro-7-nitrobenzofurazan

1. 8-Azido-ATP is a substrate for Escherichia coli (Ca²⁺ + Mg²⁺)-ATPase (E. coli F₁).2. Illumination of E. coli F₁ in the presence of 8-azido-ATP causes inhibition of ATPase activity. The presence of ATP during illumination prevents inhibition.3. 8-Azido-ATP and 4-chloro-7-nitrobenzofurazan (NbfCl) bind predominantly to the α subunit of the enzyme, but also significantly to the β subunit.4. The α subunit of E. coli F₁ seems to have some properties that in other F₁-ATPases are associated with the β subunit.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

Ca2+‐dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid β‐treated astrocytes and in a model of Alzheimer's disease

Neurotoxic effects of amyloid β peptides are mediated through deregulation of intracellular Ca²⁺ homeostasis and signaling, but relatively little is known about amyloid β modulation of Ca²⁺ homeostasis and its pathological influence on glia. Here, we found that amyloid β oligomers caused a cytoplasmic Ca²⁺ increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca²⁺ release. Furthermore, amyloid β peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP₃ receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid β oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple‐transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid β oligomer load in 12‐month‐old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid β oligomers disrupt ER Ca²⁺ homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.     

Blockers of Ca2+ channels in the plasmalemma of perfused Characeae cells

Ionic currents in the plasmalemma of perfused Nitella syncarpa cells identified as currents through Ca²⁺ channels were registered for the first time. The effect of 1,4-dihydropyridine derivatives (nifedipine, nitredipine, riodipine) and phenylalkylamines (verapamil, D600) as well as the agonist CGP-28392 on the Ca²⁺ channels in the plasmelemma of perfused cells of Nitellopsis obtusa and Nitella syncarpa have been studied. A blocking effect of 1,4-dihydropyridine derivatives and phenylalkylamines on the plasmalemma Ca²⁺ channels has been detected. Phenylalkylamines have been found to block both inward and outward Ca²⁺ currents. The activating effect of the agonist CGP-28392 on the Ca²⁺ channels of plasmalemma has been shown.     

Selectivity signatures of three isoforms of recombinant T-type Ca channels

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ (K_D1 ∼ 6 μM) and for Ca²⁺ current saturation (K_D2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.     

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca²⁺]_o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca²⁺]_o or amino acids elicits intracellular Ca²⁺ ([Ca²⁺]_i) oscillations. Here, we report the central role of predicted Ca²⁺-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca²⁺-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca²⁺]_o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca²⁺]_o and l-Phe in eliciting CaSR-mediated [Ca²⁺]_i oscillations. The heterocommunication between Ca²⁺ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca²⁺]_o signaling by positively impacting multiple [Ca²⁺]_o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca²⁺]_o and amino acids into intracellular signaling events.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.