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Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104
Submitted 18 July 2003 ; accepted in final form 4 November 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i1, Gi2, and Gi3 but not Go were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples Gi/o from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 µM) or Mas-7 (1 µM), which are compounds that directly activate Gi proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 µM), which is an inactive Mas analog. In separate experiments, Mas (10 µM) but not Mas-17 (10 µM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein Gi is a component of a signal transduction pathway for ATP release from erythrocytes.
pulmonary circulation; red blood cells; mechanical deformation; adenine nucleotides
Although the previously described studies identify stimuli for ATP release from erythrocytes, they do not propose a mechanism by which ATP could exit the cell. ATP is a large, highly charged molecule; as such, it is not likely to diffuse through the erythrocyte membrane. Therefore, it is reasonable to propose that the release of ATP from erythrocytes occurs via the activation of a signal transduction pathway. Sprague et al. reported that deformation-induced ATP release from erythrocytes required the activity of the cystic fibrosis transmembrane conductance regulator (21), protein kinase A, and adenylyl cyclase (23).
The catalytic activity of adenylyl cyclase can be stimulated or inhibited by the heterotrimeric G proteins Gs and Gi/o, respectively (8). Our laboratory identified Gs as a component of rabbit and human erythrocyte membranes (20). In addition, we reported that receptor-mediated activation of Gs using iloprost or epinephrine resulted in ATP release from both rabbit and human erythrocytes (20). These data provide support for the hypothesis that Gs is also a component of a signal transduction pathway for ATP release.
In addition to stimulation by the -subunit of the heterotrimeric G protein Gs, adenylyl cyclase activity can be regulated by the heterotrimeric G protein Gi/o (8). Once activated, the -subunit of Gi/o inhibits certain isoforms of adenylyl cyclase. It is now recognized that the 
-subunit complex, which dissociates from the -subunit upon G protein activation, can also regulate the catalytic activity of adenylyl cyclase. The -subunit complex can inhibit some isoforms of adenylyl cyclase but has been reported to stimulate others (28). Therefore, within the proposed signal transduction pathway for ATP release from erythrocytes, the activation of Gi/o could either inhibit or stimulate adenylyl cyclase activity depending on which isoforms are present in the erythrocyte membrane. Identification of the heterotrimeric G protein Gi/o as a component of a signal transduction pathway for ATP release is of particular interest, because G proteins from this family are reported to be activated by mechanical forces (9–11), which are a known stimulus for ATP release from erythrocytes (21, 22).
In the present study, we investigated the hypothesis that heterotrimeric G proteins of the Gi/o subclass are components of a signal transduction pathway for ATP release from rabbit erythrocytes. We characterized the G proteins of the Gi/o family that are present in erythrocyte membranes. In addition, we investigated the effects of pertussis toxin (PTX), which inhibits the activity of Gi/o, on deformation-induced ATP release from rabbit erythrocytes. Finally, we examined the release of ATP from rabbit erythrocytes in response to pharmacological agents that directly stimulate the activity of Gi/o.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rabbit or human blood was centrifuged at 500 g for 10 min at 4°C. The plasma, buffy coat, and uppermost erythrocytes were removed by aspiration and discarded. The remaining erythrocytes were washed three times in buffer solution that contained 140.5 mM NaCl, 21.0 mM Tris·HCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 0.1% dextrose, and 0.5% bovine serum albumin, fraction V (ICN Biomedicals; Aurora, OH) with a final pH of 7.4. The hematocrit of the washed erythrocytes was determined using an Autocrit Ultra 3 centrifuge (Becton Dickinson; Bedford, MA).
Preparation of erythrocyte membranes. Washed rabbit or human erythrocytes were diluted 1:100 with ice-cold lysis buffer (5 mM Tris·HCl and 2 mM EDTA, pH 7.4) and stirred at 4°C for 15 min. The resulting mixture was centrifuged at 23,000 g for 15 min at 4°C. The supernatant was removed and discarded. The pellets, which contained the erythrocyte membranes, were pooled and resuspended in ice-cold lysis buffer and were then centrifuged at 23,000 g for 15 min at 4°C. The supernatant was discarded and the membranes were resuspended in ice-cold lysis buffer. The resuspended membranes were aliquoted and frozen at –80°C. The protein concentrations were determined using a BCA protein assay (Pierce; Rockford, IL).
Incubation of erythrocytes with pharmacological agents that activate Gi/o. Washed rabbit erythrocytes were brought to a 20% hematocrit in a physiological salt solution (PSS) that contained 140.5 mM NaCl, 21.0 mM Tris·HCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 0.1% dextrose, and 0.5% bovine serum albumin equilibrated to 37°C for a minimum of 30 min. The erythrocyte suspension was then incubated with the Gi/o activator mastoparan (Mas, 10 µM, Bio-Mol; Plymouth Meeting, PA), its more active derivative, Mas-7 (1 µM, Bio-Mol), or an inactive derivative of Mas, Mas-17 (10 µM, Bio-Mol). Incubations of 5, 10, and 15 min were performed to determine the peak response to each pharmacological agent. The concentration of ATP released into the erythrocyte suspension, total intracellular ATP, and presence of hemoglobin in the erythrocyte suspension solution were measured in each experiment.
Incubation of erythrocytes with PTX. Washed rabbit erythrocytes, brought to a 20% hematocrit, were incubated for 2 h with 100 ng/ml PTX (holotoxin, Calbiochem; La Jolla, CA) or PSS that contained 140.5 mM NaCl, 21.0 mM Tris·HCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 0.1% dextrose, and 0.5% bovine serum albumin, the vehicle for PTX. After incubation with PTX, the erythrocytes were either incubated for 10 min with pharmacological agents that activate Gi/o or subjected to mechanical deformation as described below.
Western blot analysis. Membrane proteins were solubilized in sample buffer (2% SDS, 15% glycerol, 100 mM dithiothreitol, 62.5 mM Tris·HCl, pH 6.8, and 0.01% bromophenol blue) and resolved by electrophoresis in 10% SDS-PAGE gels (7 cm, 1:37.5 ratio of acrylamide to bis-acrylamide). After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane in transfer buffer (25 mM Tris base, 192 mM glycine, and 10% methanol). The PVDF membranes were blocked overnight in a buffer solution that contained 5% nonfat dry milk, 10 mM sodium phosphate, 150 mM sodium chloride, and 0.1% Tween 20 at pH 7.4. The PVDF membranes were then immunoblotted with either a mouse monoclonal anti-Gi1, -Gi2, or -Go antibody (Bio-Mol) or a rabbit polyclonal anti-Gi3 antibody (Bio-Mol) in a buffer solution that contained 1% nonfat dry milk, 10 mM sodium phosphate, 150 mM sodium chloride, and 0.1% Tween 20 at pH 7.4. The PVDF membranes were washed three times with 10 mM sodium phosphate, 150 mM sodium chloride, and 0.1% Tween 20 at pH 7.4 and were immunoblotted with either anti-rabbit or anti-mouse immunoglobulin secondary antibody conjugated to horseradish peroxidase (Amersham; Piscataway, NJ). Protein was visualized using enhanced chemiluminescence (Amersham).
Mechanical deformation of washed erythrocytes. Washed rabbit erythrocytes were brought to 10% hematocrit with a PSS that contained 140.5 mM NaCl, 21.0 mM Tris·HCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 0.1% dextrose, and 0.5% bovine serum albumin and were then subjected to mechanical deformation using the St. George's blood filtrometer (Carri-Med; Dorking, UK). As described previously, this device creates a calibrated pressure gradient across a vertically mounted filter with pores having an average diameter of either 8 or 5 µm (22). Deformation was achieved by passing the suspended erythrocytes through the filter. The red blood cell transit time (RCTT), which is a measure of erythrocyte deformation, as well as the concentration of ATP in the filter effluent were determined.
Measurement of ATP. ATP was measured using the luciferinluciferase technique (24). A 200-µl sample of an erythrocyte suspension was injected into a cuvette that contained 100 µl of a 1 mg/ml crude firefly-tail extract (Sigma; St. Louis, MO) and 100 µl of a 0.5 mg/ml solution of D-luciferin (Sigma). The addition of D-luciferin to the reaction mixture increases the sensitivity of the assay. The light emitted from the reaction of ATP with the crude firefly extract-D-luciferin solution was measured using a luminometer set at a wavelength of 565 nm (model TD-20/20, Turner Designs; Sunnyvale, CA). The emission peak was compared with an ATP standard curve generated on the day of the experiment. The concentration of ATP was determined by comparing the sample signal with that of the standard curve. Standard curves run in the absence of erythrocytes were not different from those run in the presence of nonstimulated erythrocytes.
To ensure that the agents with which the erythrocytes were incubated did not alter the results of the ATP assay, the effects of Mas, Mas-7, and Mas-17 on the measurements of authentic ATP were determined. Pharmacological agents at the concentrations used in these studies did not have a direct effect on the ATP measurements.
Measurement of erythrocyte lysis. To exclude the possibility of sample hemolysis, after measuring ATP in the erythrocyte suspension, erythrocytes were sedimented by centrifugation at 500 g for 10 min. The presence of hemoglobin in the supernatant was determined by light absorption at 405 nm (29).
To ensure that the method used to detect erythrocyte lysis was as sensitive as the assay used to measure ATP release, washed rabbit erythrocytes were lysed and serial dilutions were made. The concentrations of ATP and the absorbances at 405 nm were measured for each dilution. In dilutions where ATP was measured, an absorbance at 405 nm was also detected, which verified that the assay used to measure erythrocyte lysis was as sensitive as the assay used to measure ATP release.
Measurement of cAMP. Washed rabbit erythrocytes were brought to a 50% hematocrit in a buffer that contained 140.5 mM NaCl, 21.0 mM Tris·HCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 0.1% dextrose, and 0.5% bovine serum albumin (PSS). The erythrocyte suspension was then incubated with either Mas (10 µM), Mas-17 (10 µM), or vehicle (PSS) in the presence of IBMX (100 µM) for 10 min. After the incubation, the reaction was stopped by the addition of 1 ml of the erythrocyte suspension to 4 ml of ice-cold ethanol that contained 1 mM HCl. The samples were then centrifuged at 21,000 g for 10 min at 4°C. The supernatant was removed from the resulting pellet and stored overnight at –20°C to precipitate any remaining protein. Samples were centrifuged a second time at 3,700 g for 10 min at 4°C. The supernatant was removed and dried under vacuum centrifugation. The concentration of cAMP for each sample was determined in duplicate using the cAMP Enzyme Immunoassay Biotrak system (Amersham).
Statistical methods. Statistical significance between experimental periods was determined with a Student's t-test or, where appropriate, an ANOVA. When using an ANOVA, a least-significant difference test was used to identify individual differences in the event that the F-ratio indicated that changes had occurred. P values 
0.05 were considered statistically significant. Results are reported as means ± SE.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i/o in erythrocyte membranes. If the heterotrimeric G proteins Gi/o are components of a signal transduction pathway for ATP release, then these proteins must be present in the erythrocyte membrane. The heterotrimeric G proteins Gi1, Gi2, and Gi3 were identified in purified human erythrocyte membranes (3). Here we confirmed this finding in human erythrocytes and demonstrated that rabbit erythrocyte membranes also stain positively for Gi1, Gi2, and Gi3 (Fig. 1). However, neither rabbit nor human erythrocyte membranes stained positively for Go (Fig. 1).
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Effects of PTX on deformation-induced ATP release from erythrocytes. To provide support for the hypothesis that the heterotrimeric G protein Gi is involved in a signal transduction pathway for deformation-induced ATP release from erythrocytes, washed rabbit erythrocytes were incubated with PTX (100 ng/ml, 2 h) and then mechanically deformed by passage through filters with a pore size of either 8 or 5 µm using the St. George's blood filtrometer. PTX catalyzes the mono-ADP-ribosylation of the -subunit of Gi/o, thereby preventing its dissociation from the -subunit complex (15). This action of PTX uncouples both the -subunit and the -subunit complex from their effector proteins. As illustrated in Fig. 2, PTX inhibited deformation-induced ATP release from rabbit erythrocytes that resulted from passage through 8- and 5-µm pore filters. Incubating the erythrocytes with PTX had no effect on erythrocyte deformability as measured by passage through 8-µm pore filters (RCTT, 5.4 ± 0.2 vs. 5.3 ± 0.2 for control and PTX-treated erythrocytes, respectively). However, PTX did produce a small but significant decrease in erythrocyte deformability as measured by passage through 5-µm pore filters (RCTT, 8.0 ± 0.3 vs. 8.7 ± 0.4 for control and PTX-treated erythrocytes, respectively). Incubating rabbit erythrocytes with PTX did not significantly alter total intracellular concentrations of ATP in experiments using either the 8- or 5-µm pore filters; concentrations were 1.58 ± 0.31 mM/erythrocyte before PTX treatment and 1.54 ± 0.10 mM/erythrocyte after PTX treatment for 5-µm pore filters and 1.66 ± 0.39 mM/erythrocyte before PTX treatment and 1.95 ± 0.36 mM/erythrocyte after PTX treatment for 8-µm pore filters.
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Effects of pharmacological agents that directly activate Gi on ATP release from erythrocytes. To demonstrate that activation of Gi has an effect on ATP release, washed rabbit erythrocytes were incubated with either Mas or Mas-7. Mas is a cell-permeable wasp venom tetradecapeptide that directly activates Gi and thereby stimulates the dissociation of the -subunit from the -subunit complex (13). Mas-7 is an analog of Mas with approximately fivefold greater potency (13). Incubation of washed rabbit erythrocytes with either Mas (10 µM) or Mas-7 (1 µM) resulted in ATP release (Fig. 3). Importantly, incubation of washed rabbit erythrocytes with Mas or Mas-7 did not result in erythrocyte lysis: measurements of hemoglobin absorbance at 405 nm were 0.07 ± 0.01 for untreated cells; 0.08 ± 0.03 for Mas (10 µM)-treated cells; and 0.06 ± 0.01 for Mas-7 (1 µM)-treated cells. Incubation of human erythrocytes with Mas (10 µM) also resulted in an increase in ATP release (Fig. 4) in the absence of erythrocyte lysis: absorbance of hemoglobin at 405 nm measured 0.08 ± 0.03 for vehicle and 0.10 ± 0.03 for Mas-treated human erythrocytes. Importantly, washed rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 µM), which is an inactive Mas analog that is used as a negative control for the mastoparans (13); ATP values measured 0.50 ± 0.10 vs. 0.34 ± 0.05 µM per 2 x 105 erythrocytes/mm3 for untreated (n = 9) and Mas-17-treated (n = 6) erythrocytes, respectively.
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Effects of Mas on intracellular concentrations of cAMP in erythrocytes. Sprague et al. (23) identified adenylyl cyclase as a component of a signal transduction pathway for ATP release from erythrocytes. Therefore, if activation of Gi using Mas resulted in ATP release from erythrocytes, then Mas could also be expected to stimulate increases in intracellular concentrations of cAMP. Incubation of rabbit erythrocytes with Mas (10 µM) in the presence of IBMX (100 µM) resulted in an increase in intracellular concentrations of cAMP (Fig. 5). Importantly, the inactive Mas analog Mas-17 (10 µM) in the presence of IBMX (100 µM) was without effect on intracellular concentrations of cAMP (2.36 ± 0.13 vs. 2.39 ± 0.12 pmol cAMP per 3 x 105 erythrocytes/mm3 for vehicle and Mas-17-treated erythrocytes, respectively; n = 9). Incubation of washed rabbit erythrocytes with forskolin-IBMX (100:100 µM) as a positive control resulted in an increase in intracellular concentrations of cAMP to 2.36 ± 0.07 vs. 5.62 ± 0.25 pmol cAMP per 3 x 105 erythrocytes/mm3 for vehicle and forskolin-IBMX-treated erythrocytes, respectively (n = 18; P < 0.001).
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Effects of PTX on Mas-induced ATP release from erythrocytes. To ensure that the action of Mas was specific for the heterotrimeric G protein Gi, rabbit erythrocytes were preincubated with PTX (100 ng/ml, 2 h). PTX mono-ADP-ribosylates the -subunit of Gi, which prevents the dissociation of the -subunit from the -subunit complex, and thereby uncouples the -subunit and the -subunit complex from their effector proteins (15). Therefore, if Mas elicits its effects on ATP release by stimulating the dissociation of the -subunit from the -subunit complex, then preincubation of the cells with PTX would be expected to block that response. Mas-induced ATP release from washed rabbit erythrocytes was completely inhibited when the erythrocytes were preincubated with PTX (Fig. 6). Incubation of rabbit erythrocytes with PTX did not statistically alter total intracellular concentrations of ATP (1.58 ± 0.16 vs. 1.40 ± 0.12 mM/erythrocyte before and after incubation with PTX, respectively).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although previous studies identified the stimuli for the release of ATP from erythrocytes, they did not describe the mechanism by which the ATP is released. ATP is a large, highly charged molecule; its diffusion through the erythrocyte membrane is improbable. Moreover, because the release of ATP can be related to distinct physiological stimuli, it is likely that the mechanism by which ATP exits the erythrocyte involves a signal transduction pathway. Our laboratory has identified the cystic fibrosis transmembrane conductance regulator (21), adenylyl cyclase, protein kinase A (23), and the heterotrimeric G protein Gs (20) as components of a signal transduction pathway for ATP release from rabbit and human erythrocytes.
In addition to regulation by the -subunit of the heterotrimeric G protein Gs, the catalytic activity of adenylyl cyclase is also regulated by heterotrimeric G proteins of the Gi/o subtype. Here we investigated the hypothesis that heterotrimeric G proteins of the Gi/o subtype participate in a signal transduction pathway that is responsible for ATP release from erythrocytes.
The heterotrimeric G proteins Gi1, Gi2, and Gi3 but not Go were identified in both rabbit and human erythrocyte membranes using Western blot analysis (see Fig. 1). The latter finding is consistent with previous reports (3). However, the identification of Gi proteins in the erythrocyte membrane by Western blot analysis alone does not determine that the protein is a component of a signal transduction pathway for ATP release. To determine whether Gi proteins are involved in a signal transduction pathway for deformation-induced ATP release, rabbit erythrocytes were mechanically deformed by passage through filters with average pore diameters of either 8 or 5 µm in the presence and absence of PTX holotoxin. Upon binding to and entering the erythrocyte (12, 25, 30), PTX specifically mono-ADP-ribosylates the -subunit of Gi and prohibits its dissociation from the -subunit complex, thereby uncoupling both the -subunit and the -subunit complex from their effector proteins. Therefore, within the proposed signal transduction pathway for ATP release from erythrocytes, uncoupling of Gi from adenylyl cyclase would be expected to permit greater stimulation of adenylyl cyclase by the -subunit of the heterotrimeric G protein Gs and a subsequent increase in ATP release. Interestingly, in the present study, ATP release resulting from mechanical deformation of rabbit erythrocytes, induced by passage through filters with either an 8- or 5-µm pore size, were both inhibited by PTX (see Fig. 2). In experiments in which PTX-treated erythrocytes were mechanically deformed by passage through filters with a 5-µm pore size, there was a small but statistically significant decrease in erythrocyte deformability. Therefore, a portion of the inhibition measured with PTX could result from an effect of PTX on erythrocyte deformability. However, in experiments whereby PTX-treated erythrocytes were mechanically deformed by passage through filters with an 8-µm pore size, there was not a measurable difference in erythrocyte deformability. In addition, results with Mas suggest that the ability of PTX to inhibit ATP release is independent of erythrocyte deformability. The inhibition of mechanical deformation-induced ATP release from erythrocytes by PTX provides support for the hypothesis that Gi is a component of a signal transduction pathway for ATP release from erythrocytes.
Cellular responses resulting from the activation of heterotrimeric G proteins are due not only to the effects of the -subunit but also the -subunit complex, which dissociates from the -subunit upon G protein activation. The -subunit complex can also regulate cellular responses (27, 28). The -subunit as well as the -subunit complex can regulate adenylyl cyclase activity either individually or synergistically (8, 26). The -subunit of Gi inhibits the catalytic activity of adenylyl cyclase types I, III, VIII and types V and VI, which results in a decrease in intracellular concentrations of cAMP. The -subunit of Gi does not influence the catalytic activity of adenylyl cyclase types II, IV, and VII (8, 18, 19, 26), whereas the -subunit complex of activated Gi stimulates adenylyl cyclase types II, IV, and VII (6, 7, 17, 28). Interestingly, it has been demonstrated that the -subunit complex can act synergistically with the -subunit of Gs to stimulate adenylyl cyclase types II, IV, and VII (6, 7, 17, 28). Thus within the proposed signal transduction pathway for ATP release from erythrocytes, activation of Gi would be expected to inhibit ATP release only if its -subunit acted on adenylyl cyclase type I, III, or VIII or type V or VI. If adenylyl cyclase type II, IV, or VII were present in the erythrocyte membrane, then activation of Gi/o would be expected to stimulate ATP release via stimulation of adenylyl cyclase by the -subunit complex. PTX would therefore be expected to inhibit deformation-induced ATP release from erythrocytes by preventing the dissociation of the -subunit from the -subunit complex, thereby uncoupling the -subunit complex from adenylyl cyclase.
To determine whether activation of Gi proteins affects ATP release, intact rabbit erythrocytes were incubated with Mas, a tetradecapeptide that directly activates Gi proteins (13). Incubation of rabbit erythrocytes with Mas resulted in an increase in cAMP accumulation and an increase in ATP release (see Figs. 5 and 3, respectively). Human erythrocytes also released ATP when incubated with Mas (see Fig. 4). In addition, Mas-7, a more potent analog of Mas, also stimulated ATP release from rabbit erythrocytes (see Fig. 3). Mas- and Mas-7-induced increases in ATP release were not the result of erythrocyte lysis. In separate experiments, rabbit erythrocytes incubated with Mas-17, the inactive analog of Mas, did not stimulate cAMP accumulation or ATP release. To demonstrate that the stimulatory effect of Mas on ATP release was due to the activation of Gi, we determined that Mas-induced ATP release from rabbit erythrocytes was inhibited by pretreatment of erythrocytes with PTX (see Fig. 6). Taken together, these data provide strong support for the hypothesis that activation of Gi either mechanically via deformation or pharmacologically using Mas stimulates a signal transduction pathway that results in ATP release from erythrocytes.
Interestingly, heterotrimeric G proteins from the Gi family have been reported to serve as mechanosensors (9). These sensors translate extracellular mechanical forces such as fluid flow or strain into intracellular biochemical signals. It was reported (11) that the heterotrimeric G protein Gi3 was activated within 1 s when confluent human umbilical vein endothelial cell monolayers were exposed to fluid flow in a parallel-plate flow chamber. This flow-induced activation of Gi3 was inhibited when cells were incubated with PTX. In similar studies using adult rat cardiac fibroblasts, mechanical strain was reported to activate the heterotrimeric G protein Gi1 (10). Erythrocytes are subjected to mechanical stress when they traverse microvessels and/or when they are exposed to increases in the velocity of blood flow. Therefore, the heterotrimeric G protein Gi could serve as a mechanosensor in the mature erythrocyte by triggering a proposed signal transduction pathway for ATP release.
The results presented here provide support for the hypothesis that heterotrimeric G proteins of the Gi subtype are involved in a signal transduction pathway for ATP release from rabbit erythrocytes. We have identified the heterotrimeric G proteins Gi1, Gi2, and Gi3 but not Go in both human and rabbit erythrocytes. Activation of Gi with Mas resulted in ATP release possibly via -subunit activation of adenylyl cyclase. The results supporting this mechanism are strengthened by studies in which PTX inhibited both Mas- and deformation-induced ATP release from rabbit erythrocytes. Taken together, these data identify Gi as a component of a unique signal transduction pathway for the release of ATP from erythrocytes.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Heart, Lung, and Blood Institute Grants HL-64180 and HL-51298 and National Institute of General Medical Sciences Training Grant GM-08306-11.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P < 0.05, different from vehicle.
