Second Phase Of Ca2 Research Articles

Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins.

The trafficking of transient receptor potential (TRP) channels to the plasma membrane and the release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) are implicated in some aspects of chronic migraines. These exocytotic processes are inhibited by cleavage of SNAREs with botulinum neurotoxins (BoNTs); moreover, type A toxin (/A) clinically reduces the frequency and severity of migraine attacks but not in all patients for unknown reasons. Herein, neonatal rat TGNs were stimulated with allyl isothiocyanate (AITC), a TRPA1 agonist, and dose relationships were established to link the resultant exocytosis of CGRP with Ca2+ influx. The CGRP release, quantified by ELISA, was best fit by a two-site model (EC50 of 6 and 93 µM) that correlates with elevations in intracellular Ca2+ [Ca2+]i revealed by time-lapse confocal microscopy of fluo-4-acetoxymethyl ester (Fluo-4 AM) loaded cells. These signals were all blocked by two TRPA1 antagonists, HC-030031 and A967079. At low [AITC], [Ca2+]i was limited because of desensitisation to the agonist but rose for concentrations > 0.1 mM due to a deduced non-desensitising second phase of Ca2+ influx. A recombinant BoNT chimera (/DA), which cleaves VAMP1/2/3, inhibited AITC-elicited CGRP release to a greater extent than SNAP-25-cleaving BoNT/A. /DA also proved more efficacious against CGRP efflux evoked by a TRPV1 agonist, capsaicin. Nerve growth factor (NGF), a pain-inducing sensitiser of TGNs, enhanced the CGRP exocytosis induced by low [AITC] only. Both toxins blocked NGF-induced neuropeptide secretion and its enhancement of the response to AITC. In conclusion, NGF sensitisation of sensory neurons involves TRPA1, elevated Ca2+ influx, and CGRP exocytosis, mediated by VAMP1/2/3 and SNAP-25 which can be attenuated by the BoNTs.

Measuring the dynamics of glucose-stimulated oxygen consumption in individual pancreatic islets using islet-on-a-chip

Pancreatic islets secrete insulin in response to glucose in two phases involving a Ca2+ burst followed by smaller second phase of Ca2+-oscillations. This biphasic response is determined by the rate of glucose metabolism with models indicating that first phase Ca2+ influx is triggered by glycolysis while the second phase Ca2+ oscillations are controlled by oxidative phosphorylation (OXPHOS). Yet, it is presently unclear how quickly islets transition control between glycolysis and OXPHOS, and whether second phase oscillations are a direct result of metabolic fluctuations.

An Inositol 1,4,5-Triphosphate (IP3)-IP3 Receptor Pathway Is Required for Insulin-Stimulated Glucose Transporter 4 Translocation and Glucose Uptake in Cardiomyocytes

Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gβγ peptidic inhibitor βARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and βARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.

Identification and pharmacological characterization of the prostaglandin FP receptor and FP receptor variant complexes

Background and purpose: A prostamide analogue, bimatoprost, has been shown to be effective in reducing intraocular pressure, but its precise mechanism of action remains unclear. Hence, to elucidate the molecular mechanisms of this effect of bimatoprost, we focused on pharmacologically characterizing prostaglandin FP receptor (FP) and FP receptor variant (altFP) complexes.Experimental approach: FP receptor mRNA variants were identified by reverse transcription-polymerase chain reaction. The FP-altFP4 heterodimers were established in HEK293/EBNA cells co-expressing FP and altFP4 receptor variants. A fluorometric imaging plate reader was used to study Ca2+ mobilization. Upregulation of cysteine-rich angiogenic protein 61 (Cyr61) mRNA was measured by Northern blot analysis, and phosphorylation of myosin light chain (MLC) by western analysis.Key results: Six splicing variants of FP receptor mRNA were identified in human ocular tissues. Immunoprecipitation confirmed that the FP receptor is dimerized with altFP4 receptors in HEK293/EBNA cells co-expressing FP and altFP4 receptors. In the studies of the kinetic profile for Ca2+ mobilization, prostaglandin F2α (PGF2α) elicited a rapid increase in intracellular Ca2+ followed by a steady state phase. In contrast, bimatoprost elicited an immediate increase in intracellular Ca2+ followed by a second phase. The prostamide antagonist, AGN211335, selectively and dose-dependently inhibited the bimatoprost-initiated second phase of Ca2+ mobilization, Cyr61 mRNA upregulation and MLC phosphorylation, but did not block the action of PGF2α.Conclusion and implications: Bimatoprost lacks effects on the FP receptor but may interact with the FP-altFP receptor heterodimer to induce alterations in second messenger signalling. Hence, FP-altFP complexes may represent the underlying basis of bimatoprost pharmacology.

Cell cycle-dependent repetitive Ca(2+ )waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization.

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.

Sperm-triggered calcium oscillations during meiosis in ascidian oocytes first pause, restart, then stop: correlations with cell cycle kinase activity.

We have investigated the relationship between the sperm-triggered Ca2+ oscillations and the activities of two cell cycle kinases (MPF activity and MAP kinase activity) at fertilisation of Ascidiella aspersa oocytes. Maturation Promoting Factor (MPF) activity is elevated in the metaphase I (MI)-arrested unfertilised oocyte (as measured by phosphorylation of exogenous histone H1) and falls 5 minutes after fertilisation to remain at low levels for 5 minutes. The first polar body (pb1) is extruded when the MPF activity is low. The MPF activity is elevated again 15 minutes after fertilisation and finally becomes inactivated 25 minutes after fertilisation when the pb2 is extruded. MAP kinase activity increases from an initially elevated level to reach maximal activity 10 minutes after fertilisation and subsequently falls to reach low levels 25 minutes after fertilisation. Sperm trigger a series of Ca2+ oscillations that pause for 5 minutes while only the MPF activity is low and are present when both MPF and MAP kinase activity are elevated. We next attempted to determine whether the second phase of calcium oscillations is required to reactivate the MPF activity that precedes extrusion of the second polar body. To do this, we triggered a monotonic Ca2+ signal. This leads to the inactivation of MPF followed by MPF reactivation. The MPF activity then remains elevated for an extended period of time. During this period, the chromatin remains condensed and a metaphase II (MII) spindle forms. Fertilisation of these MII oocytes triggers extrusion of pb2 in 7 minutes. Interestingly, the second phase of Ca2+ oscillations is completely absent when MII oocytes are fertilised. Thus, in both MI and MII oocytes, the sperm-triggered Ca2+ oscillations follow the MPF activity. Finally we discuss our finding that the Ca2+ release system remains sensitive during the metaphase-like state (including the period when the Ca2+ oscillations pause).

Hypo-osmotic Shock of Tobacco Cells Stimulates Ca2+Fluxes Deriving First from External and then Internal Ca2+Stores

Hypo-osmotic shock of aequorin-transformed tobacco cells induces a biphasic cytosolic Ca2+ influx. Because both phases of Ca2+ entry are readily blocked by Ca2+ channel inhibitors, we conclude that the Ca2+ transients are mediated by Ca2+ channels. Evidence that the first but not second Ca2+ transient derives from external Ca2+ stores is that the first but not second influx is (i) eliminated by membrane-impermeable Ca2+ chelators, (ii) enlarged by supplementation of the medium with excess Ca2+, and (iii) reduced by the addition of competitive cations such as Mg2+ and Mn2+. Furthermore, entry of 45Ca during osmotic shock is prevented by inhibitors of the first but not second phase of Ca2+ entry. Evidence that the second wave of Ca2+ influx stems from release of intracellular Ca2+ is based on the above data plus observations that probable modulators of intracellular Ca2+ channels selectively block this phase of Ca2+ influx. Finally, a mechanism of communication between the two Ca2+ release pathways has become apparent, since perturbations that elevate or reduce the first Ca2+ transient lead to a compensating diminution/elevation of the second and vice versa. These data thus suggest that osmotic shock leads to the sequential opening of extracellular followed by intracellular Ca2+ stores and that these Ca2+ release pathways are internally compensated.

Simultaneous measurement of membrane potential, cytosolic Ca2+, and tension in intact smooth muscles

Microelectrode techniques and the fluorescent Ca2+ indicator indo-1 were used to measure membrane potential, cytosolic Ca2+ ([Ca2+]cyt), and muscle tension simultaneously in canine antral smooth muscles. Responses of muscles from the myenteric and submucosal regions were compared, since electrical activity and excitation-contraction coupling in these regions differ. The upstroke phase of electrical slow waves in both regions induced an increase in [Ca2+]cyt. In myenteric muscles the plateau phase of slow waves often caused either a further rise in [Ca2+]cyt or maintenance of the level reached during the upstroke event. In submucosal muscles, the plateau phase was significantly smaller and did not induce a second phase in the Ca2+ transient. Contractions were related to the amplitudes of Ca2+ transients. Acetylcholine (ACh; 3 x 10(-8)-10(-6) M) increased the amplitude and duration of the plateau phase of slow waves in a concentration-dependent manner. ACh also increased the second phase of Ca2+ transients and contractile responses associated with the plateau potential. In submucosal muscles ACh induced a significant increase in the plateau phase of the slow wave and increased the corresponding phase of Ca2+ transient. Nicardipine (10(-6) M) inhibited plateau phase of slow waves and the associated increases in [Ca2+]cyt and muscle tension. BAY K 8644 (10(-7) M) augmented the plateau potential and increased [Ca2+]cyt and muscle tension. These results suggest that dihydropyridine-sensitive Ca2+ currents participate in the plateau potential. Cholinergic stimulation modulates [Ca2+]cyt and therefore force by regulating the amount of Ca2+ entering cells through these channels.

Formation and actions of calcium-mobilizing messenger, inositol 1,4,5-trisphosphate.

A variety of surface membrane receptors can activate a phospholipase C, which degrades phosphatidylinositol 4,5-bisphosphate liberating a calcium mobilizing second messenger, inositol 1,4,5-trisphosphate [(1,4,5)IP3]. The coupling of surface receptors to the phospholipase C involves one or more guanine nucleotide-dependent regulatory proteins that are similar but not identical to those that regulate adenylate cyclase. (1,4,5)IP3 has been shown to release Ca2+ from a portion of the endoplasmic reticulum and is believed responsible for the initial phase of Ca2+ mobilization ascribed to internal Ca2+ release. (1,4,5)IP3 acts by binding to a specific receptor that either is a component of, or regulates, a Ca2+ ion channel. The release of Ca2+ from the (1,4,5)IP3-sensitive component of the endoplasmic reticulum may secondarily activate the second phase of Ca2+ mobilization, which involves Ca2+ entry. (1,4,5)IP3 is metabolized by two pathways. One involves the action of a 5-phosphatase that degrades (1,4,5)IP3 to inositol 1,4-bisphosphate, whereas the other involves a 3-kinase that phosphorylates (1,4,5)IP3 to produce inositol 1,3,4,5-tetrakisphosphate. The significance of this dual metabolism is not known, but it may be important in rapidly extinguishing the Ca2+-releasing activity (1,4,5)IP3.

Inhibition of inositol phospholipids metabolism and calcium mobilization by cyclic AMP-increasing agents and phorbol ester in neutrophils

Cyclic AMP-increasing agents such as PGE2 and dibutyryl cAMP inhibited the fMLP-induced inositol phospholipids metabolism mainly through the suppression of the conversion of phosphatidylinositol(PI) to phosphatidylinositol 4,5-bisphosphate(PIP2). A part of this inhibition was found to be caused by the inhibitory effect of cAMP on PI kinase using isolated plasma membranes. On the other hand, 12-O-tetradecanoyl phorbol acetate(TPA) mainly inhibited the conversion of phosphatidylinositol 4-phosphate(PIP) to PIP2 without a significant effect on the fMLP-induced breakdown of PIP2, though direct effect of TPA on PI and PIP kinases was not demonstrated in isolated plasma membranes. Concerning Ca2+ mobilization, both cAMP-increasing agents and TPA inhibited the fMLP-induced second phase of Ca2+ elevation, while they did not affect the first phase of Ca2+ rapid increase. However, Ca2+ ionophore ionomycin-induced Ca2+ elevation was only inhibitable by TPA but not PGE2. These results suggest that cAMP inhibits the fMLP-induced Ca2+ influx, while TPA stimulates Ca2+ removal from cytosol.