Biomedical subjects

A D Michel

Publications and source records attributed to A D Michel.

At least 19 recordsLinked to original sources

PubMed2001

Species- and agonist-dependent differences in the deactivation-kinetics of P2X7 receptors.

In this study we have expressed recombinant P2X7 receptors in HEK293 cells and examined the reasons for the species- and agonist-dependent differences in the time taken for the closure of the P2X7 receptor ion-channels after agonist removal. Channel closure times, measured in electrophysiological studies or by measuring cellular permeability to ethidium cations, were slower at rat than at human or mouse P2X7 channels following washout of the P2X7 agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP). In contrast, there were no species differences in channel closure times when ATP was the agonist. BzATP was more potent than ATP at the three species homologues and exhibited highest potency for rat P2X7 receptors suggesting that channel closure time was related to agonist potency. Furthermore, BzATP potency for the P2X7 receptor could be modified by changing extracellular ionic concentrations or by mutating the receptor and modifications which increased agonist potency also increased the time taken for channel closure. The dependence of channel closure time on agonist potency suggests it reflects agonist dissociation from the P2X7 receptor rather being an intrinsic property of the ion-channel. Consistent with this, our previous studies have shown that agonist potency increases after repeated agonist applications and in this study channel closure time at rat P2X7 receptors increased after repeated agonist applications. Overall these results suggest that the species differences in channel closure times reflect differences in agonist dissociation rates which arise as a consequence of the marked species differences in agonist potency.

Adenosine Triphosphate↗

PubMed2001

Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells.

1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.

Adenine Nucleotides↗

PubMed2001

Serum constituents can affect 2'-& 3'-O-(4-benzoylbenzoyl)-ATP potency at P2X(7) receptors.

1. 2'-& 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) is the prototypic agonist for P2X(7) receptors. In this study we demonstrate that bovine serum albumin (BSA) can affect the potency of BzATP at P2X receptors. 2. BzATP potency (pEC(50)) to stimulate ethidium accumulation in cells expressing recombinant P2X7 receptors varied between 6.5 and 4, depending upon the species orthologue studied and ionic conditions employed. BSA (0.1 - 1 mg ml(-1)) and foetal bovine serum (FBS, 1 - 10% v v(-1)) inhibited responses to BzATP but only when the BzATP pEC(50) exceeded 5. 3. BSA did not block ATP-stimulated ethidium accumulation, suggesting its effects were independent of P2X(7) receptor blockade. 4. BSA did not cause breakdown of nucleotides, although FBS (10% v v(-1)) exhibited appreciable nucleotidase activity and caused significant breakdown of ATP. 5. In the presence of BSA, lipids such as 11-((5-dimethylaminonaphthalene-1-sulphonyl)amino)undecanoic acid (DAUDA) and arachidonic acid (AA) markedly increased BzATP potency. Lipids had no affect on ATP potency in the presence of BSA and had little effect on responses to BzATP in the absence of BSA. 6. These results suggested that the reduction in BzATP potency by BSA was due to BzATP binding to BSA and that lipids prevented this binding. Consistent with this hypothesis, BzATP inhibited binding of the fluorescent lipid, DAUDA, to BSA. 7. In conclusion, BSA and lipids can markedly affect BzATP potency at P2X(7) receptors but this is probably a consequence of BzATP binding to BSA. This finding has important implications when using BzATP in vivo or in the presence of albumin.

Adenosine Triphosphate↗

PubMed2001

Complexities of measuring antagonist potency at P2X(7) receptor orthologs.

The ability of P2 antagonists to affect agonist-stimulated fluorescent dye accumulation in cells expressing human, rat, or mouse P2X(7) receptors was examined. Several compounds, including pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), which was previously thought to be a weak P2X(7) receptor antagonist, possessed high potency (nanomolar IC(50)) at human and rat P2X(7) receptors. However, there were species differences in antagonist potency with PPADS, pyridoxal 5'-phosphate (P5P), and periodate-oxidized ATP (OxATP) exhibiting 20- to 500-fold higher potency for human than for mouse P2X(7) receptors. HMA (5-(N,N-hexamethylene)amiloride) was also selective for human over rat P2X(7) receptors but potentiated responses at mouse P2X(7) receptors. Coomassie Brilliant Blue G (CBB) was a nonselective antagonist with high potency at mouse P2X(7) receptors (IC(50) approximately 100 nM). All compounds were noncompetitive antagonists, and potency could only be quantified by measuring IC(50) values. These values were similar when determined against EC(50) concentrations of ATP or 2'- and 3'-O-4(-benzoylbenzoyl)-ATP and, for most compounds, only slightly (3- to 5-fold) affected by agonist concentration. However, IC(50) values for KN62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) and suramin, varied up to 25-fold depending upon agonist concentration. Furthermore, IC(50) values for KN62 and OxATP were 10-fold lower at 22 degrees C than at 37 degrees C, whereas IC(50) values for PPADS, P5P, suramin, and OxATP were up to 20-fold lower in NaCl than in sucrose buffer. Potency estimates for CBB and PPADS decreased 5-fold in the presence of bovine serum albumin, possibly due to protein binding. Given the species differences, and the effects of assay conditions on antagonist potency, caution must be exercised when interpreting results obtained with the available antagonists.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Functional characterization of the P2X(4) receptor orthologues.

1. The aim of this study was to functionally characterize the recombinant mouse P2X(4) receptor and to compare its pharmacological properties with those of the human and rat orthologues. 2. Whole cell recordings were made from rafts of HEK-293 cells stably expressing recombinant mouse, rat or human P2X(4) receptors, using Cs-aspartate containing electrodes (3 - 8 MOmega) in a HEPES-buffered extracellular medium. 3. The agonist potency of ATP at the three species orthologues was similar, with mean EC(50) values of 2.3 microM, 1.4 microM and 5.5 microM, respectively. 4. Adenosine-5'-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X(4) receptors (EC(50)=2.6 and 3.0 microM), but was significantly less potent at the rat orthologue (EC(50)=20.0 microM). alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X(4) and 24% at the human P2X(4) receptor. 5. In contrast to the other species orthologues, alpha,beta-meATP failed to elicit a significant agonist response at rat P2X(4) receptors, and was found to act as an antagonist, with an IC(50) of 4.6 microM, against 10 microM ATP. 6. Mouse P2X(4) receptors were found to be sensitive to the antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (IC(50)=10.5 microM), as were human P2X(4) receptors (IC(50)=9.6 microM). The rat receptor however, showed a low sensitivity to PPADS (IC(50)>100 microM). 7. All three orthologues were relatively suramin-insensitive (IC(50)>100 microM) and insensitive to 1-[N, O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC(50)>3 microM). 8. Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X(4) receptor, and differ markedly from the rat receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Apparent species differences in the kinetic properties of P2X(7) receptors.

1. Apparent species differences in the responses of recombinant P2X(7) receptors to repeated application of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) have been investigated. 2. Repeated application of 100 microM BzATP resulted in a progressive increase in current magnitude (current growth) at mouse and human, but not rat P2X(7) receptors. 3. Current growth was thought to reflect progressive dilation of the P2X(7) ion-channel to a pore permeable to large molecules (MW<900), suggesting that channel dilation was not occurring at the rat P2X(7) receptor. However, 100 microM BzATP produced a rapid influx of YO-PRO-1 (MW375) in cells expressing rat or human P2X(7) receptors. 4. There were, however, species differences in agonist potency such that 100 microM BzATP was a supra-maximal concentration at rat, but not human or mouse, P2X(7) receptors. Importantly, when sub-maximal concentrations of BzATP or ATP were examined, current growth occurred at rat P2X(7) receptors. 5. The rate of current growth and YO-PRO-1 accumulation increased with agonist concentration and appeared more rapid at rat and human, than at mouse P2X(7) receptors. 6. The potency of BzATP and ATP was 1.5 - 10 fold lower in naïve cells than in cells repeatedly exposed to ATP. 7. This study demonstrates that current growth occurs at mouse, rat and human P2X(7) receptors but only when using sub-maximal concentrations of agonist. Previously, current growth was thought to reflect the progressive increase in pore diameter of the P2X(7) receptor ion channel, however, the results of this study suggest a progressive increase in agonist potency may also contribute.

Adenosine Triphosphate↗

PubMed2000

Antagonist effects on human P2X(7) receptor-mediated cellular accumulation of YO-PRO-1.

We have examined the interaction of P2 antagonists with the human P2X(7) receptor by studying their effect on 2' and 3'-O-benzoyl-benzoyl-ATP (DbATP) stimulated cellular accumulation of the fluorescent, DNA binding dye, YO-PRO-1 (MW=375Da). In suspensions of HEK293 cells expressing human recombinant P2X(7) receptors, DbATP produced time and concentration-dependent increases in YO-PRO-1 fluorescence. This response presumably reflects YO-PRO-1 entry through P2X(7) receptor channels and binding to nucleic acids. When studies were performed in a NaCl-free, sucrose-containing buffer, full concentration-effect curves to DbATP could be constructed. The P2 antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and periodate oxidized ATP (oATP), reduced the potency of DbATP and decreased its maximum response. 1-[N,O-bis(1, 5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62) and its analogue, KN04, reduced the potency of DbATP. Schild slopes for KN62 and KN04 were shallow and exhibited a plateau at concentrations of compound greater than 1 microM, indicating that these compounds were not competitive antagonists. Calmidazolium and a monoclonal antibody to human P2X(7) receptors attenuated DbATP-stimulated YO-PRO-1 accumulation but they were not competitive antagonists and only produced 2 - 3 fold decreases in the potency of DbATP. The effects of PPADS and KN62 were partially reversible whereas those of oATP were not. PPADS protected cells against the irreversible antagonist effects of oATP suggesting a common site of action. In contrast KN62 was not effective suggesting that it may bind at a different site to oATP and PPADS. This study has demonstrated that P2X(7) receptor function can be quantified by measuring DbATP stimulated YO-PRO-1 accumulation and has provided additional information about the interaction of P2 receptor antagonists with the human P2X(7) receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Ionic effects on human recombinant P2X7 receptor function.

The actions of monovalent and divalent ions on the P2X7 receptor have been assessed by measuring their effect on responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoyl-benzoyl)-ATP (DbATP), in HEK293 cells expressing the human recombinant P2X7 receptor. In these cells, DbATP increased the cellular accumulation of the DNA binding, fluorescent dye, YO-PRO-1. The potency of DbATP to elicit this effect was decreased by both calcium and magnesium ions. In addition, when the pH was increased above 8 or reduced below 6.5, the potency of DbATP was less than obtained at pH 7.5. Monovalent ions also affected the P2X7 receptor such that the potency of DbATP was 19-fold higher in NaCl-free buffer containing 280 mM sucrose (pEC50=6.48) than in 140 mM NaCl containing buffer (pEC50=5.19). Monovalent cations differentially affected the potency of DbATP. Thus, when the chloride concentration was maintained at 140 mM, pEC50 values for DbATP were 6.14, 5.87 and 5.19 when the counter cation was 140 mM choline, potassium or sodium, respectively. Monovalent anions also differentially affected the potency of DbATP and in the presence of 140 mM sodium ions, pEC50 values for DbATP were 6.14, 6.07, 5.19 and 4.53, respectively, when the counter anion was 140 mM aspartate, glutamate, chloride or iodide. The inhibitory effect of monovalent anions on P2X7 receptor function was also observed in electrophysiological studies. Thus in sodium glutamate containing buffer the potency of DbATP (pEC50=5.55) was approximately 22-fold higher than in NaCl containing buffer (pEC50=4.20). This study has demonstrated that P2X7 receptor function can be markedly affected by a wide range of ions and that physiological concentrations of sodium and chloride ions, as well as divalent cations, contribute to the low potency of ATP as an agonist at this receptor.

Adenosine Triphosphate↗

PubMed1999

Activation of adenylate cyclase by human recombinant sst5 receptors expressed in CHO-K1 cells and involvement of Galphas proteins.

1. The coupling of the human somatostatin sst5 receptor recombinantly expressed in Chinese hamster ovary (CHO-K1) cells to adenylate cyclase was investigated using receptor selective ligands. 2. Forskolin (10 microM)-stimulated adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation was inhibited by somatostatin-14 and a number of receptor-selective agonists with a rank order of agonist potency typical of the sst5 receptor. L-362,855 and BIM-23056 behaved as full agonists. At higher somatostatin-14 concentrations there was sub-maximal inhibition resulting in a bell-shaped concentration-effect relationship. Pertussis toxin (PTx; 100 ng ml(-1), 18 h) pre-treatment abolished agonist-mediated inhibition of cyclic AMP accumulation and markedly enhanced stimulation of cyclic AMP at higher agonist concentrations. 3. The concentration of prostaglandin E2 (PGE2) in the incubation media was raised 14 fold by 1 microM somatostatin-14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors. 4. Pre-treatment with cholera toxin (ChTx; 20 microg ml(-1), 18 h) markedly inhibited sst5 receptor-mediated increases in cyclic AMP formation in intact cells. Somatostatin-14-stimulated cyclic AMP accumulation was also observed in sst5 receptor containing CHO-K1 membranes and was inhibited by the synthetic peptide Galphasacetyl-354-372-amide (100 microM) by 65.9+/-3.5%, implicating a Galphas protein involvement in this response. 5. Activation of Galphas proteins by somatostatin-14 could be demonstrated with [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding and subsequent immunoprecipitation of 35S labelled Galphas proteins with anti-Galphas serum. 6. These data show that the sst5 receptor is very efficiently coupled in a negative manner to adenylate cyclase. However, at higher agonist concentrations the receptor can also mediate activation of adenylate cyclase by a mechanism apparently involving Galphas protein activation.

Adenylate Cyclase Toxin↗

PubMed1999

Pharmacological characterization of ATP- and LPS-induced IL-1beta release in human monocytes.

1. We have utilized the human monocytic cell line, THP-1, and freshly isolated adherent human monocytes with the compounds pyridoxalphosphate-6-azophenyl-2',4'-disuphonic acid (PPADS), oxidized ATP, and 1-(N, O-bis[5-isoquinolinesufonyll]-N-methyl-L-tyrosyl)-4-phenylpiper azi ne (KN-62) to pharmacologically characterize the P2 receptor involved in ATP-induced release of interleukin 1beta (IL-1beta). We have also investigated the involvement of P2 receptors in lipopolysaccharide (LPS)-induced IL-1beta release from both cell types. 2. ATP caused release of IL-1beta from LPS primed THP-1 cells in both a time- and concentration-dependent manner, with a minimal effective ATP concentration of 1 mM. Stimulation of cells with 5 mM ATP resulted in detectable concentrations of IL-1beta in cell supernatants within 30 min. 3. The ATP analogue benzoylbenzoyl ATP (DBATP), a P2X7 receptor agonist, was approximately 10 fold more potent than ATP at eliciting IL-1beta release. 4. KN-62 (1 micro M), PPADS (100 microM) or oxidized ATP (100 uM) significantly inhibited 5 mM ATP-induced IL-1beta release by 81, 90 and 66% respectively, but failed to significantly inhibit LPS-induced IL-1beta release in both THP-1 cells and in freshly isolated human monocytes. 5. In both THP-1 cells and freshly isolated human monocytes, addition of the ATP degrading enzyme apyrase (0.4 U ml(-1)) to cell supernatants prior to LPS activation failed to significantly inhibit the LPS-induced IL-1beta release. In addition there was no correlation between extracellular ATP concentrations and IL-1beta release in THP-1 cells when studied over a 6 h time period. 6. In conclusion our data confirm the involvement of P2X7 receptors in ATP-induced IL-1beta release in human monocytes. However no evidence was obtained which would support the involvement of either endogenous ATP release or P2X7 receptor activation as the mechanism by which LPS-induces IL-1beta release in either the THP-1 cell line or in freshly isolated human monocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Blockade of human P2X7 receptor function with a monoclonal antibody.

A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

Adenosine Triphosphate↗

PubMed1998

Cloning and functional characterisation of the mouse P2X7 receptor.

We have isolated a 1785-bp complementary DNA (cDNA) encoding the murine P2X7 receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80%, and 85% homology to the human and rat P2X7 subunits, respectively. Functional properties of the heterologously expressed murine P2X7 homomeric receptor broadly resembled those of the P2X7 receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X7 receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.

Amino Acid Sequence↗

PubMed1998

Cibacron blue allosterically modulates the rat P2X4 receptor.

We have used whole-cell patch clamp electrophysiology to characterise the actions of the P2 antagonist, cibacron blue, on the rat recombinant P2X4 receptor, stably expressed in human embryonic kidney 293 (HEK293) cells. In single cells, adenosine triphosphate (ATP) evoked inward currents, but the response was subject to considerable run down which precluded obtaining quantitative data. However, when recordings were made from cells that were part of a group of 20-40 electrically coupled cells (cell rafts), run-down of current was not observed and reproducible responses could be obtained. When studied using cell rafts, cibacron blue was a weak antagonist of the rat P2X4 receptor (IC50 > 300 microM) when co-applied with ATP. However, when cell rafts were preincubated with low concentrations of cibacron blue (3-30 microM) for 5 min prior to ATP addition, cibacron blue increased responses to ATP by increasing its potency (up to 4-fold) without affecting the maximum current. Potentiation of ATP-evoked currents was also observed following washout of high, inhibitory concentrations of cibacron blue (300 microM). In contrast to these effects on P2X4 receptors, cibacron blue inhibited the ATP-induced response in both single cells and rafts of HEK293 cells expressing the P2X2 receptor (IC50 approximately 600-800 nM). The effects of cibacron blue on the P2X4 receptor were quantitatively similar to those of Zn2+ which also increased ATP-evoked currents by decreasing the EC50 of ATP (up to 3.5-fold). These data are consistent with the concept that cibacron blue, like zinc, allosterically regulates the function of the P2X4 receptor.

Adenosine Triphosphate↗

PubMed1998

Effects of antagonists at the human recombinant P2X7 receptor.

1. We have used whole-cell patch clamping methods to examine the properties of the recombinant human P2X7 (P2Z) receptor stably expressed in HEK-293 cells. 2. In an extracellular solution with lowered concentrations of divalent cations (zero Mg2+ and 0.5 mM Ca2+), both ATP and the nucleotide analogue, 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (Bz-ATP) evoked concentration-dependent whole-cell inward currents with maxima of 4658+/-671 and 5385+/-990 pA, respectively, at a holding potential of -90 mV. Current-voltage relationships determined using 100 microM Bz-ATP reversed at -2.7+/-3.1 mV, and did not display significant rectification. 3. Repeated applications of 300 microM Bz-ATP produced inward currents with similar rise-times (approx. 450 ms, 5-95% current development) but with progressively slower 95-5% decay times, with the eighth application of this agonist yielding a decay time of 197% of the first application. 4. Concentration-effect curves to ATP and Bz-ATP produced estimated EC50 values of 780 and 52.4 microM, respectively. Consecutive concentration-effect curves to Bz-ATP produced curves with similar maxima and EC50 values. 5. The non-selective P2 antagonists, pyridoxal-phosphate-6-azophenyl-, 2',4'-disulphonic acid (PPADS) and suramin, both produced concentration-dependent increases in maximal inward currents to Bz-ATP, with IC50 concentrations of approximately 1 microM and 70 microM, respectively. The profile of antagonism produced by PPADS was not that of a competitive antagonist. 6. The isoquinolene derivatives 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpi perazine (KN-62) and calmidazolium both produced antagonism which was not competitive, with IC50 concentrations of approximately 15 and 100 nM, respectively. HMA (5-(N,N-hexamethylene)- amiloride) was also an effective antagonist at a concentration of 10 microM. The group IIb metal, copper, also displayed antagonist properties at the human P2X7 receptor, reducing the maximum response to Bz-ATP by about 50% at a concentration of 1 microM. 7. These data demonstrate that the human recombinant P2X7 receptor displays functional behaviour which is similar to the recombinant rat P2X7 receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.

Adenosine Triphosphate↗

PubMed1998

Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells.

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.

Adenosine Triphosphate↗

PubMed1997

Functional evidence for multiple purinoceptor subtypes in the rat medial vestibular nucleus.

Extracellular recording techniques were used in brain slices to characterize excitatory responses produced by purine nucleotides in the rat medial vestibular nucleus, an area where functional purinoceptors have not previously been described. In the continued presence of the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine, which alone caused a small increase in the spontaneous firing rate, the P2 purinoceptor agonists alpha,beta-methyleneadenosine 5'-triphosphate (alphabeta meATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) caused concentration-dependent increases in spontaneous firing rate, with EC50 values of 41.8 and 1.7 microM, respectively. Only approximately 35% of all neurons studied displayed excitatory responses to these agents. Responses waned in the continued presence of high concentrations of the latter, but not the former agonist. Furthermore, in the continued presence of a maximal concentration of alphabeta meATP, ADPbetaS produced further increases in the firing rate of these neurons. The P2 antagonist, suramin, ablated responses to alphabeta meATP, but did not affect responses to ADPbetaS, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid antagonized responses to both agonists. The nucleotide analogue alpha,beta-methyleneadenosine 5'-diphosphate, which displays affinity for putative P2X receptors in brain, also produced concentration-dependent increases in firing frequency, which were also markedly antagonized in the presence of suramin, this agonist being only slightly less potent than alphabeta meATP. In conclusion, a subpopulation of rat medial vestibular neuronal responses mediated by both P2X and P2Y purinoceptors can be distinguished. Comparison of their properties with those of recombinantly expressed P2X and P2Y receptors suggests that these endogenous P2 purinoceptor types differ in several important aspects from heterologously expressed recombinant receptors identified from cloning studies.

Adenosine Diphosphate↗

PubMed1997

Properties of the pore-forming P2X7 purinoceptor in mouse NTW8 microglial cells.

1. We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X7 (P2Z) receptor in the NTW8 mouse microglial cell line. 2. At room temperature, in an extracellular solution containing 2 mM Ca2+ and 1 mM Mg2+, 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate (Bz-ATP; 300 microM), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of -90 mV, of 549 +/- 191 and 644 +/- 198 pA, respectively. Current-voltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong rectification. 3. In an extracellular solution containing zero Mg2+ and 500 microM Ca2+ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909 +/- 138 and 1818 +/- 218 pA, Bz-ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15-20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration-effect curves to Bz-ATP, ATP and 2-methylthio-ATP yielded EC50 values of 58.3, 298 and 505 microM, respectively. These affects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl- 2', 4'-disulphonic acid (PPADS; 30 microM) but not suramin (100 microM). 4. In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 microM) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 microM) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 32 degrees C, current augmentation also occurred in normal divalent cation containing solution. 5. These data demonstrate that mouse microglial NTW8 cells possess a purinoceptor with pharmacological characteristics resembling the P2X7 receptor. We suggest that the current augmentation phenomenon observed reflects formation of the large cytolytic pore characteristic of this receptor. We have demonstrated that pore formation can occur under normal physiological conditions and can be modulated pharmacologically, both positively and negatively.

Adenosine Triphosphate↗

PubMed1997

Somatostatin5 receptor-mediated [35S]guanosine-5'-O-(3-thio)triphosphate binding: agonist potencies and the influence of sodium chloride on intrinsic activity.

We studied the activation of the human somatostatin5 receptor recombinantly expressed in CHO-K1 cells by using some newly available agonists and antagonists. Somatostatin-28 bound to this receptor with a higher affinity than somatostatin-14 and was more potent in increasing [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding. Somatostatin-14-induced [35S]GTPgammaS binding to membranes from this cell line was decreased in a concentration-related manner by increasing concentrations of GDP and sodium chloride. At 50 mM (low) sodium, agonist EC50 values for stimulating [35S]GTPgammaS binding were lower than those at 150 mM (high) sodium and were closer to their respective affinity estimates (dissociation equilibrium constants) for binding to the receptor in the absence of sodium. Both agonist binding to the high affinity state of the receptor and agonist-induced [35S]GTPgammaS binding were abolished by pertussis toxin pretreatment. The putative somatostatin5 receptor-selective ligand L-362,855, unlike somatostatin-14 and somatostatin-28, showed differential intrinsic activity for stimulation of [35S]GTPgammaS binding, behaving as a partial agonist in high sodium and a full agonist in low sodium. In contrast, BIM-23056 did not behave as an agonist under any conditions studied but was able to antagonize somatostatin-14-induced [35S]GTPgammaS binding. We conclude that measurement of [35S]GTPgammaS binding mediated by somatostatin receptor activation in the presence of different concentrations of sodium chloride provides a useful functional assay for assessing the relative agonist efficacies of novel ligands identified from radioligand binding studies.

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