Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.     

Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.     

Adenosine Receptors Modulate [Ca2+]i in Hippocampal Astrocytes In Situ

Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca²⁺]_i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca²⁺]_i that were blocked by the adenosine receptor antagonist 8-( p -sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P_2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca²⁺]_i, whereas the P₂ agonist adenosine 5'- O -(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P₁ purinergic receptors coupled to increases in [Ca²⁺]_i, whereas a small minority appear to contain P₂ purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca²⁺]_i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.     

Mitochondrial calcium transport is regulated by P2Y1- and P2Y2-like mitochondrial receptors

Ischemia-reperfusion injury remains a major clinical problem in liver transplantation. One contributing factor is mitochondrial calcium (mCa(2+)) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5'-triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y(1)- and P2Y(2)-like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y(1) and P2Y(2) receptors ligands: 2-methylthio-adenosine 5'-diphosphate (2meSADP) and uridine 5'-triphosphate (UTP), respectively, were used, and mitochondrial calcium uptake was measured. 2meSADP and UTP had a maximum effect at concentrations in the range of the known P2Y(1) and P2Y(2) receptors. The P2Y inhibitor phosphate-6-azophenyl-2',4'-disulfonate (PPADS) blocked the effects of both ligands. The phospholipase C (PLC) antagonist U73122 inhibited the effect of both ligands while its inactive analog U73343 had no effect. These data strongly support the hypothesis that mitochondrial Ca(2+) uptake is regulated in part by adenine nucleotides via a P2Y-like receptor mechanism that involves mitochondrial PLC activation.     

Purinergic signalling and intercellular Ca2+ wave propagation in the organ of Corti

Extracellular ATP is a key neuromodulator of visual and auditory sensory epithelia. In the rat cochlea, pharmacological dissection indicates that ATP, acting through a highly sensitive purinergic/IP(3)-mediated signaling pathway with (little or) no involvement of ryanodine receptors, is the principal paracrine mediator implicated in the propagation of calcium waves through supporting and epithelial cells. Measurement of sensitivity to UTP and other purinergic agonists implicate P2Y(2) and P2Y(4) as the main P2Y receptor isoforms involved in these responses. Ca2+ waves, elicited under highly reproducible conditions by carefully controlling dose (1 microM) and timing of focal agonist application (0.2s), extended over radial distance greater than 160 microm from the source, identical to those activated by damaging single outer hair cells. Altogether, these results indicate that intercellular calcium waves are a robust phenomenon that confers a significant ability for cell-cell communication in the mammalian cochlea. Further ongoing research will reveal the roles that such Ca2+ waves play in the inner ear.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

Dual roles of P2 purinergic receptors in insulin-stimulated leptin production and lipolysis in differentiated rat white adipocytes

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3'-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP > UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca(2+) concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y(2). On the other hand, cAMP was generated by treatment with BzATP and ATPgammaS, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y(11) receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y(11) subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y(11) receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.     

Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides

Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with the idea that P2Y receptor activation increases the rate of exchange of GTP and GTP analogs for GDP on the relevant guanine nucleotide regulatory protein. Once the active enzymic species is formed, hydrolysis of guanine nucleotide reverts the enzyme to the inactive state.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.     

Characterization of P2 receptors in thymic epithelial cells.

The presence of P2 receptors was investigated in three distinct preparations of murine thymic epithelial cells (TEC): 2BH4 murine cell line, IT45-R1 rat cell line, and a primary murine cell derived from the Nurse cell lympho-epithelial complex. In all preparations, application of ATP to the extracellular milieu triggered intracellular calcium signals indicating the presence of P2 receptor(s) in these cells. After an initial peak of calcium concentration, a plateau phase that could last more than 10 min was frequently observed. Ion replacement and channel blockage experiments indicated that the initial peak was associated with the release of calcium from intracellular stores, while the plateau phase was associated with an influx from the extracellular medium. ATP and UTP induced similar calcium signals, suggesting the presence of P2Y2 receptors in all three cell types. The murine 2BH4 cells also expressed P2X7/P2Z receptor, since under exposure to millimolar concentrations of ATP, a continuous rise in intracellular calcium concentration was observed and their plasma membranes became permeabilized to the fluorescent dyes Lucifer yellow and ethidium bromide. In addition, this permeabilization phenomenon was blocked by the P2Z-specific antagonist, oxidized ATP. RT-PCR assays confirmed the presence of mRNAs for the P2Y2 molecule in all TEC, while mRNA for the P2X7 molecule was detected only in 2BH4 cells. Our data indicate that P2Y2 purinergic receptors are widely expressed by thymic epithelial cells, whereas the expression of the P2X7 receptor appears to be more restricted, raising the possibility that its expression is related only to a particular epithelial microenvironment within/the thymus.     

A permissive role of pertussis toxin substrate G-protein in P2-purinergic stimulation of phosphoinositide turnover and arachidonate release in FRTL-5 thyroid cells. Cooperative mechanism of signal transduction systems

Extracellular ATP and other purinergic agonists were found to inhibit cAMP accumulation by depressing adenylate cyclase as an "inhibitory action" and/or to stimulate arachidonate release in association with phospholipase C or A2 activation and Ca2+ mobilization as "stimulatory actions" in FRTL-5 cells. The stimulatory actions of a group of P2-agonists represented by ATP were partially inhibited by the pretreatment of the cells with islet-activating protein (IAP), pertussis toxin, even when an about 41-kDa membrane protein(s) was completely ADP-ribosylated. Only the IAP-sensitive part of the stimulatory actions was antagonized by 1,3-diethyl-8-phenylxanthine (DPX), an adenosine antagonist. GTP and 8-bromoadenosine 5'-triphosphate (Br-ATP) at two to three orders of higher concentrations than ATP also exerted the stimulatory actions, although they were entirely insensitive to both IAP and DPX. Ligand binding experiments with, [35S]ATP gamma S and [3H]DPX showed that ATP occupies both DPX-sensitive and insensitive receptor sites, whereas GTP does only ATP-displaceable DPX-insensitive sites. Thus, lack of sensitivity of GTP action to DPX was associated with its inability to occupy the DPX-sensitive sites. Adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and P1-agonists such as AMP and N6-(L-2-phenylisopropyl-adenosine (PIA) did not show any stimulatory action. Nevertheless, the agonists remarkably enhanced the stimulatory actions of GTP or Br-ATP. Such permissive actions of PIA and others were sensitive to both IAP and DPX, as were shown for a part of the stimulatory actions of ATP as well as the "inhibitory actions" of both PIA and ATP. We conclude that an IAP substrate G-protein(s) which mediates the inhibitory action of purinergic agonists via a DPX-sensitive purinergic receptor(s) may not directly link to the phospholipase C or A2 system but enhance the system which links to a DPX-insensitive P2-receptor, in an indirect or permissive manner.     

Disturbances in purinergic [Ca2+]i signaling pathways in a transformed rat thyroid cell line

It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines.In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells.Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells.     

Purinergic receptor-induced Ca2+ signaling in the neuroepithelium of the vomeronasal organ of larval Xenopus laevis

Purinergic signaling has considerable impact on the functioning of the nervous system, including the special senses. Purinergic receptors are expressed in various cell types in the retina, cochlea, taste buds, and the olfactory epithelium. The activation of these receptors by nucleotides, particularly adenosine-5′-triphosphate (ATP) and its breakdown products, has been shown to tune sensory information coding to control the homeostasis and to regulate the cell turnover in these organs. While the purinergic system of the retina, cochlea, and taste buds has been investigated in numerous studies, the available information about purinergic signaling in the olfactory system is rather limited. Using functional calcium imaging, we identified and characterized the purinergic receptors expressed in the vomeronasal organ of larval Xenopus laevis. ATP-evoked activity in supporting and basal cells was not dependent on extracellular Ca²⁺. Depletion of intracellular Ca²⁺ stores disrupted the responses in both cell types. In addition to ATP, supporting cells responded also to uridine-5′-triphosphate (UTP) and adenosine-5′-O-(3-thiotriphosphate) (ATPγS). The response profile of basal cells was considerably broader. In addition to ATP, they were activated by ADP, 2-MeSATP, 2-MeSADP, ATPγS, UTP, and UDP. Together, our findings suggest that supporting cells express P2Y₂/P2Y₄-like purinergic receptors and that basal cells express multiple P2Y receptors. In contrast, vomeronasal receptor neurons were not sensitive to nucleotides, suggesting that they do not express purinergic receptors. Our data provide the basis for further investigations of the physiological role of purinergic signaling in the vomeronasal organ and the olfactory system in general.     

32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor

A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a P2Y-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Calcium signaling properties of a thyrotroph cell line,mouse TαT1 cells

TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner.     

Real-time imaging reveals that P2Y2 and P2Y12 receptor agonists are not chemoattractants and macrophage chemotaxis to complement C5a is phosphatidylinositol 3-kinase (PI3K)- and p38 mitogen-activated protein kinase (MAPK)-independent

Adenosine 5'-triphosphate (ATP) has been implicated in the recruitment of professional phagocytes (neutrophils and macrophages) to sites of infection and tissue injury in two distinct ways. First, ATP itself is thought to be a chemotactic "find me" signal released by dying cells, and second, autocrine ATP signaling is implicated as an amplifier mechanism for chemotactic navigation to end-target chemoattractants, such as complement C5a. Here we show using real-time chemotaxis assays that mouse peritoneal macrophages do not directionally migrate to stable analogs of ATP (adenosine-5'-(γ-thio)-triphosphate (ATPγS)) or its hydrolysis product ADP (adenosine-5'-(β-thio)-diphosphate (ADPβS)). HPLC revealed that these synthetic P2Y(2) (ATPγS) and P2Y(12) (ADPβS) receptor ligands were in fact slowly degraded. We also found that ATPγS, but not ADPβS, promoted chemokinesis (increased random migration). Furthermore, we found that photorelease of ATP or ADP induced lamellipodial membrane extensions. At the cell signaling level, C5a, but not ATPγS, activated Akt, whereas both ligands induced p38 MAPK activation. p38 MAPK and Akt activation are strongly implicated in neutrophil chemotaxis. However, we found that inhibitors of phosphatidylinositol 3-kinase (PI3K; upstream of Akt) and p38 MAPK (or conditional deletion of p38α MAPK) did not impair macrophage chemotactic efficiency or migration velocity. Our results suggest that PI3K and p38 MAPK are redundant for macrophage chemotaxis and that purinergic P2Y(2) and P2Y(12) receptor ligands are not chemotactic. We propose that ATP signaling is strictly autocrine or paracrine and that ATP and ADP may act as short-range "touch me" (rather than long-range find me) signals to promote phagocytic clearance via cell spreading.     

Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.

Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) was also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. We further show that the BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.