Biomedical subjects

Colin W Taylor

Publications and source records attributed to Colin W Taylor.

12 recordsLinked to original sources

PubMed2003

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis.

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a tetrameric intracellular Ca(2+) channel, which mediates the release of Ca(2+) from the endoplasmic reticulum in response to many different extracellular stimuli. We present a 3D structure of the type 1 IP(3)R obtained by electron microscopy and single-particle analysis that reveals its domain organization. The IP(3)R has a flower-like appearance with fourfold symmetry and is made up of three distinct domains connected by slender links. By relating the organization of the structural domains to secondary-structure predictions and biochemical data we develop a model in which structural domains are mapped onto the amino acid sequence to deduce the location of the channel region and the cytoplasmic inositol 1,4,5-trisphosphate-binding and modulatory subdomains. The structure of the IP(3)R is compared with that of other tetrameric cation channels. The channel domain is similar in size and shape to its counterparts in the ryanodine receptor and the Shaker voltage-gated K(+) channel.

Animals↗

PubMed2003

Nitric oxide co-ordinates the activities of the capacitative and non-capacitative Ca2+-entry pathways regulated by vasopressin.

In A7r5 vascular smooth muscle cells vasopressin, via arachidonic acid, regulates two Ca(2+)-entry pathways. Capacitative Ca(2+) entry (CCE), activated by empty Ca(2+) stores, is inhibited by arachidonic acid, and non-capacitative Ca(2+) entry (NCCE) is stimulated by it. This reciprocal regulation ensures that all Ca(2+) entry is via NCCE in the presence of vasopressin, while CCE mediates a transient Ca(2+) entry only after removal of vasopressin. We demonstrate that type III NO synthase (NOS III) is expressed in A7r5 cells and that NO inhibits CCE. Inhibition of CCE by vasopressin requires NOS III and the requirement lies downstream of arachidonic acid. Activation of soluble guanylate cyclase by NO and subsequent activation of protein kinase G are required for inhibition of CCE. Stimulation of NCCE by vasopressin also requires NOS III, but the stimulation is neither mimicked by cGMP nor blocked by inhibitors of soluble guanylate cyclase or protein kinase G. We conclude that arachidonic acid formed in response to vasopressin stimulates NOS III. NO then directly stimulates Ca(2+) entry through NCCE and, via protein kinase G, it inhibits CCE. The additional amplification provided by the involvement of guanylate cyclase and protein kinase G ensures that CCE will always be inhibited when vasopressin activates NCCE.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed2003

Parathyroid hormone increases the sensitivity of inositol trisphosphate receptors by a mechanism that is independent of cyclic AMP.

1 In fura 2-loaded HEK-293 cells stably expressing human type 1 parathyroid hormone (PTH) receptors, PTH potentiated the Ca(2+) mobilization evoked by carbachol by >4 fold without itself increasing the intracellular [Ca(2+)]. 2 PTH potentiated the Ca(2+) release evoked by a cell-permeant analogue of inositol 1,4,5-trisphosphate (InsP(3)BM). 3 Prolonged incubation with InsP(3)BM emptied the Ca(2+) stores as effectively as PTH in combination with a maximal concentration of carbachol, indicating that PTH did not increase the size of the InsP(3)-sensitive Ca(2+) pool. 4 Responses to PTH were unaffected by disruption of the cytoskeleton. 5 The EC(50) for carbachol-evoked Ca(2+) release and InsP(3) formation were indistinguishable (approximately 40 microM), consistent with even the highest concentrations of carbachol generating insufficient InsP(3) to release the entire InsP(3)-sensitive Ca(2+) pool. 6 Inhibition of cyclic AMP-dependent protein kinase A (PKA), using H89 or CMIQ, did not affect potentiation of carbachol-evoked Ca(2+) signals by PTH. 7 SQ22536 or DDA, inhibitors of adenylyl cyclase, inhibited PTH-evoked cyclic AMP formation and IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, increased the amount of cyclic AMP detected after stimulation by PTH. None of these drugs affected the potentiation of Ca(2+) signals by maximal or submaximal concentrations of PTH. 8 We conclude that PTH potentiates the Ca(2+) release evoked by receptors that stimulate InsP(3) formation by sensitizing InsP(3) receptors through a cyclic AMP-independent mechanism.

Calcium↗

PubMed2002

Controlling calcium entry.

Ca(2+) enters cells through an assortment of Ca(2+)-permeable channels that respond to different stimuli and couple to different cellular responses. Several different Ca(2+) entry pathways can be activated by receptors that stimulate phospholipase C (PLC). Both limbs of this signaling pathway (IP(3) and diacylglycerol), PLC itself, and its substrate (PIP(2)) contribute to the coordinate regulation of these Ca(2+) entry pathways.

Animals↗

PubMed2002

Modulation of IP(3)-sensitive Ca(2+) release by 2,3-butanedione monoxime.

We describe the actions of 2,3-butanedione monoxime (BDM) on calcium responses in secretory cells. Our studies were prompted by the widespread use of BDM as a myosin-ATPase inhibitor. Application of 10 mM BDM almost completely inhibited agonist-evoked amylase secretion from mouse pancreatic acinar cells. This action might be interpreted as indicating a role for myosin in secretion. However, BDM alone elicited a calcium response in single cells and this calcium signal was sufficient to activate calcium-dependent chloride currents. Furthermore, in some cases, BDM potentiated agonist-evoked calcium signals but almost always blocked agonist-evoked calcium oscillations. These effects of BDM were not due to an action on calcium influx pathways but rather to direct effects on IP(3)-sensitive stores. We conclude that BDM cannot be used for unequivocal identification of the involvement of myosin motors in a cellular response. Further, our evidence suggests that BDM can act directly to modify the opening of IP(3) receptors.

Acetylcholine↗

PubMed2002

Determinants of adenophostin A binding to inositol trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP(3)) receptors from cerebellum and recombinant type 1 IP(3) receptors expressed in Sf9 cells had indistinguishable affinities for IP(3) ( K (d)=6.40+/-0.48 nM) and adenophostin A ( K (d)=0.89+/-0.05 nM). In cytosol-like medium, each of the three mammalian IP(3) receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP(3). It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [(3)H]IP(3) from type 1 IP(3) receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [(3)H]IP(3) binding to the 1-604 protein, but abolished binding to the 224-604 protein. The 1-604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP(3), suggesting that C-terminal residues selectively inhibit IP(3) binding. The 224-604S1(+) fragment bound IP(3) and adenophostin A with increased affinity, but as with the 1-604 fragment it bound adenophostin A with only 2-fold greater affinity than IP(3). High-affinity binding of adenophostin A may be partially determined by its 2'-phosphate interacting more effectively than the 1-phosphate of IP(3) with residues within the IP(3)-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP(3) for the minimal IP(3)-binding domain. In addition we suggest that C-terminal residues, which impede access of IP(3), may selectively interact with adenophostin A to allow it unhindered access to the IP(3)-binding domain.

Adenosine↗

PubMed2002

Interactions of inositol 1,4,5-trisphosphate (IP(3)) receptors with synthetic poly(ethylene glycol)-linked dimers of IP(3) suggest close spacing of the IP(3)-binding sites.

The distances between the inositol 1,4,5-trisphosphate (IP(3))-binding sites of tetrameric IP(3) receptors were probed using dimers of IP(3) linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated (45)Ca(2+) release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP(3) receptors. The shortest dimers, with PEG linkers of an effective length of 1.5 nm or less, were the most potent, being 3-4-fold more potent than IP(3). In radioligand binding experiments using cerebellar membranes, the shortest dimers bound with highest affinity, although the longest dimer (8 nm) also bound with almost 4-fold greater affinity than IP(3). The affinity of monomeric IP(3) with only the PEG attached was 2-fold weaker than IP(3), confirming that the increased affinity of the dimers requires the presence of both IP(3) motifs. The increased affinity of the long dimer probably results from the linked IP(3) molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP(3) to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP(3) to purified receptors. IP(3) and the IP(3) dimers, irrespective of their length, bound with similar affinity to a monomeric IP(3)-binding domain of the type 1 IP(3) receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP(3)-binding sites of an IP(3) receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 x 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP(3)-binding sites of the IP(3) receptor.

Animals↗

PubMed2002

Fast biphasic regulation of type 3 inositol trisphosphate receptors by cytosolic calcium.

In cytosol-like medium (CLM) with a free [Ca(2+)] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP(3)) (30 microm) evoked (45)Ca(2+) release from type 3 IP(3) receptors only after a latency of 48 +/- 6 ms; this latency could not be reduced by increasing the IP(3) concentration. In CLM containing a low free [Ca(2+)] ( approximately 4 nm), 300 microm IP(3) evoked (45)Ca(2+) release after a latency of 66 +/- 11 ms; this was reduced to 14 +/- 3 ms when the [Ca(2+)] was 1 mm. Preincubation with CLM containing 100 microm Ca(2+) caused a rapid (half-time = 33 +/- 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP(3) (300 microm). Hepatic (type 2) IP(3) receptors were not inhibited by Ca(2+) once they had bound IP(3), but 100 microm Ca(2+) rapidly inhibited type 3 IP(3) receptors whether it was delivered before addition of IP(3) or at any stage during a response to IP(3). Ca(2+) increases the affinity of IP(3) for hepatic receptors by slowing IP(3) dissociation, but Ca(2+) had no effect on IP(3) binding to type 3 receptors. The rate of inhibition of type 3 IP(3) receptors by Ca(2+) was faster than the rate of IP(3) dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP(3)) agonist. Dissociation of agonist is not therefore required for Ca(2+) to inhibit type 3 IP(3) receptors. We conclude that type 2 and 3 IP(3) receptors are each biphasically regulated by Ca(2+), but by different mechanisms. For both, IP(3) binding causes a stimulatory Ca(2+)-binding site to be exposed allowing Ca(2+) to bind and open the channel. IP(3) binding protects type 2 receptors from Ca(2+) inhibition, but type 3 receptors are inhibited by Ca(2+) whether or not they have IP(3) bound. Increases in cytosolic [Ca(2+)] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP(3) has dissociated.

Animals↗

PubMed2002

Reciprocal regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells: only the latter operates during receptor activation.

In A7r5 vascular smooth muscle cells, Arg(8)-vasopressin (AVP) stimulates phospholipase C leading to activation of two distinct Ca(2+) entry pathways. The capacitative Ca(2+) entry (CCE) pathway is activated by depletion of Ca(2+) stores, is permeable to Mn(2+), Ba(2+) and Ca(2+), and is selectively blocked by Gd(3+)(1 microM). A7r5 cells also express a non-capacitative Ca(2+) entry (NCCE) pathway, which is activated by arachidonic acid that is released by the sequential activities of phospholipase C and diacylglycerol lipase. This pathway is permeable to Sr(2+), Ba(2+) and Ca(2+) and selectively blocked by (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate ("LOE-908"). We use these selective tools to show that AVP, via the same signalling pathway that leads to activation of NCCE, also inhibits CCE and that the inhibition is not due to depolarization of the plasma membrane. Using the selective inhibitors to resolve the contributions of each Ca(2+) entry pathway during stimulation with AVP, we establish that reciprocal regulation of CCE and NCCE by arachidonic acid ensures that only NCCE is active in the presence of AVP, whereas CCE is active only after its removal. NCCE and CCE are therefore activated in a strict temporal sequence: NCCE first and then CCE. Because Ca(2+) passing through different Ca(2+) entry pathways can selectively regulate different responses, reciprocal regulation of CCE and NCCE may allow a stimulus to first evoke a response and then recruit actively a different response when the stimulus is removed.

Amino Acid Sequence↗

PubMed2002

A novel Ca2+-induced Ca2+ release mechanism mediated by neither inositol trisphosphate nor ryanodine receptors.

Members of both major families of intracellular Ca(2+) channels, ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, are stimulated by substantial increases in cytosolic free Ca(2+) concentration ([Ca(2+)]c). They thereby mediate Ca(2+)-induced Ca(2+) release (CICR), which allows amplification and regenerative propagation of intracellular Ca(2+) signals. In permeabilized hepatocytes, increasing [Ca(2+)]c to 10 microM stimulated release of 30+/-1% of the intracellular stores within 60 s; the EC(50) occurred with a free [Ca(2+)] of 170+/-29 nM. This CICR was abolished at 2 degrees C. The same fraction of the stores was released by CICR before and after depletion of the IP3-sensitive stores, and CICR was not blocked by antagonists of IP3 receptors. Ryanodine, Ruthenium Red and tetracaine affected neither the Ca(2+) content of the stores nor the CICR response. Sr(2+) and Ba(2+) (EC(50)=166 nM and 28 microM respectively) mimicked the effects of increased [Ca(2+)] on the intracellular stores, but Ni(2+) blocked the passive leak of Ca(2+) without blocking CICR. In rapid superfusion experiments, maximal concentrations of IP3 or Ca(2+) stimulated Ca(2+) release within 80 ms. The response to IP3 was complete within 2 s, but CICR continued for tens of seconds despite a slow [half-time (t(1/2))=3.54+/-0.07 s] partial inactivation. CICR reversed rapidly (t(1/2)=529+/-17 ms) and completely when the [Ca(2+)] was reduced. We conclude that hepatocytes express a novel temperature-sensitive, ATP-independent CICR mechanism that is reversibly activated by modest increases in [Ca(2+)], and does not require IP3 or ryanodine receptors or reversal of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase. This mechanism may both regulate the Ca(2+) content of the intracellular stores of unstimulated cells and allow even small intracellular Ca(2+) signals to be amplified by CICR.

Adenosine Triphosphate↗

PubMed2002

Regulation of Ca2+ entry pathways by both limbs of the phosphoinositide pathway.

All inositol 1,4,5-trisphosphate (InsP3) receptors are biphasically regulated by cytosolic Ca2+. For type 2 InsP3 receptors, InsP3 binding controls whether a stimulatory Ca2+-binding site (exposed after InsP3 binding) or an inhibitory Ca2+-binding site (exposed only in the absence of InsP3) is accessible. Ca2+ therefore inhibits these InsP3 receptors only after InsP3 has dissociated. The capacitative Ca2+ entry (CCE) pathway is activated by depletion of Ca2+ stores, but the local increase in cytosolic [Ca2+] as Ca2+ flows through these channels could cause long-lasting inhibition of InsP3 receptors and so termination of the signal that activates CCE. However, the duration of the openings of CCE channels is matched to the behaviour of InsP3 receptors such that during the brief openings of CCE channels, active InsP3 receptors are unlikely to lose enough InsP3 for them to become inhibited. In A7r5 vascular smooth muscle cells, CCE and a non-capacitative Ca2+ entry (NCCE) pathway, which is activated by arachidonic acid released from diacylglycerol by diacylglycerol lipase, can be distinguished by their different permeation properties and sensitivity to selective blockers. Arachidonic acid also inhibits CCE and so ensures that during receptor activation only the NCCE pathway mediates Ca2+ entry, while CCE contributes only after removal of the agonist.

Animals↗

PubMed2001

Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore.

We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch clamping to record cytosolic Ca(2+) and plasma membrane Ca(2+)-dependent Cl(-) currents, we find that paclitaxel abolishes cytosolic Ca(2+) oscillations and in more than half of the cells it also induces a rapid, transient cytosolic Ca(2+) response. This response is not affected by removal of extracellular Ca(2+) indicating that paclitaxel releases Ca(2+) from an intracellular Ca(2+) store. Using saponin-permeabilized cells, we show that paclitaxel does not affect Ca(2+) release from an inositol trisphosphate-sensitive store. Furthermore, up to 15 min after paclitaxel application, there is no significant effect on either microtubule organization or on endoplasmic reticulum organization. The data suggest a non-endoplasmic reticulum source for the intracellular Ca(2+) response. Using the mitochondrial fluorescent dyes, JC-1 and Rhod-2, we show that paclitaxel evoked a rapid decline in the mitochondrial membrane potential and a loss of mitochondrial Ca(2+). Cyclosporin A, a blocker of the mitochondrial permeability transition pore, blocked both the paclitaxel-induced loss of mitochondrial Ca(2+) and the effect on Ca(2+) spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca(2+) signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca(2+) signal cascade.

Animals↗