Stewart O. Sage

ACTIVATION OF RECEPTOR-OPERATED CATION CHANNELS VIA P2X1 NOT P2T PURINOCEPTORS IN HUMAN PLATELETS

We have investigated the purinoceptor subtypes responsible for calcium signaling in human platelets, which previous studies have shown to involve both Ca influx via receptor-operated cation channels and release of Ca from intracellular stores. Fura-2 measurements of [Ca] in stirred platelet suspensions showed that both ADP (40 μM) and the non-hydrolyzable ATP analogue αβ-meATP (α,β-methyleneadenosine 5′-triphosphate, 10 μM) activated a rapid Ca influx whereas only ADP mobilized Ca from internal stores. In “nystatin” whole-cell patch clamp recordings, ATP, ADP, and the non-hydrolyzable ATP analogues, α,β-meATP and ATPS (adenosine 5′-O-(3-thiotriphosphate), all activated a cation channel permeable to both monovalent and divalent cations with a single-channel conductance of 11 picosiemens in NaCl saline. The current response to ATP (40 μM) was activated within 20 ms and desensitized with a time constant of 47-107 ms in the continued presence of agonist, which are characteristics of P receptors in other tissues. We conclude that human platelets possess a P purinoceptor, which mediates a rapid phase of ADP- or ATP-evoked Ca entry via a cation channel, whereas one or more separate ADP-selective P purinoceptors evoke release of calcium from intracellular stores.

Coupling between inositol 1,4,5-trisphosphate receptors and human transient receptor potential channel 1 when intracellular Ca2+ stores are depleted

In the present study we have investigated the role of inositol 1,4, 5-trisphosphate (IP(3)), functional IP(3) receptors (IP(3)Rs) and the human homologue of the Drosophila transient receptor potential (Trp) channel, human Trp1 (hTrp1), in store-mediated Ca(2+) entry (SMCE) in human platelets. Inhibition of IP(3) recycling using Li(+), or the inhibition of IP(3)Rs using xestospongin C, both resulted in the inhibition of SMCE activation following Ca(2+) store depletion using thapsigargin. Co-immunoprecipitation experiments indicated that endogenously expressed hTrp1 couples with IP(3)R type II, but not types I or III, in platelets with depleted intracellular Ca(2+) stores, but not in control, undepleted cells. These results provide strong evidence for the activation of SMCE by conformational coupling involving de novo association between IP(3)Rs and a plasma membrane channel in normal human cells.

Endogenously Expressed Trp1 Is Involved in Store-mediated Ca2+ Entry by Conformational Coupling in Human Platelets

Physical interaction between transient receptor potential (Trp) channels and inositol 1,4,5-trisphosphate receptors (IP3Rs) has been presented as a candidate mechanism for the activation of store-mediated Ca2+ entry. The role of a human homologue of Drosophila transient receptor potential channel, hTrp1, in the conduction of store-mediated Ca2+ entry was examined in human platelets. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 557–571 of hTrp1, inhibited both store depletion-induced Ca2+ and Mn2+ entry in a concentration-dependent manner. Stimulation of platelets with the physiological agonist thrombin activated coupling between the IP3 receptor type II and endogenously expressed hTrp1. This event was reversed by refilling of the internal Ca2+ stores but maintained after removal of the agonist if the stores were not allowed to refill. Inhibition of IP3 recycling using Li+ or inhibition of IP3Rs with xestospongin C or treatment with jasplakinolide, to stabilize the cortical actin filament network, abolished thrombin-induced coupling between hTrp1 and IP3R type II. Incubation with the anti-hTrp1 antibody inhibited thrombin-evoked Ca2+ entry without affecting Ca2+ release from intracellular stores. These results provide evidence for the involvement of hTrp1 in the activation of store-mediated Ca2+ entry by coupling to IP3R type II in normal human cells.

The actin cytoskeleton in store-mediated calcium entry

Store‐mediated Ca2+ entry is the main pathway for Ca2+ influx in platelets and many other cells. Several hypotheses have considered both direct and indirect coupling mechanisms between the endoplasmic reticulum and the plasma membrane. Here we pay particular attention to new insights into the regulation of store‐mediated Ca2+ entry: the role of the cytoskeleton in a secretion‐like coupling model. In this model, Ca2+ entry may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, that shows close parallels to the events mediating secretion. As with secretion, the actin cytoskeleton plays an inhibitory role in the activation of Ca2+ entry by preventing the approach and coupling of the endoplasmic reticulum with the plasma membrane, making cytoskeletal remodelling a key event in the activation of Ca2+ entry. We also review recent advances investigating the regulation of store‐mediated Ca2+ entry by small GTPases and phosphoinositides, which might be involved in the store‐mediated Ca2+ entry pathway through roles in the remodelling of the cytoskeleton.

Calcium‐activated potassium channels in the endothelium of intact rat aorta.

1. Single K+ channel currents and membrane potential were recorded in the endothelium of excised intact rat aorta. 2. Two types of K+ channel were found in excised patches, KCh and KAp. With Na+ and K+ as the main external and internal cations, outward conductances were 6.7 pS (KCh) and 2.8 pS (KAp). In symmetric 150 mM K+, the inward conductances were 18 and 9.1 pS. 3. Activation by Ca2+ was concentration dependent. KCh channels were activated by [Ca2+] > 0.1 microM and KAp by [Ca2+] > 0.5 microM. 4. Apamin at concentrations > 1 nM inhibited KAp Channels. Block was complete at 10 nM. KAp channels were insensitive to charybdotoxin. KCh channels were inhibited by charybdotoxin at concentrations > 50 nM, but were insensitive to apamin. 5. d‐Tubocurarine (dTC) evoked flickering activity of KAp channels at concentrations > 5 microM and complete block at 100 microM. At these doses, dTC did not affect KCh channels, but at concentrations > 1 mM it decreased the single channel amplitude. 6. Hyperpolarization evoked by acetylcholine was unaffected by apamin or dTC at low concentrations ( < or = 100 microM), but inhibited by high concentrations of charybdotoxin ( > 50 nM) or dTC ( > 1 mM). 7. These data suggest that KCh channels are novel Ca(2+)‐activated K+ channels responsible for the ACh‐evoked hyperpolarization in the endothelium of rat aorta.

A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets.

Store-mediated Ca(2+) entry (SMCE) is a major pathway for Ca(2+) influx in many cells, yet how depletion of the intracellular Ca(2+) stores leads to the activation of Ca(2+) entry across the plasma membrane is not well understood. Recent work in platelets favors a secretion-like conformational coupling mechanism involving proteins in the plasma membrane (PM) and in the membrane of the Ca(2+) store, located in the endoplasmic reticulum (ER). The activation and maintenance of SMCE in platelets has been shown to depend on remodeling of the actin cytoskeleton, which may be required to allow trafficking of the ER toward the PM to permit coupling to occur and to stabilize this coupling once achieved. The coupling itself has been shown to involve one isoform of the inositol 1,4,5-trisphosphate receptor (IP(3)RII) and the Ca(2+)-permeable channel protein, human Trp1 (hTrp1).

The tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and genistein reduce thrombin-evoked tyrosine phosphorylation and Ca2+ entry in human platelets

Platelet activation is associated with the phosphorylation of a number of platelet proteins at tyrosine residues. The significance of this is unknown. Here we have investigated the effects of two tyrosine kinase inhibitors, methyl 2,5‐dihydroxycinnamate and genistein, on thrombin‐evoked protein tyrosine phosphorylation and Ca2+ signal generation in fura‐2‐loaded human platelets. Both compounds inhibited thrombin‐evoked tyrosine phosphorylation and reduced the elevation of [Ca2+], in the presence, but not the absence, of external Ca2+. This suggested a selective inhibition of thrombin‐evoked Ca2+ entry but not release from internal stores. Both compounds also reduced thrombin‐evoked Mn2+ entry. In contrast, selective blockade of protein kinase C with Ro 31/8220‐002 potentiated the thrombin‐evoked Ca2+ signal. These data are compatible with a role for protein tyrosine phosphorylation contributing to thrombin‐evoked Ca2+ entry in human platelets.

Activation of store-mediated calcium entry by secretion-like coupling between the inositol 1,4,5-trisphosphate receptor type II and human transient receptor potential (hTrp1) channels in human platelets.

Physical coupling between inositol 1,4,5-trisphosphate (IP(3)) receptors and transient receptor potential (Trp) channels has been demonstrated in both transfected and normal cells as a candidate mechanism for the activation of store-mediated Ca(2+) entry (SMCE). We have investigated the properties of the coupling between the type II IP(3) receptor and naturally expressed human Trp1 (hTrp1) in human platelets. Treatment with xestospongin C, an inhibitor of IP(3) receptor function, abolished SMCE and coupling between the IP(3) receptor and hTrp1. The coupling was activated by depletion of the intracellular Ca(2+) stores, and was reversed by refilling of the stores. We have also examined the role of actin filaments in the activation and maintenance of the coupling. Stabilization of the cortical actin network with jasplakinolide prevented the coupling, indicating that, as with secretion, the actin filaments at the cell periphery act as a negative clamp which prevents constitutive coupling. In addition, the actin cytoskeleton plays a positive role, since disruption of the actin network inhibited the coupling when the Ca(2+) stores were depleted. These results provide strong evidence for the activation of SMCE by a secretion-like coupling mechanism involving a reversible association between IP(3) receptors and hTrp1 in normal human cells.

Shedding of procoagulant microparticles from unstimulated platelets by integrin-mediated destabilization of actin cytoskeleton

Platelet activation by potent, Ca2+‐mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from αIIbβ3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin‐mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo.

TRPC channels and store-operated Ca2+ entry

Store-operated calcium entry (SOCE) is a major mechanism for Ca(2+) influx. Since SOCE was first proposed two decades ago many techniques have been used in attempting to identify the nature of store-operated Ca(2+) (SOC) channels. The first identified and best-characterised store-operated current is I(CRAC), but a number of other currents activated by Ca(2+) store depletion have also been described. TRPC proteins have long been proposed as SOC channel candidates; however, whether any of the TRPCs function as SOC channels remains controversial. This review attempts to provide an overview of the arguments in favour and against the role of TRPC proteins in the store-operated mechanisms of agonist-activated Ca(2+) entry.