Physiology Article#00014

<body bgcolor="#FFFFFF"> <div align="left"><table border="3"> <tr> <td><img src="images/h_phy.gif" width="225" height="51"></td> </tr> </table> </div><table border="0"> <tr> <td valign="top"><a name="top">(c) Neuroscience-Net<br> Volume 2, Article #10014 </a></td> <td align="right" width="500"><a name="top">Received April 17, 1997<br> Accepted for Publication June 31, 1997<br> Published August 25, 1997<br> </a></td> </tr> </table> <p align="left"><a href="http://www.neuroscience.com/theoretical_neuroscience.html"><font size="4"><strong><img src="file:///C:/WINDOWS/Desktop/neuroscience/images/b_back.gif" border="0" width="76" height="37"></strong></font></a></p> <p align="center"><font size="4"><strong>P</strong><sub><strong>2U</strong></sub><strong> Receptor Stimulation Increases Intracellular Ca</strong><sup><strong>2+</strong></sup><strong> in Hybrid N18TG2 X Mesencephalon (MES-23.5) Cells Via Two Distinct Mechanisms</strong></font></p> <p align="center"><font size="2">Himanshu N. Parikh, Li-Xin Liu and Louis A Chiodo, Department of Pharmacology,<br> Texas Tech University Health Sciences Center, Lubbock, Texas</font></p> <p><font size="2">Send correspondence to:</font></p> <p><font size="2">Himanshu Parikh<br> Department of Pharmacology<br> Texas Tech University Health Science Center<br> 3601, 4th Street<br> Lubbock, TX 79430<br> </font><a href="mailto:phrhp@ttuhsc.edu"><font size="2">phrhp@ttuhsc.edu</font></a><font size="2"><br> (806) 743-2425 voice<br> (806) 743-2744 fax</font></p> <blockquote> <p><font size="2"><strong>KEY WORDS:</strong> </font><font size="3">Keywords: fura-2, intracellular calcium, purinergic receptors, MES-23.5 cells</font></p> </blockquote> <hr> <p align="left"><font size="6">Abstract<br> </font><font size="3">(Neuroscience-Net, Volume 2, Article #10014; August 25, 1997)</font></p> <p align="left"><font size="3">In the present study, we have characterized the signal transduction pathway associated with activation of P<sub>2</sub> receptors endogenously present in a cultured hybrid N18TG2 X mesencephalon (MES-23.5) cells and responsible for increases in intracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>i</sub>) in response to extracellular adenosine triphosphate (ATP) application. The agonist potency profile for this receptor was UTP ³ ATP >> 2-methylthio-adenosine 5'- triphosphate (2-MeSATP) > a, b-methylene adenosine 5'-triphosphate (</font><font size="1" face="Symbol">a</font><font size="1"><font MeATP), typical of P<SUB>2U<sup> purinergic receptor type. Extracellular application of UTP or ATP (1-100 micromolar) to MES-23.5 cells resulted in a rapid, transient increase in [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup> that was inhibited by >50% when extracellular Ca</sup><sup><sup>2+</sup></sup><sup> was removed. These findings demonstrated that P</sup>2U<sup> receptor activation increased [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup> by two mechanisms: mobilization of Ca</sup><sup><sup>2+</sup></sup><sup> from internal stores and increased Ca</sup><sup><sup>2+</sup></sup><sup> entry into the cell. Enhanced entry of Ca</sup><sup><sup>2+</sup></sup><sup> as a result of P</sup>2U<sup> receptor-induced intracellular Ca</sup><sup><sup>2+</sup></sup><sup> mobilization suggested the presence of Ca</sup><sup><sup>2+</sup></sup><sup> release-activated Ca</sup><sup><sup>2+</sup></sup><sup> channels (CRAC) in MES-23.5 cells. In direct support of this hypothesis, whole-cell patch recordings demonstrated a sustained inward current in response to bath application of thapsigargin (which is known significantly increase [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup> in these cells) or ATP. Ca</sup><sup><sup>2+</sup></sup><sup> entry and mobilization in MES-23.5 cells was pertussis toxin sensitive in that pretreatment of cells completely blocked the increase in [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup>, In addition, U-73122, a phospholipase C inhibitor, also inhibited P</sup>2U<sup> receptor mediated mobilization of [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup>. These results demonstrate the presence of functional P</sup>2U<sup> purinergic receptors on MES-23.5 cells. These receptors are coupled to phospholipase C by a pertussis toxin sensitive G-protein and their activation results in the mobilization of Ca</sup><sup><sup>2+</sup></sup><sup> from internal stores and the subsequent activation of CRAC Ca</sup><sup><sup>2+</sup></sup><sup> entry. </sup></font><font size="3"><sup></font></sup></font></p> <p align="left"><font size="3"><sup></sup></font> </p> <p align="center"><a name="P<SUB>2U</SUB> Receptor Stimulation Increases Intracellular Ca<sup>2+</sup> in Hybrid N18TG2 X Mesencephalon (MES-23.5) Cells Via Two Distinct Mechanisms"><font size="4"><sup><strong>P</strong></sup><strong>2U</strong><sup><strong> Receptor Stimulation Increases Intracellular Ca</strong></sup><sup><sup><strong>2+</strong></sup></sup><sup><strong> in Hybrid N18TG2 X Mesencephalon (MES-23.5) Cells Via Two Distinct Mechanisms</strong></sup></font></a></p> <p align="center"><font size="2"><sup>Himanshu N. Parikh, Li-Xin Liu and Louis A Chiodo, Department of Pharmacology,<br> Texas Tech University Health Sciences Center, Lubbock, Texas</sup></font></p> <p><font size="2"><sup>Send correspondence to:</sup></font></p> <p><font size="2"><sup>Himanshu Parikh<br> Department of Pharmacology<br> Texas Tech University Health Science Center<br> 3601, 4th Street<br> Lubbock, TX 79430<br> </sup></font><a href="mailto:phrhp@ttuhsc.edu"><font size="2"><sup>phrhp@ttuhsc.edu</sup></font></a><font size="2"><sup><br> (806) 743-2425 voice<br> (806) 743-2744 fax</sup></font></p> <blockquote> <p><font size="2"><sup><strong>KEY WORDS:</strong> </sup></font><font size="3"><sup>Keywords: fura-2, intracellular calcium, purinergic receptors, MES-23.5 cells</sup></font></p> </blockquote> <hr> <ul> <li><a href="TN00014_ab.html" target="_parent"><font size="3"><sup>Abstract</sup></font></a></li> <li><a href="#intro"><font size="3"><sup>Introduction</sup></font></a></li> <li><a href="#Methods"><font size="3"><sup>Materials and Methods</sup></font></a></li> <li><a href="#results"><font size="3"><sup>Results</sup></font></a></li> <li><a href="#discuss"><font size="3"><sup>Discussion</sup></font></a></li> <li><a href="#references"><font size="3"><sup>References</sup></font></a></li> <li><a href="#figures"><font size="3"><sup>Figures and Legends</sup></font></a><font size="3"><sup><br> </sup></font></li> </ul> <p align="left"><a name="intro"><font size="6"><sup><strong>Introduction</strong></sup></font></a></p> <p align="left"><font size="3"><sup>Burnstock in early 1970 (Burnstock, 1972) suggested a possible role of ATP as a synaptic transmitter and the existence of specific ATP or purinergic (P2) receptors. Extracellular ATP mediates a variety of biological processes including neurotransmission, platelet aggregation, smooth muscle contraction and secretion of norepinephrine and dopamine in PC12 cells (Gordon, 1986; Olsson and Pearson, 1990; Burnstock, 1993, Dubyak and El-Motaasim, 1993; Inoue et. al. 1989; Fasolatto et. al. 1990; and Sela et. al. 1991). Recent evidence also suggest that dopamine and 5-hydroxytryptamine (Nakazawa and Ohno, 1996) and dopamine receptor agonists, apomorphine, (+)-SKF-38393, quinpirole and dopamine receptor antagonist (-)-sulpiride and (+)-SCH-23390 (Inoue et al., 1992) selectively facilitate a cationic current activated by extracellular ATP in rat PC12 cells. Increase in [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup> seems to be a common feature in all the cellular responses to ATP and this increase in [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup>, is caused in some cases by the influx of ions and in others by the activation of phospholipase C and the subsequent mobilization of [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup> stores. Extracellular ATP also has a neurotransmitter like properties in the CNS and PNS which are modulated by P</sup>2<sup> purinoreceptors. Uridine triphosphate (UTP), is also stored in granules of some cells such as platelets and released into the extracellular space upon stimulation (Gordon, 1986; Seifer and Schultz, 1989; and O'Connor et. al. 1991) exerting the effects similar to that that of ATP on target cells. UTP also stimulates both Ca</sup><sup><sup>2+</sup></sup><sup> mobilization and Ca</sup><sup><sup>2+</sup></sup><sup> influx (Koizumi et al., 1995). It appears that both ATP and UTP share the same receptors in human airway epithelial cells (Pfeilschifter, 1990) and in rat renal mesangial cell (Brown et. al. 1991). Although Ca</sup><sup><sup>2+</sup></sup><sup> appears to play an important role in biological responses to ATP and UTP, this increase in [Ca</sup><sup><sup>2+</sup></sup><sup>]</sup>i<sup> are mediated by several distinct mechanisms. </sup></font></p> <p><font size="3"><sup>Pharmacological classification of P</sup>2<sup> receptor types has involved establishment of the rank order of potency that characterizes the ability of naturally occurring or synthetic ligands to interact with particular receptors. These are termed P</sup>2X<sup>, P</sup>2Y<sup>, P</sup>2U<sup>, P</sup>2Z<sup>, P</sup>2T<sup> and P</sup>2D<sup> (Fredholm, B. B. et al., 1994). The P</sup>2X<sup> purinoreceptor is a ligand gated ion channel with a rank order potencies for ATP and its analogs as follows: </sup></font><font size="1" face="Symbol"><sup>a<font MeATP ³ <font size="1">bg</sup></font><font size="3"><sup></font>-</sup></font><font size="2"><sup>MeATP</sup></font><font size="3"><sup> > ATP³2-MeSATP. The P</sup>2Y<sup> receptor is a G protein coupled purinoreceptor with the following rank order potencies: 2-MeSATP >> ATP >> </sup></font><font size="1" face="Symbol"><sup>a</sup></font><font size="1"><sup><font MeATP. The P<SUB>2U</sup><sup><sup> receptor is also a G protein coupled purinoreceptor but displaying a different rank order for agonist potency than P</sup></sup><sup>2Y</sup><sup><sup> receptors: UTP ³ ATP >> 2-MeSATP. The P</sup></sup><sup>2Z</sup><sup><sup> receptor has been described as a non-selective pore forming purinoreceptor for which ATP</sup></sup><sup><sup><sup>4-</sup></sup></sup><sup><sup> is the selective agonist (Gordon, 1986). The G protein-coupled purinoreceptor P</sup></sup><sup>2T</sup><sup><sup> is selectively activated by 2-ADP (Gordon, 1986) and the P</sup></sup><sup>2D</sup><sup><sup> receptor is a G protein coupled purinoreceptor displaying a rank order potencies for agonist as follows: Ap</sup></sup><sup>4</sup><sup><sup>A > Ap</sup></sup><sup>5</sup><sup><sup>A > MeATP >> 2-MeSATP. </sup></sup></font><font size="3"><sup><sup></font></sup></sup></font></p> <p><font size="3"><sup><sup>P</sup></sup><sup>2U</sup><sup><sup> purinoreceptors have been identified in various cell types including NG108-15 cells (Lin et al., 1993) and C6-2B rat glioma cells (Munshi et al., 1993). P</sup></sup><sup>2U</sup><sup><sup> purinoreceptors are typically coupled to IP</sup></sup><sup>3</sup><sup><sup> formation via the activation of phospholipase C and mobilize Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> from IP</sup></sup><sup>3</sup><sup><sup>-sensitive intracellular stores (Murrin and Boarder, 1992; Majid et al., 1993; Raha et al., 1993; Barry and Cheek, 1994; de Souza et al., 1995). Activation of phospholipase A2, which occurs secondarily to activation of inositol lipid signaling pathway, is also a prominent sequel of P</sup></sup><sup>2U</sup><sup><sup> purinergic receptor activation (Okajima et al., 1989; Xing and Mattera, 1992; Lazarowski et al., 1994). P</sup></sup><sup>2U</sup><sup><sup> receptors appear to be capable of activating either pertussis toxin sensitive or insensitive pathways in a cell type specific manner. Lustig et al., (1993) isolated cDNA from NG108-15 cells encoding a metabotropic P</sup></sup><sup>2U</sup><sup><sup> nucleotide purinoreceptor (pP2R). Recently UTP/ATP receptor has been cloned from human airways (HP2U) and colonic epithelium (Parr et al., 1994) and from rat heart (Godecke and Schrader, 1994). Both HP2U and pP2R cloned purinoreceptor were activated by UTP and ATP resulting in the increase in IP</sup></sup><sup>3</sup><sup><sup> and increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>, an effect that was partially blocked by pertussis toxin and they both showed predicted structural features characteristic of most known G-protein coupled receptors. In the present study, we have described the initial characterization of P</sup></sup><sup>2U</sup><sup><sup> receptor signal transduction pathway present in dopaminergic neuroblastoma MES-23.5 cells.</sup></sup></font></p> <p align="left"><a name="Methods"><font size="6"><sup><sup><strong>Materials and Methods</strong></sup></sup></font></a></p> <p><font size="3"><sup><sup>MES-23.5 cells were originally obtained from G. D. Crawford. Cells were suspended at ~1 X 105 cells/ml in a growth medium (pH 7.3) consisting of DMEM (high glucose), 2% Hyclone fetal bovine serum, L-glutamine (GIBCO, Gaithersburg, MD) and bacteriostatic agent penicillin (100 u/ml)-streptomycin (Sigma Biochemicals, St. Louise, MO). The cell suspension was plated on poly-D lysine coated 13 mm round coverslips and were subsequently incubated at 37 </sup></sup><sup><sup><sup>o</sup></sup></sup><sup><sup>C in a humidified (90%) atmosphere of 10% CO</sup></sup><sup>2</sup><sup><sup> and 90% air. The growth medium was changed every two days and cell density was determined by using hemocytometer. </sup></sup></font></p> <p><font size="3"><sup><sup>Fura-2 Loading: Coverslips with attached MES-23.5 cells were placed in a 35 mm petri dish containing 700 microliter of fura-2 acetoxymethyl ester (fura-2 AM) (Molecular Probes Inc.) at a final concentration of 2 micromolar, dissolved in anhydrous DMSO and 0.1% pluoronic F-127 (BASF). The cells were returned to the incubator (37 </sup></sup><sup><sup><sup>o</sup></sup></sup><sup><sup>C, 10% CO</sup></sup><sup>2</sup><sup><sup>) for 30 minutes. After the incubation, cells were washed three times with basic external recording solution which contained (in mM) NaCl 135; KCl 5.4; CaCl</sup></sup><sup>2</sup><sup><sup> 1.8; MgSO4 0.8; glucose 20; HEPES 5; 0.5 micromolar tetrodotoxin, (adjusted to pH 7.3 with Tris base 315-325 mOsmoles). They were then incubated in the same solution for 55-70 minutes to allow complete hydrolysis of the ester bond on the intracellularly incorporated fura-2 AM. </sup></sup></font></p> <p><font size="3"><sup><sup>Fluorescence Microscopy and Image Analysis: Coverslips with attached, fura-2 loaded cells were placed in a flow chamber (flow rate 3ml/min) attached to the stage of a Nikon Diaphot 300 (Nikon, Japan) inverted microscope. A Nikon UV-F 40X (Nikon Fluor, NA 1.3) objective was used for the epifluorescence measurements together with a 75W xenon lamp. The images were obtained via an intensified CCD camera (Hamamatsu) linked to an image-processing system (Universal Imaging, Image-1/MetaFluor version 2.0). Images were obtained at two different excitation wavelengths (340 and 380 nm) via excitation filters mounted on a computer-controlled Lambda-10 optical filter changer (Sutter Instruments) and fluorescence emission was filtered with a 500 nm long pass filter (Chroma Filters). The frame capture period was 40 msec and only four frames were averaged (for each wavelength) once every second. The averaged images obtained at both 340 and 380 nm were ratioed on a pixel-by-pixel basis and [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> was calculated according to the equation presented by Grynkiewicz et al., (1985): </sup></sup></font></p> <p><font size="3"><sup><sup>[Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> = K</sup></sup><sup>d</sup><sup><sup> * F</sup></sup><sup>max</sup><sup><sup>/F</sup></sup><sup>min</sup><sup><sup>*(R - R</sup></sup><sup>min</sup><sup><sup>)/(R</sup></sup><sup>max</sup><sup><sup> - R) </sup></sup></font></p> <p><font size="3"><sup><sup>R</sup></sup><sup>min</sup><sup><sup> is the ratio of fluorescence intensities at 340 and 380 nm obtained at zero [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> and R</sup></sup><sup>max</sup><sup><sup> is the ratio at saturating [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>, K</sup></sup><sup>d</sup><sup><sup> is the dissociation constant for fura-2 and F</sup></sup><sup>min</sup><sup><sup> and F</sup></sup><sup>max</sup><sup><sup> are the fluorescence intensities at 380 nm minus and plus Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>, respectively. The apparent dissociation constant for fura-2 used in all calibrations was 224 nM (Grynkiewicz et al., 1985). In situ calibration of fura-2 was carried out by bathing the cells in a Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> -free external solution with 10 micromolar ionomycin and 5 mM EGTA to determine R</sup></sup><sup>min</sup><sup><sup> and 2 mM Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> in external solution with 10 mM ionomycin to determine R</sup></sup><sup>max</sup><sup><sup> as suggested by Malgaroli et al., (1987).</sup></sup></font></p> <p><font size="3"><sup><sup>Pertussis Toxin Preincubation: Cultures were pretreated with pertussis toxin as follows: 500 ml of the medium was removed from the well of a 24 well plate and replaced with fresh medium with 1000 ng/ml of pertussis toxin giving a final concentration 500 ng/ml. The cultures were then returned to the incubator for about 3-5 hours. Prior to loading the cells with fura-2 AM , the coverslip was washed three times with external recording solution and image analysis were carried out as outlined above. </sup></sup></font></p> <p><font size="3"><sup><sup>Whole-Cell Electrophysiological Recordings: Coverslips with attached MES-23.5 cells were transferred from 24-well plates into a recording chamber, where cells were continuously perfused at a flow rate of 2 ml/min with an external recording solution containing (in mM): NaCl 135, NaCl, 140; KCL, 2.8; CaCl</sup></sup><sup>2</sup><sup><sup>, 10; MgCl, 2; glucose, 11; HEPES-NaOH, 10 (adjusted to pH 7.3 with Tris base, osmolarity adjusted to 310-320 mOsmoles). The recording chamber was affixed to the stage of an inverted microscope (Zeiss, model ICM 405, New York, NY) and visualized with Hoffman differential interference contrast optics (X 400). The recording headstage and electrode assembly were held on an articulated arm of a three-position joystick-controlled micromanipulator (MM-8000F, ASI Inc.) during recording. The cells were voltage-clamped in the whole-cell configuration used an EPC-7/List patch clamp amplifier (Adams-List Associates, Ltd., Westbury, NY). Patch electrodes were fabricated from 1.65 O.D. micro-hematocrit capillary tubes pulled in a two-stage process on a Narishigie vertical puller (model PA-81) and polished on a Narishigie microforge (model MF-83). The holding potential and all voltage steps were controlled by PCLAMP software and hardware (Axon Instruments, Foster City, CA) with the CLAMPEX program. The patch pipette solution contained the following (in mM): KCl 140, MgCl</sup></sup><sup>2</sup><sup><sup> 2, CaCl</sup></sup><sup>2</sup><sup><sup> 1, HEPES 10, ATP 2, cAMP 0.25, and BAPTA 0.5 (adjusted to pH 7.3 with KOH, osmolarity adjusted to 310-320 mOsmoles). All the solutions were filtered through a 0.45 mM membrane filter. The patch electrode resistance ranged between 1-4 megohms. Seal resistances were greater than 5 gigohms and after membrane rupture the series resistance was between 4.1 and 13 megohms and the level of capacitance compensation ranged between 8-22 pF. All recordings were carried out at room temperature (18 - 22 </sup></sup><sup><sup><sup>o</sup></sup></sup><sup><sup>C). Once whole-cell recordings were established the cells studied were held at a membrane potential of 0 mV and either ATP or thapsigargin were bath applied. </sup></sup></font></p> <p><font size="3"><sup><sup>Chemicals and Drugs: ATP disodium salt, 2-MeSATP, a, b, Met ATP, suramin hydrochloride were obtained from RBI Biochemicals (Natick, MA). UTP was purchased from Pharmaceia Biotechnology (UK), thapsigargin, ionomycin and U-73122 were purchased from Calbiochem Corporation (La Jolla, CA). Pertussis toxin, tetrodotoxin and nifedipine were purchased from Sigma Biochemicals (St. Louis, MO). Fura-2 AM and pluoronic F-120 were purchased from Molecular Probes (Eugene, OR). All the solutions were prepared in plasticware and filtered by 0.22 micron filter before use.</sup></sup></font></p> <p><font size="3"><sup><sup>Data Analysis: Group differences in peak [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> observed were determined using an analysis variance with post-hoc comparisons performed using the Bonferroni test. </sup></sup></font></p> <p align="left"><a name="results"><font size="6"><sup><sup><strong>Results</strong></sup></sup></font></a></p> <p><font size="3"><sup><sup>The effects of extracellular UTP and ATP on [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> in MES-23.5 cells were measured using Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> sensitive dye fura-2. The basal level of [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> did not vary in MES-23.5 cells incubated for five minutes in external solution (mean ± SEM 71 ± 3.7 nM, n = 472). Bath application of ATP (1-100 micromolar) produced a dose-dependent increase in free [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> (Fig. 1). When cells were treated with 100 micromolar ATP, free [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> increased rapidly from resting levels to (mean ± SEM 452 ± 66 nM, n = 113). UTP also resulted in a dose-dependent increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>, when bath applied (Fig. 2). When cells were exposed to 100 micromolar UTP there was rapid increase in free [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>, to 435 ± 24 nM (n = 61) (mean ( SEM) within 8-10 seconds. Extracellular UTP and ATP both induced a dose-dependent transient increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> after a latency of approximately 10 sec in Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>-free medium (Fig. 1-2). However, in the absence of any extracellular Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> the increase in free [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> was reduced by 45-50% in cells exposed to UTP and ATP (Fig. 3). The application of ATP (100 micromolar) in a Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>-free medium increased [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> to (mean ± SEM 232 ± 20 nM, n = 40, Fig. 1). UTP in a Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>-free medium also displayed a dose-dependent increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> (Fig. 2b). Similarly, bath application of 100 micromolar UTP in Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>-free medium increased [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> to (mean ± SEM 255 ± 15 nM, n = 75, Fig. 2). In contrast, when MES-23.5 cells were treated with UTP (10 micromolar) and thapsigargin (10 micromolar) (which depletes [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> stores by inhibiting sarcoplasmic reticulum Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>-adenosine triphosphateses (Thastrup et al., 1990) in Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> free medium, the normal UTP elevation of [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> observed under this condition was completely eliminated. This suggested a possible involvement of CRAC channels in the mobilization seen under control conditions. This result further demonstrates that UTP did activate Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> entry in MES-23.5 cells secondary to the mobilization of Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> from intracellular stores. </sup></sup></font></p> <p><font size="3"><sup><sup>To characterize P</sup></sup><sup>2</sup><sup><sup> receptor subtype that mediated the response to UTP and ATP in MES-23.5 cells, the effect of non-selective P</sup></sup><sup>2X</sup><sup><sup> receptor agonist </sup></sup></font><font size="1" face="Symbol"><sup><sup>a</sup></sup></font><font size="1"><sup><sup><font MeATP, non-selective P<sub>2Y</sup></sup><sup><sup><sup> receptor agonist 2-MeSATP, and selective P</sup></sup></sup><sup><sup>2X</sup></sup><sup><sup><sup> receptor antagonist suramin were determined in the presence and absence of extracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>. After a short delay, 2-MeSATP and (10 micromolar, 8-10 sec) in presence of extracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>, transiently and significantly increased free [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> to 126 (17 nM (n = 28, P < 0.1) while </sup></sup></sup></font><font size="1" face="Symbol"><sup><sup><sup>a</sup></sup></sup></font><font size="1"><sup><sup><sup><font MeATP (10 micromolar) had no effect 82 ( 1 nM (n="23," Fig. 4). When 10 micromolar 2-MeSATP was bath applied in Ca<sup>2+</sup></sup></sup><sup><sup>-free medium, the increase in free [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> was (mean ± SEM 123 ± 15 nM, n = 20, P < 0.01). Further, suramin (10 micromolar, bath application), a P</sup></sup><sup>2X</sup><sup><sup> purinoreceptor antagonists, in Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> free medium and in the presence of ATP, completely anatagonized ATP effects (Fig. 5). In contrast, suramin, in the presence of Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> and ATP, incompletely antagonized ATP effects. These data suggests that the increase in free [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> was due to the entry of Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> from external medium and also release from internal stores and the by activation of typical P</sup></sup><sup>2U</sup><sup><sup> receptor that have agonist potency UTP ~ ATP >> 2-MeSATP > </sup></sup></font><font size="1" face="Symbol"><sup><sup>a</sup></sup></font><font size="1"><sup><sup><font MeATP.</font></sup></sup></font></p> <p><font size="3"><sup><sup>The role of G proteins in the observed mobilization of [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> in MES-23.5 cells was also determined. Pretreatment of cells with pertussis toxin abolished the UTP induced mobilization of [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> in these cells (Fig. 5) suggesting that P</sup></sup><sup>2U</sup><sup><sup> receptor mobilizes [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> via pertussis toxin sensitive G-protein. To determine whether P</sup></sup><sup>2U</sup><sup><sup> receptor mediated increases in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> was associated with the activation of PIP2-specific phospholipase C, MES-23.5 cells were pretreated with U-73122, a potent inhibitor of phospholipase C (Smallridge et al., 1992; Yule and Williams, 1992). When the MES-23.5 cells were exposed to 10 micromolar UTP (in presence of U-73122), there was no increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> (mean ± SEM 76 ± 3 nM, n = 42) compared to control (mean ± SEM 76 ± 2 nM, n = 38; Fig. 6). These cells were then exposed to 10 micromolar thapsigargin and an increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>, was observed demonstrating that the internal Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> stores were intact after U-73122 treatment. </sup></sup></font></p> <p><font size="3"><sup><sup>MES-23.5 cells are known to be devoid of voltage-activated Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> currents. It was therefore hypothesized that Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> entry from the extracellular environment was due to the activation of a Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> current sensitive to the mobilization of Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> from intracellular stores. Whole-cell voltage-clamp studies (n = 9) demonstrated the extracellular application of ATP (100 micromolar) or thapsigargin (10 micromolar) activated a sustained inward current which obtained a peak magnitude between 400 and 200 pA (Fig 6). Since both these agents were shown above to mobilize [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> at these concentrations the current was termed a Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> release-activated channel current (ICRAC) </sup></sup></font></p> <p align="left"><a name="discuss"><font size="6"><sup><sup><strong>Discussion</strong></sup></sup></font></a></p> <p><font size="3"><sup><sup>The results of the present study demonstrate the existence of P</sup></sup><sup>2</sup><sup><sup> receptors on MES-23.5 cells which regulate [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>. The rank order potencies for P</sup></sup><sup>2</sup><sup><sup> receptor agonists with respect to increasing [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> was: UTP ~ ATP > 2-MesATP >> </sup></sup></font><font size="1" face="Symbol"><sup><sup>a</sup></sup></font><font size="1"><sup><sup><font MeATP. This indicated that the receptors present on these cells were of the P<SUB>2U</sup></sup><sup><sup><sup> subtype (Fredholm et al. 1994). It was shown that the magnitude of the increase observed in response to either UTP or ATP was augmented by as much as 50% by the presence of extracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> demonstrating the increase in [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> resulted from two distinct sources: intracellular stores and calcium entry. The Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> release from intracellular stores may result from IP</sup></sup></sup><sup><sup>3</sup></sup><sup><sup><sup> formation linked to P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> purinoreceptors as suggested by many investigators in various cell lines (Alex Brown et al., 1991; Murrin and Boarder, 1992; Majid et al., 1993; Raha et al., 1993; Barry and Cheek, 1994; de Souza et al., 1995). To further characterize the signal transduction pathway in MES-23.5, the cells were treated with pertussis toxin and effect so UTP were tested in presence and absence of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>. PTX completely blocked the mobilization of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> in these cells demonstrating that P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> receptors in MES-23.5 cells mobilizes [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> via pertussis toxin sensitive G protein. To further support our hypothesis that extracellular UTP interacts with cell surface P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> receptors that are coupled to a second messenger pathway involving the generation of IP</sup></sup></sup><sup><sup>3</sup></sup><sup><sup><sup> and the mobilization of [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>], the effects of phospholipase C inhibitor U-73122 were tested. U-73122, completely blocked mobilization [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> when treated with UTP In the absence of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>, suggesting that the release of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> from internal stores are mediated by IP</sup></sup></sup><sup><sup>3</sup></sup><sup><sup><sup>.</sup></sup></sup></font><font size="3"><sup><sup><sup></font></sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>Surmain has typically been considered a P</sup></sup></sup><sup><sup>2X</sup></sup><sup><sup><sup> receptor antagonist though recently evidence has demonstrated some receptor blocking actions at P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> receptors (Hartley and Kozlowski, 1997). This is consistent with our observations that suramin antagonized the [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> elevations produced by ATP. The weak actions of suramin alone in the presence of normal extracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> are not completely understood but this compound has been shown to stimulate Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> release in striated muscle (Emmick et al. 1994). Other actions of surmain remain to be investigated. </sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>It has been reported earlier that ATP, but not the UTP activates the nonspecific Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> channels that mediates Na</sup></sup></sup><sup><sup><sup><sup>+</sup></sup></sup></sup><sup><sup><sup> dependent depolarization in PC12 cells and many other tissues which may be responsible for the massive Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> influx (Nakazawa et al., 1990; de Souza et al., 1995). In addition when the effects of UTP were tested in presence of thapsigargin, in absence of extracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>, there was no mobilization of intracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> suggesting release of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> from internal stores. Similarly, when the effects of UTP were tested in the presence of nifedipine, the increase in [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> was almost 50% reduced, suggesting that increase in [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> could arise from two sources, 1) the release of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> from internal stores and 2) the Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> influx. </sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>It has been proposed by number of investigators the evidence of multiple signaling pathways mediated by P</sup></sup></sup><sup><sup>2</sup></sup><sup><sup><sup> receptors in a number of cell types including macrophages (Alonso-Torre and Trautmann, 1993) in which activation of P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> purinergic receptor by UTP leads to release of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> from intracellular stores followed by Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> influx. In the present study, we examined for the presence of CRAC calcium channels which were activated by mobilization of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> from intracellular stores. Application of ATP was shown to induce a prolonged inward current which was similar to that observed with bath application of thapsigargin. Since both treatments are known to elevate [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup>, this served as a clear demonstration of CRAC channels. At this time it is not possible to determine the precise transduction mechanism involved in activating the CRAC channels. Several possibilities have been suggested including Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> itself, a co-released factor, and small G proteins (see Berridge, 1995; Fasolato et al, 1994). Further studies will be necessary to determine the precise mechanisms involved in MES-23.5 cells. </sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> receptors have been functionally described in various human organs and cell types (Burnstock, 1990) including Ehrlic ascites tumor cells (Dubyak and De Young, 1985), and pituitary cells (Davidson et al., 1990). P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> receptor mRNA is widely distributed in human tissues including heart, liver, lung, kidney (Lustig et al., 1993) and also in kidney proximal tubule cells, salivary duct gland cell line HSG-PA and a primary cultures of nasal epithelium (Parr et al., 1994). Isolation, identification and molecular characterization of signal transduction mechanisms of the receptors for extracellular nucleotides present in human airway and epithelia will allow studies on the expression of this receptor in normal and diseased state and may facilitate identification and development of drugs for therapy.</sup></sup></sup></font></p> <p align="left"><a name="references"><font size="6"><sup><sup><sup><strong>References</strong></sup></sup></sup></font></a></p> <p><font size="3"><sup><sup><sup>Alex Brown, H., Lazarowski, E. R., Boucher, R. C., and Kendall Harden, T. (1991) Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5' nucleotide receptor in Human airway epithelial cell. Mol. Pharm., 40, 648-655.</sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>Barry V. A., and Cheek, T. R. 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Endocrinology., 131, 1883-1888.</sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>Seifert, R., and Schultz, G. (1989) Involvement of pyrimidinoreceptors in regulation of cell functions by uridine and uracil nucleotides. Trends Pharmacol. Sci. 10, 365-369.</sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>Sela, D., Ram, E., and Atlas, D. (1991) ATP receptor: A putative receptor operated channel in PC-12 cells. J. Biol. Chem., 266, 17990-17994.</sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>Thastrup, O., P. J. Cullen, Drobak, B. K., et al., (1990) Thapsigargin, a tumor promotor, discharges [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> stores by specific inhibition of the endoplasmic reticulum Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> -ATPase. Proc. Natn. Acad. Sci. U. S. A., 872, 2466-2470.</sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>Yule, D. I., and Williams, J. A. (1992) U-73122 inhibits Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> oscillations in response to CCK and Carbachol but not to JMV-80 in rat pancreatic acinar cell. J. Biol. Chem., 267, 13830-13835</sup></sup></sup></font></p> <p><font size="3"><sup><sup><sup>Xing, M., and Mattera, R. (1992) Phosphorylation dependent regulation of phospholipase A2 and Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> in HL60 granulocytes. J. Biol. Chem., 267, 6602-6610.</sup></sup></sup></font></p> <p align="left"><a name="figures"><font size="6"><sup><sup><sup><strong>Figure Legends</strong></sup></sup></sup></font></a></p> <div align="left"><table border="0" cellpadding="2"> <tr> <td valign="top"><a href="images/utp04971.gif" target="_top"><font size="3"><sup><sup><sup><img src="images/utp04971_T.gif" border="1" width="72" height="52"></sup></sup></sup></font></a></td> <td><a href="images/utp04971.gif" target="_top"><font size="3"><sup><sup><sup>Figure 1</sup></sup></sup></font></a><font size="3"><sup><sup><sup>.--- Dose-response curves illustrating the effect of bath application of ATP on the [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> measured using fura-2. It may be observed that the cells begin to respond at a concentration of 1 micromolar and a maximal effect is obtained at 10 mM. The role of extracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> is also demonstrated in that the removal of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> from the bath solution results in approximately a 50% reduction in the [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> observed. All data represents the mean ± SEM of the cells studied. Number in parentheses indicates number of cells studied. *p < 0.01, analysis of variance. Each data point represents peak [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> during the treatment. </sup></sup></sup></font></td> </tr> <tr> <td valign="top"><a href="images/utp04972.gif" target="_top"><font size="3"><sup><sup><sup><img src="images/utp04972_T.gif" border="1" width="72" height="51"></sup></sup></sup></font></a></td> <td><a href="images/utp04972.gif" target="_top"><font size="3"><sup><sup><sup>Figure 2</sup></sup></sup></font></a><font size="3"><sup><sup><sup>.--- Dose-response curves illustrating the effect of bath application of UTP on the [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> measured using fura-2. It may be observed that the cells begin to respond at a concentration of 1 micromolar and a maximal effect is obtained at 5 micromolar. The role of extracellular Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> is also demonstrated in that the removal of Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup> from the bath solution results in approximately a 50% reduction in the [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> observed. All data represents the mean + SEM of the cells studied. Number in parentheses indicates number of cells studied. *p < 0.01, analysis of variance. Each data point represents peak [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> during the treatment. </sup></sup></sup></font></td> </tr> <tr> <td valign="top"><a href="images/utp04973.gif" target="_top"><font size="3"><sup><sup><sup><img src="images/utp04973_T.gif" border="1" width="72" height="53"></sup></sup></sup></font></a></td> <td><a href="images/utp04973.gif" target="_top"><font size="3"><sup><sup><sup>Figure 3</sup></sup></sup></font></a><font size="3"><sup><sup><sup>.--- Example of the increases in [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> produced by the stimulation of P</sup></sup></sup><sup><sup>2U</sup></sup><sup><sup><sup> receptors on MES-23.5 cells. Cells were pre-loaded with fura-2 as described in the text. UTP (100 micromolar) resulted in a rapid increase in [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup>. It may be observed that in a Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>-free external solution the magnitude of the increase observed is significantly reduced. Each trace is taken from a single cell.</sup></sup></sup></font></td> </tr> <tr> <td valign="top"><a href="images/utp4974r.gif" target="_top"><font size="3"><sup><sup><sup><img src="images/utp4974r_T.gif" border="1" width="72" height="47"></sup></sup></sup></font></a></td> <td><a href="images/utp4974r.gif" target="_top"><font size="3"><sup><sup><sup>Figure 4</sup></sup></sup></font></a><font size="3"><sup><sup><sup>.--- Bar graph representation of the ability of different P</sup></sup></sup><sup><sup>2</sup></sup><sup><sup><sup> receptor agonists to increase [Ca</sup></sup></sup><sup><sup><sup><sup>2+</sup></sup></sup></sup><sup><sup><sup>]</sup></sup></sup><sup><sup>i</sup></sup><sup><sup><sup> in MES-23.5 cells. It may be seen that </sup></sup></sup></font><font size="1" face="Symbol"><sup><sup><sup>a</sup></sup></sup></font><font size="1"><sup><sup><sup><font MeATP had no effect relative to control (CONT) while 2-MesATP produced a moderate but significant increase in [Ca<sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>. Both UTP and ATP produced similar maximal increases in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>. It may again be seen that experiments conducted in Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>-conditions results in a significant reduction of the level of [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> observed indicating a role for Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> entry in this process. Data represent the mean ± SEM concentration observed after bath application of 100 micromolar of the different agonists. Number in parentheses indicates the number of individual cells in each group. Each data point represents peak [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> during the treatment. * p<0.01 relative to control, analysis of variance. </sup></sup></font><font size="3"><sup><sup></font></sup></sup></font></td> </tr> <tr> <td valign="top"><a href="images/utp04975.gif" target="_top"><font size="3"><sup><sup><img src="images/utp04975_T.gif" border="1" width="72" height="63"></sup></sup></font></a></td> <td><a href="images/utp04975.gif" target="_top"><font size="3"><sup><sup>Figure 5</sup></sup></font></a><font size="3"><sup><sup>.--- The role of second messenger systems in the P</sup></sup><sup>2U</sup><sup><sup> receptor mediated increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>. It may be observed that pretreatment of cells with pertussis toxin (PTX) completely blocked the ability of 100 mM UTP to increase [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>. This demonstrates the involvement of a pertussis-toxin sensitive G protein in the signal transduction. To determine whether or not phospholipase C may be involved in this response additional groups were pretreated with the phospholipase C inhibitor U-73121 (10 micromolar) and then exposed to 100 micromolar UTP. This treatment also abolished the increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> normally observed. Thapsigargin (10 micromolar) was however able to increase [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> in U-73122 treated cells indicating that the intracellular Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> stores were still intact. Suramin (SUR) alone produced a moderate increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> in cell only if extracellular Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> was present. In addition, SUR antagonized the ability of ATP (100 micromolar) to increase [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> in both Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> and Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>-free media. Data represent the mean ± SEM concentration observed. Number in parentheses indicates the number of individual cells in each group. Each data point represents peak [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> during the treatment. * p<0.01 relative to control, analysis of variance.</sup></sup></font></td> </tr> <tr> <td valign="top"><a href="images/utp04976.gif" target="_top"><font size="3"><sup><sup><img src="images/utp04976_T.gif" border="1" width="72" height="40"></sup></sup></font></a></td> <td><a href="images/utp04976.gif" target="_top"><font size="3"><sup><sup>Figure 6</sup></sup></font></a><font size="3"><sup><sup>.--- Whole-cell current recording of the ICRACR current present in MES-23.5 cells. This cell was clamped at a membrane potential of 0 mV and ATP 100 micromolar was applied via the bath. It may be observed that ATP produced a sustained inward current that peaked at approximately -40 pA. The same current could be activated by bath application of thapsigargin (results not shown). </sup></sup></font></td> </tr> <tr> <td valign="top"><a href="images/utp04977.gif" target="_top"><font size="3"><sup><sup><img src="images/utp04977_T.gif" border="1" width="72" height="41"></sup></sup></font></a></td> <td><a href="images/utp04977.gif" target="_top"><font size="3"><sup><sup>Figure 7</sup></sup></font></a><font size="3"><sup><sup>.--- A model representing the P</sup></sup><sup>2U</sup><sup><sup> receptor mediated increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> in MES-23.5 cells. The P</sup></sup><sup>2U</sup><sup><sup> receptor is coupled to intracellular events via a pertussis toxin (PTX) sensitive G protein. Since treatment with the phospholipase C inhibitor U-73122 completely inhibits the P</sup></sup><sup>2U</sup><sup><sup>-mediated increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>, the G protein signal transduction pathway involves the activation of phospholipase C (PLC). PLC activation would lead to the generation of inositol (1,4,5)-trisphosphate (IP</sup></sup><sup>3</sup><sup><sup>) and 1,2-diacylglycerol (DAG). We believe that IP</sup></sup><sup>3</sup><sup><sup> mobilizes Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> from IP</sup></sup><sup>3</sup><sup><sup>-sensitive intracellular Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> stores which data presented herein indicates accounts for approximately 50% of the increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> observed. This increase in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup> is then believed to serve as the signal for activation of Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup> release-activated channels (CRAC) which produces a further rise in [Ca</sup></sup><sup><sup><sup>2+</sup></sup></sup><sup><sup>]</sup></sup><sup>i</sup><sup><sup>. </sup></sup></font></td> </tr> </table> </div><p align="center"><font size="3"><sup><sup></sup></sup></font> </p> <div align="center"><center><table border="0"> <tr> <td align="center"><a href="http://www.neuroscience.com/theoretical_neuroscience.html" target="_top"><font size="3"><sup><sup><img src="images/b_back.gif" alt="Back" border="0" width="76" height="37"></sup></sup></font></a><font size="3"><sup><sup> </sup></sup></font></td> <td align="center"><a href="#top" target="_top"><font size="3"><sup><sup><img src="images/b_top.gif" alt="Top" border="0" width="76" height="37"></sup></sup></font></a><font size="3"><sup><sup> </sup></sup></font></td> <td><a href="newbody.html"><font size="3"><sup><sup><img src="images/b_body.gif" alt="Body" border="0" width="76" height="37"></sup></sup></font></a><font size="3"><sup><sup> </sup></sup></font></td> </tr> <tr> <td><a href="http://www.neuroscience.com/articles.html" target="_top"><font size="3"><sup><sup><img src="images/b_ita1.gif" alt="Index to Articles" border="0" width="179" height="54"></sup></sup></font></a><font size="3"><sup><sup> </sup></sup></font></td> <td><a href="http://www.neuroscience.com/newart.html" target="_top"><font size="3"><sup><sup><img src="images/b_naw.gif" alt="New Articles This Week" border="0" width="179" height="54"></sup></sup></font></a><font size="3"><sup><sup> </sup></sup></font></td> <td><a href="http://www.neuroscience.com/index.html" target="_top"><font size="3"><sup><sup><img src="images/b_home1.gif" alt="Return Home" border="0" width="179" height="54"></sup></sup></font></a></td> </tr> </table> </center></div> </body>