Biomedical subjects

G C Churchill

Publications and source records attributed to G C Churchill.

17 recordsLinked to original sources

PubMed2001

Pharmacological characterization of the putative cADP-ribose receptor.

cADP-ribose (cADPR), a naturally occurring metabolite of NAD(+), has been shown to be an important regulator of intracellular Ca(2+) release. Considerable evidence suggests that cADPR is the endogenous modulator of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). Indeed, cADPR-mediated Ca(2+) release is subject to functional regulation by other modulators of CICR, including Ca(2+), caffeine and calmodulin. However, the underlying basis behind the effect of such agents on cADPR activity (in particular whether they regulate cADPR binding), as well as the precise nature of the cADPR receptor remains unclear. In the present study, use of (32)P-radiolabelled cADPR has enabled a detailed pharmacological characterization of cADPR-binding sites in sea urchin egg homogenates. We report that cADPR binds specifically to a single class of high affinity receptor. Retainment of binding to membranes after a high-salt wash suggests the involvement of either an integral membrane protein (possibly the RyR itself) or a peripheral protein tightly associated to the membrane. Insensitivity of [(32)P]cADPR binding to either FK506 or rapamycin suggests that this does not concern the FK506-binding protein. Significantly, binding is highly robust, being relatively insensitive to both endogenous and pharmacological modulators of RyR-mediated CICR. In turn, this suggests that such agents modulate cADPR-mediated Ca(2+) release primarily by tuning the 'gain' of the CICR system, upon which cADPR acts, rather than influencing the interaction of cADPR with its target receptor. The exception to this is calmodulin, for which our results indicate an additional role in facilitating cADPR binding.

Adenosine Diphosphate Ribose↗

PubMed2001

NAADP induces Ca2+ oscillations via a two-pool mechanism by priming IP3- and cADPR-sensitive Ca2+ stores.

In sea urchin eggs, Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) potently self-inactivates but paradoxically induces long-term Ca2+ oscillations. We investigated whether NAADP-induced Ca2+ oscillations arise from the recruitment of other Ca2+ release pathways. NAADP, inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) all mobilized Ca2+ from internal stores but only NAADP consistently induced Ca2+ oscillations. NAADP-induced Ca2+ oscillations were partially inhibited by heparin or 8-amino-cADPR alone, but eliminated by the presence of both, indicating a requirement for both IP3- and cADPR-dependent Ca2+ release. Thapsigargin completely blocked IP3 and cADPR responses as well as NAADP-induced Ca2+ oscillations, but only reduced the NAADP-mediated Ca2+ transient. Following NAADP-mediated release from this Ca2+ pool, the amount of Ca2+ in the Ca2+-induced Ca2+ release stores was increased. These results support a mechanism in which Ca2+ oscillations are initiated by Ca2+ release from NAADP-sensitive Ca2+ stores (pool 1) and perpetuated through cycles of Ca2+ uptake into and release from Ca2+-induced Ca2+ release stores (pool 2). These results provide the first direct evidence in support of a two-pool model for Ca2+ oscillations.

Adenosine Diphosphate Ribose↗

PubMed2001

Prolonged inactivation of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release mediates a spatiotemporal Ca2+ memory.

Although numerous extracellular stimuli are coupled to increases in intracellular Ca(2+), different stimuli are thought to achieve specificity by eliciting different spatiotemporal Ca(2+) increases. We investigated the effect of nicotinic acid adenine dinucleotide phosphate (NAADP) inactivation on spatiotemporal Ca(2+) signals in intact sea urchin eggs. The photorelease of NAADP but not inositol 1,4,5-trisphosphate or cyclic ADP-ribose resulted in self-inactivation. When NAADP was released first locally and subsequently globally, the spatial pattern of the first response shaped that of the second. Specifically, the local release of NAADP created a Ca(2+) gradient that was reversed during the subsequent global release of NAADP. Neither cyclic ADP-ribose nor inositol 1,4,5-trisphosphate showed a similar effect. In contrast to homogenates, NAADP inactivation was reversible in intact eggs with resensitization occurring in approximately 20 min. Because initial NAADP responses affect later responses, NAADP can serve as a mechanism for a Ca(2+) memory that has both spatial and temporal components. This NAADP-mediated Ca(2+) memory provides a novel mechanism for cells to control spatiotemporal Ca(2+) increases.

Animals↗

PubMed2001

Coordination of Ca2+ signalling by NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes intracellular Ca2+ stores in several cell types. Ample evidence suggests that NAADP activates intracellular Ca2+ channels distinct from those that are sensitive to inositol trisphosphate and ryanodine/cyclic ADP-ribose. Recent studies in intact cells have demonstrated functional coupling ('channel chatter') between Ca2+ release pathways mediated by NAADP, inositol trisphosphate and cyclic ADP-ribose. Thus, NAADP is probably an important determinant in shaping cytosolic Ca2+ signals.

Calcium Signaling↗

PubMed2001

Ca(2+) regulation of gap junctional coupling in lens epithelial cells.

The quantitative effects of Ca(2+) signaling on gap junctional coupling in lens epithelial cells have been determined using either the spread of Mn(2+) that is imaged by its ability to quench the fluorescence of fura 2 or the spread of the fluorescent dye Alexa Fluor 594. Gap junctional coupling was unaffected by a mechanically stimulated cell-to-cell Ca(2+) wave. Furthermore, when cytosolic Ca(2+) concentration (Ca) increased after the addition of the agonist ATP, coupling was unaffected during the period that Ca was maximal. However, coupling decreased transiently approximately 5-10 min after agonist addition when Ca returned to resting levels, indicating that this transient decrease in coupling was unlikely due to a direct action of Ca on gap junctions. An increase in Ca mediated by the ionophore ionomycin that was sustained for several minutes resulted in a more rapid and sustained decrease in coupling (IC(50) ~300 nM Ca(2+), Hill coefficient of 4), indicating that an increase in Ca alone could regulate gap junctions. Thus Ca increases that occurred during agonist stimulation and cell-to-cell Ca(2+) waves were too transient to mediate a sustained uncoupling of lens epithelial cells.

Adenosine Triphosphate↗

PubMed2001

Distinct pharmacology of 2-hydroxycarbazole-induced Ca2+ release in the sea urchin egg.

2-hydroxycarbazole, a compound structurally related to the Ca2+-mobilizing marine toxin 9-methyl-7-bromoeudistomin, has recently been proposed to activate both type 1 and type 2 ryanodine receptors in skeletal and cardiac muscle, respectively. This study was undertaken to evaluate the activity of this compound in the sea urchin egg homogenate, a model system used to characterize intracellular Ca2+ mobilization mechanisms. 2-Hydroxycarbazole was found to potently release Ca2+ in a concentration-dependent manner via a specific mechanism displaying apparent desensitization. Use of selective inhibitors of the Ca2+-mobilizing messengers inositol 1,4,5-trisphosphate, cyclic adenosine diphosphate ribose, and nicotinic acid adenine dinucleotide phosphate, as well as desensitization of homogenates to each of these molecules, failed to inhibit the response to 2-hydroxycarbazole. However, the response to 2-hydroxycarbazole was competitively antagonized by caffeine. Investigation of the Ca2+ stores accessed by 2-hydroxycarbazole revealed Ca2+ release from a thapsigargin-insensitive pool. Finally, 2-hydroxycarbazole failed to enhance [3H]ryanodine binding, suggesting the operation of a nonryanodine receptor mechanism. These results demonstrate that 2-hydroxycarbazole is acting to modulate a Ca2+ release mechanism with distinct pharmacological properties to those previously reported in the sea urchin egg.

Adenosine Diphosphate↗

PubMed2000

Unique kinetics of nicotinic acid-adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel and potent Ca(2+)-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [(32)P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca(2+). Binding of [(32)P]NAADP was lost in preparations that did not mobilize Ca(2+) in response to NAADP, indicating that [(32)P]NAADP probably binds to a receptor mediating Ca(2+) mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [(32)P]NAADP binding, did not result in displacement of bound [(32)P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels.

Adenosine Diphosphate Ribose↗

PubMed2000

Spatial control of Ca2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients.

Intracellular Ca(2+) is able to control numerous cellular responses through complex spatiotemporal organization. Ca(2+) waves mediated by inositol trisphosphate or ryanodine receptors propagate by Ca(2+)-induced Ca(2+) release and therefore do not have an absolute requirement for a gradient in either inositol trisphosphate or cyclic ADP-ribose, respectively. In contrast, we report that although Ca(2+) increases induced by nicotinic acid adenine dinucleotide phosphate (NAADP) are amplified by Ca(2+)-induced Ca(2+) release locally, Ca(2+) waves mediated by NAADP have an absolute requirement for an NAADP gradient. If NAADP is increased such that its concentration is spatially uniform in one region of an egg, the Ca(2+) increase occurs simultaneously throughout this area, and only where there is diffusion out of this area to establish an NAADP gradient is there a Ca(2+) wave. A local increase in NAADP results in a Ca(2+) increase that spreads by NAADP diffusion. NAADP diffusion is restricted at low but not high concentrations of NAADP, indicating that NAADP diffusion is strongly influenced by binding to immobile and saturable sites, probably the NAADP receptor itself. Thus, the range of action of NAADP can be tuned by its concentration from that of a local messenger, like Ca(2+), to that of a global messenger, like IP(3) or cyclic ADP-ribose.

Adenosine Diphosphate Ribose↗

PubMed2000

Widespread distribution of binding sites for the novel Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate, in the brain.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing agent in invertebrate eggs that has recently been shown to be active in certain mammalian and plant systems. Little, however, is known concerning the properties of putative NAADP receptors. Here, for the first time, we report binding sites for NAADP in brain. In contrast to sea urchin egg homogenates, [(32)P]NAADP bound reversibly to multiple sites in brain membranes. The rank order of potency of NAADP, 2',3'-cyclic NAADP and 3'-NAADP in displacing [(32)P]NAADP was, however, the same in the two systems and in agreement with their ability to mobilize Ca(2+) from homogenates. These data indicate that [(32)P]NAADP likely binds to receptors mediating Ca(2+) mobilization. Autoradiography revealed striking heterogeneity in the distribution of [(32)P]NAADP binding sites throughout the brain. Our data strongly support a role for NAADP-induced Ca(2+) signaling in the brain.

Animals↗

PubMed2000

Cyclic ADP ribose as a calcium-mobilizing messenger.

This Perspective by Galione and Churchill is one in a series on intracellular calcium release mechanisms. The authors review the evidence for cyclic adenosine diphosphate ribose (cADPR) being a second messenger involved in regulating intracellular calcium. In addition, the physiological stimuli and responses mediated by cADPR are discussed. The Perspective is accompanied by a movie showing a calcium wave triggered by cADPR.

Adenosine Diphosphate Ribose↗

PubMed2000

NAADP-induced calcium release in sea urchin eggs.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores described. It acts on a mechanism distinct from inositol trisphosphate and ryanodine receptors, the two major Ca2+ release channels characterised. NAADP-gated Ca2+ release channels do not appear to be regulated by Ca2+ and may be better suited for triggering Ca2+ signals rather than propagating them. They exhibit a remarkable pharmacology for a putative intracellular Ca2+ release channel in that they are selectively blocked by potassium and L-type Ca2+ channel antagonists. Furthermore, in contrast to microsomal Ca2+ stores expressing IP3Rs and RyRs, those sensitive to NAADP are thapsigargin-insensitive, suggesting that they may be expressed on a different part of the endoplasmic reticulum. Perhaps the most unusual feature of the NAADP-gated Ca2+ release mechanisms is its inactivation properties. Unlike the mechanisms regulated by IP3 and cADPR in sea urchin eggs which after induction of Ca2+ release appear to become refractory to subsequent activation, very low concentrations of NAADP are able to inactivate NAADP-induced Ca2+ release fully at concentrations well below those required to activate Ca2+ release. The mechanism and physiological significance of this most unusual desensitisation phenomenon are unclear. More recently, NAADP has been shown to mobilise Ca2+ in ascidian oocytes, brain microsomes and pancreatic acinar cells suggesting a more widespread role in Ca2+ signalling. A possible role for this novel Ca2+ release mechanism in sea urchin egg fertilisation is discussed.

Animals↗

PubMed1999

Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells.

Many hormones and neurotransmitters evoke Ca2+ release from intracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3) and cyclic adenosine 5'-diphosphate-ribose (cADPR) are established Ca2+-mobilizing messengers that activate Ca2+ release through intracellular InsP3 and ryanodine receptors, respectively. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca2+ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca2+-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP), an endogenous metabolite of beta-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1-2s) and long-lasting (0.2-1 min) Ca2+ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP3, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca2+ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.

Adenosine Diphosphate Ribose↗

PubMed1999

Imaging of intracellular calcium stores in single permeabilized lens cells.

Intracellular Ca2+ stores in permeabilized sheep lens cells were imaged with mag-fura 2 to characterize their distribution and sensitivity to Ca2+-releasing agents. Inositol 1,4,5-trisphosphate (IP3) or cyclic ADP-ribose (cADPR) released Ca2+ from intracellular Ca2+ stores that were maintained by an ATP-dependent Ca2+ pump. The IP3 antagonist heparin inhibited IP3- but not cADPR-mediated Ca2+ release, whereas the cADPR antagonist 8-amino-cADPR inhibited cADPR- but not IP3-mediated Ca2+ release, indicating that IP3 and cADPR were operating through separate mechanisms. A Ca2+ store sensitive to IP3, cADPR, and thapsigargin appeared to be distributed throughout all intracellular regions. In some cells a Ca2+ store insensitive to IP3, cADPR, thapsigargin, and 2,4-dinitrophenol, but not ionomycin, was present in a juxtanuclear region. We conclude that lens cells contain intracellular Ca2+ stores that are sensitive to IP3, cADPR, and thapsigargin, as well as a Ca2+ store that appears insensitive to all these agents.

Adenosine Diphosphate Ribose↗

PubMed1998

Roles of Ca2+, inositol trisphosphate and cyclic ADP-ribose in mediating intercellular Ca2+ signaling in sheep lens cells.

To further characterize how gap junction-dependent Ca2+ waves propagate between sheep lens cells, we examined the possible roles of inositol 1,4,5-trisphosphate (IP3), Ca2+ and cyclic ADP-ribose (cADPR) in mediating intercellular Ca2+ waves. Second messengers were microinjected into a single cell in a monolayer of sheep lens cells while monitoring cytosolic Ca2+ with fura-2 and fluorescence microscopy. All three compounds initiated intercellular Ca2+ waves, but more cells responded following the injection of either IP3 or cADPR than responded following the injection of Ca2+. When either IP3 or cADPR was co-injected with the Ca2+ chelator EGTA, cytosolic Ca2+ in the injected cell decreased but cytosolic Ca2+ in the adjacent cells increased, indicating that the intercellular messenger was IP3 or cADPR, rather than Ca2+. The phospholipase C inhibitor U73122 eliminated mechanically initiated intercellular Ca2+ waves, indicating that mechanical initiation probably requires IP3 production. In U73122-treated cells, injected IP3 initiated an intercellular Ca2+ wave in which the number of cells responding increased as the amount of IP3 injected increased, indicating that the distance traveled by the Ca2+ wave was dependent on cell-to-cell diffusion of IP3. In contrast, the ability of cADPR both to increase cytosolic Ca2+ in the injected cell and to initiate intercellular Ca2+ waves was greatly attenuated by U73122. In conclusion, Ca2+, IP3 and cADPR can all mediate intercellular Ca2+ waves by passing through gap junction channels, but both IP3 and cADPR are more effective intercellular messengers than Ca2+.

Animals↗

PubMed1997

Stimulation of P2U purinergic or alpha 1A adrenergic receptors mobilizes Ca2+ in lens cells.

PURPOSE: To identify agonists that elevate cytosolic Ca2+ (Cai2+) in lens cells and to characterize their mechanism of action. METHODS: Digital imaging and the Ca(2+)-reporting dye fura-2 were used to study the effects of agonists and their antagonists of Cai2+ in sheep lens primary cell cultures. RESULTS: Exposing cells to adenosine triphosphate (ATP) and epinephrine increased Cai2+, whereas dopamine, 5-hydroxytryptamine, acetylcholine, histamine, kassinin, bradykinin, and glutamate did not elevate Cai2+. The ATP response was mediated by P2U purinergic receptors based on inhibition by the P2 antagonist suramin and the agonist rank potency order ATP = UTP = ATP gamma S > ADP > AMP > > adenosine; adenine, AMP-CPP, and AMP-PCP were inactive. The epinephrine response was mediated by alpha 1 adrenergic receptors based on the greater potency of the alpha 1 adrenergic selective antagonist prazosin compared to that of the alpha 2 adrenergic selective antagonist yohimbine. More specifically, the epinephrine response was mediated by the alpha 1A adrenergic receptor subtype based on the greater potencies exhibited by the alpha 1A subtype selective competitive antagonists WB 4101 and 5-methylurapidil compared to the alpha 1B and alpha 1D selective antagonists spiperone and BMY 7378, respectively. The agonist-mediated Cai2+ increase was dependent on intracellular Ca2+ stores and was inhibited by the phospholipase C inhibitor U73122. ATP or epinephrine could desensitize the cells to either agonist because of both the depletion of intracellular Ca2+ stores and the downregulation of a common intermediate in the signal transduction pathway. CONCLUSIONS: Ca2+ is mobilized from intracellular stores in the sheep lens by ATP and epinephrine acting through P2U purinergic and the alpha 1A adrenergic receptors, respectively. This confirms previous reports of P2U receptors in lens and provides the first report of alpha 1A adrenergic receptors in the lens.

Adenosine Triphosphate↗

PubMed1996

Mechanical stimulation initiates cell-to-cell calcium signaling in ovine lens epithelial cells.

Although abnormalities in calcium regulation have been implicated in the development of most forms of cataract, the mechanisms by which Ca2+ is regulated in the cells of the ocular lens remain poorly defined. Cell-to-cell Ca2+ signaling was investigated in primary cultures of ovine epithelial cells using the Ca(2+)-reporter dye fura-2 and fluorescence microscopy. Mechanical stimulation of a single cell with a micropipette initiated a propagated increase in cytosolic free Ca2+ that spread from the stimulated cell through 2-8 tiers of surrounding cells. During this intercellular Ca2+ wave, cytosolic Ca2+ increased 2- to 12-fold from resting levels of approximately 100 nM. Nanomolar extracellular Ca2+ did not affect the cell-to-cell propagation of the Ca2+ wave, but reduced the magnitude of the cytosolic Ca2+ increases, which was most evident in the mechanically-stimulated cell. Depletion of intracellular Ca2+ stores with thapsigargin eliminated the propagated intercellular Ca2+ wave, but did not prevent the cytosolic Ca2+ increase in the mechanically-stimulated cell, which required extracellular Ca2+ and was attenuated by the addition of the Ca2+ channel blockers Ni2+, Gd3+ and La3+ to the medium. These results are most easily explained by a mechanically-activated channel in the plasma membrane of the stimulated cell. The propagated increase in cytosolic Ca2+ appeared to be communicated to adjacent cells by the passage of an intracellular messenger other than Ca2+ through gap junction channels. However, if the plasma membrane of the mechanically-stimulated cell was ruptured such that there was loss of cytosolic contents, the increase in cytosolic Ca2+ in the surrounding cells was elicited by both a messenger passing through gap junction channels and by a cytosolic factor(s) diffusing through the extracellular medium. These results demonstrate the existence of intercellular Ca2+ signaling in lens cells, which may play a role in regulating cytosolic Ca2+ in the intact lens.

Animals↗

PubMedDate not supplied

Interactions between calcium release pathways: multiple messengers and multiple stores.

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles. In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.

Animals↗