Search PubMed⌕ Search

Biomedical subjects

T J Shuttleworth

Publications and source records attributed to T J Shuttleworth.

At least 19 recordsLinked to original sources
PubMed2000

I(ARC), a novel arachidonate-regulated, noncapacitative Ca(2+) entry channel.

Along with the inositol trisphosphate-induced release of stored Ca(2+), a receptor-enhanced entry of Ca(2+) is a critical component of intracellular Ca(2+) signals generated by agonists acting at receptors coupled to the activation of phospholipase C. Although the simple emptying of the intracellular Ca(2+) stores is known to be capable of activating Ca(2+) entry via the so-called "capacitative" mechanism, recent evidence suggests that Ca(2+) entry at physiological agonist concentrations, where oscillatory Ca(2+) signals are typically observed, does not conform to such a model. Instead, a noncapacitative Ca(2+) entry pathway regulated by arachidonic acid appears to be responsible for Ca(2+) entry under these conditions. Using whole-cell patch clamp techniques we demonstrate that low concentrations of arachidonic acid activate a Ca(2+)-selective current that is superficially similar to the store-operated current I(CRAC), but which also demonstrates certain distinct features. We have named this novel current I(ARC) (for arachidonate-regulated calcium current). Importantly, I(ARC) can be readily activated in cells whose Ca(2+) stores have been maximally depleted. I(ARC) represents a novel Ca(2+) entry pathway that is entirely separate from those activated by store depletion and is specifically activated at physiological levels of stimulation.

Arachidonic Acid↗
PubMed2000

A Ca(2+)-independent activation of a type IV cytosolic phospholipase A(2) underlies the receptor stimulation of arachidonic acid-dependent noncapacitative calcium entry.

The oscillatory [Ca(2+)](i) signals typically seen following physiologically relevant stimulation of phospholipase C-linked receptors are associated with a receptor-activated entry of Ca(2+), which plays a critical role in driving the oscillations and influencing their frequency. We have recently shown that this receptor-activated entry of Ca(2+) does not conform to the widely accepted "capacitative" model and, instead, reflects the activity of a distinct, novel Ca(2+) entry pathway regulated by arachidonic acid (Shuttleworth, T. J., and Thompson, J. L. (1998) J. Biol. Chem. 273, 32636-32643). We now show that the generation of arachidonic acid under these conditions results from the activity of a type IV cytosolic phospholipase A(2) (cPLA(2)). Although cPLA(2) activation commonly involves a Ca(2+)-dependent translocation to the membrane, at these low agonist concentrations cPLA(2) activation was independent of increases in [Ca(2+)](i), and no detectable translocation to the membrane occurs. Nevertheless, stimulation of cPLA(2) activity was confined to the membrane fraction, where an increase in phosphorylation of the enzyme was observed. We suggest that, at the low agonist concentrations associated with oscillatory [Ca(2+)](i) signals, cPLA(2) activation involves an increased phosphorylation of a discrete pool of the total cellular cPLA(2) that is already localized within the membrane fraction at resting [Ca(2+)](i).

Arachidonic Acid↗
PubMed1999

Discriminating between capacitative and arachidonate-activated Ca(2+) entry pathways in HEK293 cells.

We have recently questioned whether the capacitative or store-operated model for receptor-activated Ca(2+) entry can account for the influx of Ca(2+) seen at low agonist concentrations, such a those typically producing [Ca(2+)](i) oscillations. Instead, we have identified an arachidonic acid-regulated, noncapacitative Ca(2+) entry mechanism that appears to be specifically responsible for the receptor-activated entry of Ca(2+) under these conditions. However, it is unclear whether these two systems reflect the activity of distinct entry pathways or simply different mechanisms of regulating a common pathway. We therefore used the known selectivity of the Ca(2+)-stimulated type VIII adenylyl cyclase for Ca(2+) entry occurring via the capacitative pathway (Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444) to attempt to discriminate between these two entry mechanisms in HEK293 cells. Consistent with the earlier reports, we found that thapsigargin induced an approximate 3-fold increase in adenylyl cyclase activity that was unrelated to global changes in [Ca(2+)](i) or to the release of Ca(2+) from internal stores but was specifically dependent on the induced capacitative entry of Ca(2+). In marked contrast, the arachidonate-induced entry of Ca(2+) completely failed to affect adenylyl cyclase activity despite producing a substantially greater rate of entry than that induced by thapsigargin. These data demonstrate that the arachidonate-activated entry of Ca(2+) occurs via an entirely distinct influx pathway.

Adenylyl Cyclases↗
PubMed1999

Vertebrate salt glands: short- and long-term regulation of function.

Excess salt loads in most non-mammalian vertebrates are dealt with by a variety of extra-renal salt-secreting structures collectively described as salt glands. The best studied of these are the supra-orbital nasal salt glands of birds. Two distinct types of response to osmoregulatory disturbances are shown by this structure: a progressive adaptive response on initial exposure to a salt load that results in the induction and enhancement of the secretory performance or capabilities of the gland; and the rapid activation of existing osmoregulatory mechanisms in the adapted gland in response to immediate osmoregulatory imbalance. Not only is the time-frame of these two types of response very different, but the responses usually involve fundamentally different processes: e.g., the growth and differentiation of osmoregulatory structures and their components in the former case, compared with the rapid activation of ion channels, pumps etc. in the latter. Despite marked differences in the nature and time-frame of these responses, they both are apparently triggered by neuronally released acetylcholine, which acts at muscarinic receptors on the secretory cells to induce an inositol phosphate-dependent increase in cytosolic-free calcium concentrations ([Ca2+]i). Therefore, the question arises as to how the cells produce the appropriate distinct response using a single common signal (i.e., an increase in [Ca2+]i). Examination of the features of this signaling pathway in the two conditions described, reveals that they each are uniquely tuned to generate a response with the characteristics appropriate for the cells' requirements. This tuning of the signal involves often rather subtle changes in the overall signaling pathway that are part of the adaptive differentiation process.

Acetylcholine↗
PubMed1999

What drives calcium entry during [Ca2+]i oscillations?--challenging the capacitative model.

An increased entry of Ca2+ across the plasma membrane plays a key role in the generation and maintenance of the [Ca2+]i signals seen in cells following activation of receptors coupled to the PLC/InsP3 signaling pathway. In recent years, considerable efforts have been made to define the nature and control of this agonist-enhanced Ca2+ entry. To date, these studies have largely focussed on the so-called 'capacitative' or store-operated model and, although many important details remain unclear, the critical role this mechanism plays in maintaining the sustained elevated 'plateau' type of [Ca2+]i response seen at high agonist concentrations is now well established. Far less well understood is the nature of the enhanced Ca2+ entry associated with the more complex [Ca2+]i signals typical of stimulation at more physiological levels of agonist. Where such entry has been considered, it too has generally been assumed to result from a capacitative or 'store-operated' mechanism. Significantly, however, direct evidence in support of this assumption is lacking. This review attempts to critically examine this assumption and presents the argument that several key characteristics of capacitative or store-operated mechanisms of agonist-activated Ca2+ entry are incompatible with its operation during these types of [Ca2+]i signal.

Animals↗
PubMed1998

Muscarinic receptor activation of arachidonate-mediated Ca2+ entry in HEK293 cells is independent of phospholipase C.

Receptor-enhanced entry of Ca2+ in non-excitable cells is generally ascribed to a capacitative mechanism in which the activation of the entry pathway is specifically dependent on the emptying of agonist-sensitive intracellular Ca2+ stores. Although such entry can be clearly demonstrated under conditions of maximal or near-maximal stimulation, it is uncertain whether such a mechanism can operate during the oscillatory [Ca2+]i signals that are frequently seen following stimulation with low concentrations of agonists. In this study, we report that the stimulation of human m3 muscarinic receptors stably transfected into HEK293 cells results in the appearance of a novel arachidonate-mediated Ca2+ entry pathway. We show that the generation of arachidonic acid and the activation of this pathway are specifically associated with stimulation at the low agonist concentrations that typically give rise to oscillatory [Ca2+]i signals. At such agonist concentrations, however, the generation of arachidonic acid is independent of the simultaneous activation of the phospholipase C-inositol 1,4,5-trisphosphate pathway. We further show that the arachidonate-mediated Ca2+ entry demonstrates characteristics that distinguish it from the corresponding capacitative pathway in the same cells and therefore is likely to represent an entirely distinct pathway that is specifically responsible for the receptor-enhanced entry of Ca2+ during [Ca2+]i oscillations.

Arachidonic Acid↗
PubMed1997

Intracellular Ca2+ signalling in secretory cells.

The secretion of ions and fluid plays a critical role in a variety of physiological activities that are vital to homeostatic mechanisms in animals. Control of such secretory activity is achieved by a range of neurotransmitters and hormones many of which act intracellularly by generating the second messenger inositol 1,4,5-trisphosphate (InsP3) and increasing cytosolic free calcium ion concentrations ([Ca2+]i). These increases are achieved by a combination of the InsP3-induced release of Ca2+ from specific intracellular stores and the activation of Ca2+ entry from the extracellular environment. The [Ca2+]i signal represents a balance between the adequate activation of components of the secretory mechanism and the avoidance of [Ca2+]i levels that are toxic to the cell. Resting [Ca2+]i is maintained low by the action of Ca2+ pumps on the intracellular stores and plasma membrane, with the result that gradients for Ca2+ movement into the cytosol from either of these two sources are very large and there is considerable potential for achieving rapid increases in [Ca2+]i. Consequently, for successful Ca2+ signalling, it is imperative that these two mechanisms of raising [Ca2+]i (i.e. Ca2+ release and Ca2+ entry) are closely integrated. Current models emphasize the activation of Ca2+ entry as a downstream result of the emptying of the intracellular stores ("capacitative' model). Whilst this may be true for situations of maximal stimulation, recent experiments on the oscillatory [Ca2+]i responses typical of more physiological levels of stimulation indicate a previously unsuspected, independent activation of Ca2+ entry involving arachidonic acid. This arachidonic-acid-activated entry plays a key role, along with InsP3, in inducing the repetitive release of Ca2+ from the stores to produce the [Ca2+]i oscillations. In this way, the two components responsible for the elevation of [Ca2+]i are intimately related and their dual effects closely coordinated, resulting in the finely tuned control of agonist-induced changes in [Ca2+]i.

Animals↗
PubMed1996

Arachidonic acid activates the noncapacitative entry of Ca2+ during [Ca2+]i oscillations.

Current models for agonist-activated Ca2+ entry in nonexcitable cells focus on the capacitative mechanism where entry is activated as a downstream result of the sustained depletion of agonist-sensitive stores without any direct requirement for inositol phosphates. This mechanism has been shown to be important for the sustained Ca2+ signals seen in a variety of nonexcitable cells under conditions of maximal stimulation. In contrast, relatively little attention has been given to Ca2+ entry under more physiological levels of agonist where, for example, oscillating Ca2+ responses are common. In recent studies using cells from the exocrine avian nasal gland, we have shown that agonist-activated Ca2+ entry under these conditions demonstrates properties that are inconsistent with current versions of the capacitative model. We now report that activation of this novel noncapacitative Ca2+ entry is via a distinct signaling pathway involving an agonist-induced, phospholipase A2-mediated generation of arachidonic acid.

Alkaloids↗
PubMed1996

Evidence for a non-capacitative Ca2+ entry during [Ca2+] oscillations.

Current models for the agonist-induced activation of Ca2+ entry from the extracellular medium in non-excitable cells generally emphasize a capacitative mechanism whereby Ca2+ entry is activated simply as a result of the emptying of intracellular Ca2+ stores, without any direct involvement of inositol phosphates. To date, the activation and control of Ca2+ entry have generally been studied under conditions where the agonist-sensitive stores undergo a profound and sustained depletion. However, responses under more normal physiological conditions typically involve the cyclical release and refilling of the stores associated with oscillations in [Ca2+], and the nature and control of entry under these conditions has received relatively little attention. In this study, using isolated cells from the exocrine avian nasal gland as a model system, we show that: (a) the agonist-enhanced rate of Mn2+ quench is independent of the cyclical emptying and refilling of the agonist-sensitive Ca2+ pool during oscillations; (b) the Ca2+ entry pathway is maintained in an activated state for extended periods following inhibition of oscillations under conditions in which agonist-sensitive stores can be shown to be full; (c) no Ca2+ entry could be detected in oscillating cells in experiments that followed a definitive protocol for the demonstration of capacitative entry; and (d) on initial exposure to low agonist concentrations, activation of Ca2+ entry preceded any detectable release of Ca2+ from the stores. We conclude that the essential characteristics of the control of Ca2+ entry during oscillations are incompatible with current capacitative models.

Animals↗
PubMed1996

Ca2+ entry modulates oscillation frequency by triggering Ca2+ release.

As in many cells, the frequency of agonist-induced cytosolic Ca2+ concentration ([Ca2+]1) oscillations in exocrine avian nasal gland cells is dependent on the rate of Ca2+ entry. Experiments reveal that the initiation of each oscillatory spike is independent of the relative fullness of the stores and, furthermore, the oscillating pool is normally fully refilled by the end of each [Ca2+]1 spike. Therefore, contrary to current models, the interspike interval (which essentially sets the frequency) does not reflect the time taken to recharge the oscillating stores. Instead, the data show that it is the previously demonstrated role that Ca2+ entry plays in triggering the repetitive release of Ca2+ from the oscillating stores, rather than the recharging of those stores, that provides the basis for the observed effects of Ca2+ entry rate on oscillation frequency.

Animals↗
PubMed1996

Clustered distribution of calcium sensitivities: an indication of hetero-tetrameric gating components in Ca2+-activated K+ channels reconstituted from avian nasal gland cells.

Calcium-activated potassium channels (maxi K+ channels) isolated from avian nasal salt gland cells were reconstituted into lipid bilayers and characterized. The 266 pS channel is blocked discretely by charybdotoxin from the external solution at nanomolar concentrations and by Ba2+ from the cytosolic side at micromolar concentrations. Fast tetraethylammonium (TEA) block is seen as apparent reductions in amplitude of the unitary currents. From the extent of the reductions, TEA binding affinity was calculated to be 0.16 mM from the external solution and 37 mm from internal solution. The overall channel properties conform to those of maxi K+ channels in other epithelial tissues. The calcium sensitivity of the channel was found to be variable from channel to channel, extending over a wide range of concentrations from 1 to 1,000 microM. Examination of the pooled calcium titration curves, revealed that these curves are grouped into five clusters, and the probability distribution of the clusters matches a binomial distribution. The Hill coefficient derived from the titration curves varies from 1 to 5 and is linearly correlated to calcium binding with a slope of 1 per 10-fold change in Kd. Clustered titration curves with such a characteristic suggest that the gating components and the calcium binding sites of the maxi K+ channels in the avian nasal gland are hetero-tetrameric and may result from random mixing of two distinct subunits possessing high and low calcium sensitivities, respectively.

Animals↗
PubMed1995

A re-evaluation of the apparent effects of luminal Ca2+ on inositol 1,4,5-trisphosphate-induced Ca2+ release.

It has been suggested that the release of Ca2+ from intracellular stores by inositol 1,4,5-trisphosphate (InsP3) is modulated by the luminal Ca2+ content of the stores and that such an effect could underlie the apparent 'quantal' nature of InsP3-induced release. Although initial studies failed to find evidence in support of such a modulation, several subsequent reports have indicated luminal Ca2+ effects that become apparent only after a greater than 70-75% depletion of Ca2+ stores. In these studies, Ca2+ release was expressed as a percentage of an A23187-releasable pool which comprised both InsP3-sensitive and InsP3-insensitive components. In model calculations we have found that the presence of even a minor InsP3-insensitive component in the total Ca2+ pool significantly distorts interpretation of the data. We show that the published results can be accurately duplicated without any requirement for a shift in the true InsP3 sensitivity of Ca2+ release if either: (a) the InsP3-insensitive component does not remain a constant proportion of the total pool during depletion (i.e. depletion disproportionally affects the InsP3-sensitive component); or (b) during generation of InsP3-response curves, additional Ca2+ is released from the InsP3-insensitive component as the InsP3-sensitive component is progressively emptied. Examination indicates that either, or both, of these conditions apply in the published reports and we conclude that the demonstrated effects of luminal Ca2+ may be artifacts.

Calcium↗
PubMed1995

Activation by ATP of a P2U 'nucleotide' receptor in an exocrine cell.

1. We employed the perforated patch whole-cell technique to investigate the effects of ATP and other related nucleotides on membrane conductances in avian exocrine salt gland cells. 2. ATP (10 microM-1 mM) evoked an increase in maxi-K+ and Cl- conductances with a reversal potential of -35 mV. At lower concentrations of ATP (< or = 100 microM) responses were generally oscillatory with a sustained response observed at higher concentrations (> or = 200 microM). 3. Both oscillatory and sustained responses were abolished by the removal of bath Ca2+. In cells preincubated in extracellular saline containing reduced Ca2+, the application of ATP resulted in a transient increase in current. 4. As increasing concentrations of ATP (and related nucleotides) evoked a graded sequence of events with little run-down we were able to establish a rank order of potency in single cells. The order of potency of ATP analogues and agonists of the various P2-receptor subtypes was UTP > ATP = 2-methylthio-ATP > ADP. Adenosine (1 microM-1 mM), AMP (1 microM-1 mM), alpha,beta-methylene-ATP (1 microM-1 mM) and beta,gamma-methylene-ATP (1 microM-1 mM) were without effect. 5. In conclusion, although unable to preclude a role for a P2Y-receptor, our results suggest that ATP binds to a P2U-receptor increasing [Ca2+]i and subsequently activating Ca(2+)-sensitive K+ and Cl- currents.

Adenosine↗
PubMed1994

Ca2+ influx drives agonist-activated [Ca2+]i oscillations in an exocrine cell.

In current models describing agonist-induced oscillations in [Ca2+]i, Ca2+ entry is generally assumed to have a simple sustaining role, replenishing Ca2+ lost from the cell and recharging intracellular Ca2+ stores. In cells from the avian nasal gland, a model exocrine cell, we show that inhibition of Ca2+ entry by La3+, SK&F 96365, or by membrane depolarization, rapidly blocks [Ca2+]i oscillations but does so without detectable depletion of agonist-sensitive Ca2+ stores. As the rate of Mn2+ quenching during [Ca2+]i oscillations is constant, Ca2+ entry is not directly contributing to the [Ca2+]i changes and, instead, appears to be involved in inducing the repetitive release of Ca2+ from internal stores. Together, these data contradict current models in that (i) at the low agonist concentrations where [Ca2+]i oscillations are seen, generated levels of Ins(1,4,5)P3 are themselves inadequate to result in a regenerative [Ca2+]i signal, and (ii) Ca2+ entry is necessary to actually drive the intrinsic oscillatory mechanism.

Animals↗
PubMed1994

Vasoactive intestinal peptide stimulates a cAMP-mediated Cl- current in avian salt gland cells.

VIP plays an integral role in both protein and fluid secretion in many exocrine glands. By employing the perforated patch-clamp whole-cell recording technique we investigated the effects of VIP on membrane potential and transmembrane currents in avian exocrine salt gland cells. Prior to application of VIP, salt gland cells had a resting membrane potential close to -45 mV. When challenged with VIP (1-100 nM) a sustained depolarization to ECl- was induced which was mimicked by the application of cell-permeable cAMP analogues or forskolin (1 microM). By employing the voltage-clamp recording configuration a sustained increase in current was observed with a reversal potential which approximated ECl-. Ionic substitution experiments confirmed that the current was a Cl- conductance which was inhibited by the Cl- channel blockers flufenamic acid and niflumic acid and by the inhibitory cAMP isomer, adenosine-3',5'-cyclic monophosphothioate, Rp-isomer. Based on this, and the fact that the kinetic properties of the Cl- current activated by VIP are similar to those activated by cAMP, we propose that VIP-receptor interaction results in the activation of a cAMP-dependent Cl- current.

Animals↗
PubMed1994

Muscarinic-receptor activation stimulates oscillations in K+ and Cl- currents which are acutely dependent on extracellular Ca2+ in avian salt gland cells.

By utilizing the perforated-patch variant of the whole-cell patch-clamp recording technique, in order to maintain the integrity of the normal cellular buffering systems, we demonstrate that carbachol (CCh) stimulates simultaneous oscillations in a Ca(2+)- and voltage-activated K+ current and a linear Ca(2+)-activated Cl- current in an exocrine avian salt gland cell preparation. Similar conductance changes, although sustained rather than oscillatory, are stimulated by the Ca2+ ionophore A23187. The outward K+ current can be inhibited by tetraethylammonium chloride (TEA) whereas the Cl- current is inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino) (NPPB) and N-phenylanthranilic acid (DPC). The oscillations in current stimulated by CCh are acutely dependent on extracellular Ca2+ and are not affected by the application of low doses of caffeine. In addition, the application of caffeine at all doses fails to mimic the current transients stimulated by CCh. As both caffeine and A23187 are unable to stimulate oscillations under the perforated-patch conditions we suggest that in avian salt gland cells the primary oscillatory mechanism probably involves a one-pool mechanism of Ca2+ release which is intimately related to the activation of a Ca2+ influx pathway.

Animals↗
PubMed1994

Temporal relationships between Ca2+ store mobilization and Ca2+ entry in an exocrine cell.

Consideration of the principal current models for agonist-induced activation of Ca2+ entry in electrically non-excitable cells suggests that it may be possible to distinguish between them on the basis of predicted differences in the temporal relationship(s) between intracellular Ca2+ release and the activation of Ca2+ entry. Measurements of changes in [Ca2+]i and Mn2+ quench in individual exocrine cells from the avian nasal gland indicate that, whereas Ins(1,4,5)P3-induced release of intracellular Ca2+ occurs within 3-5 s, the increase in Mn2+ quench is delayed by some 20-30 s. Mn2+ quench rate is similarly increased by thapsigargin, and is blocked by SK&F 96365, indicating that the increased Mn2+ quench observed genuinely reflects agonist-enhanced activity of the divalent cation entry pathway normally traversed by Ca2+. Additional experiments indicate that the observed delay is not due to inhibition of this pathway by elevated [Ca2+]i. Furthermore, the delay cannot be explained by the time required for Ins(1,3,4,5)P4 generation, which is essentially maximal within 10 s of agonist addition. It is concluded that the observed delay in the activation of the Ca2+ entry pathway is best explained by 'capacitative' models where increased entry requires the generation, and transmission to the plasma membrane, of an unknown messenger as a direct result of the depletion of intracellular Ca2+ stores.

Animals↗