Erythrocytes have been suggested to participate in the regulation of vascular resistance in the lung (1), striated muscle (2) and isolated cerebral arterioles (3). The finding that erythrocytes of humans and rabbits (1, 4) as well as hamsters (2) and rats (3) release ATP in response to physiological stimuli such as reduced oxygen tension (2, 3, 5) and mechanical deformation (1,4,6) is consistent with the hypothesis that erythrocyte-derived ATP is a determinant of vascular resistance in vivo. It was reported that the cystic fibrosis transmembrane conductance regulator (CFTR) (4) as well as protein kinase A (PKA) (6), adenylyl cyclase (6) and the heterotrimeric G protein, Gs, (7) are components of the membrane of erythrocytes and function in a signal-transduction pathway relating deformation to ATP release.

In addition to Gs, heterotrimeric G proteins of the Gi subclass have been identified as components of the membrane of human erythrocytes (8). G proteins of the Gi subclass have been reported to be activated by mechanical force (9), a known stimulus for ATP release from erythrocytes (1,4,6). Gi was originally described as an inhibitor of the activity of adenylyl cyclase activity (10). It is now clear, however, that although some isoforms that enzyme are inhibited via the activity of the a subunit (10), some bg dimers, released upon dissociation of this heterotrimeric G protein can, in the presence of a subunit of Gs, stimulate the activity of other isoforms of adenylyl cyclase (11-14). Here we investigated the hypothesis that stimulation of heterotrimeric G proteins of the Gi subclass results in ATP release from human erythrocytes. In addition, we characterized the b subunits in the human erythrocyte membranes to determine if subunits capable of stimulating adenylyl cyclase (subunits 1, 2, 3, or 4) are present (11-14).


MATERIAL AND METHODS
Preparation of washed red blood cells

Human blood was obtained by venipuncture without use of a tourniqet. Blood (35 ml) was collected in a syringe containing heparin (500 units) and centrifuged at 500 x 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 (in mM;21.0 Tris-HCL, 4.7 KCl, 2.0 CaCl2, 140.5 NaCl, 1.2 MgS04 with 2.5% dextrose and 0.5% bovine serum albumin, fraction V, final pH adjusted to 7.4). After the last centrifugation, the hematocrit of the erythrocytes was determined. The protocol for blood collection was approved by the Institutional Review Committee of Saint Louis University.

Preparation of Erythrocyte Membranes

Washed erythrocytes were diluted 1:100 with ice-cold lysis buffer (5 mM Tris-HCL, 2 mM EDTA, pH 7.4), and stirred at 4°C for 20 min. The resulting lysate was centrifuged at 23,000 x g for 15 min at 4°C. The hemoglobin-containing supernatant was removed and discarded. The pellet containing crude erythrocyte membranes was re-suspended in ice-cold lysis buffer and centrifuged at 23,000 x g for 15 min at 4°C for a second time. The resultant membrane pellet was aliquoted and frozen at -80°C. The protein concentration of the membrane preparation was determined using a BCA protein assay (Pierce, Rockford, III).

Western Blot Analysis

Membrane proteins were solubilized in sample buffer (2% SDS, 15% glycerol, 100 mM dithiothreitol, 62.5 mM Tris-HCL pH 6.8,0.01% bromphenol blue) and resolved by electrophoresis in 10% SDS-polyacrylamide gels (1:37.5 acrylamide to bis-acrylamide). Following electrophoresis, the proteins were transferred to a PVDF membrane in transfer buffer (25 mM Tris-base, 192 mM glycine and 10% methanol). The PVDF membranes were incubated for 1 h in buffer containing 5% non-fat dry milk, 10mM NaP04,150mM NaCl and 0.1%Tween-20atpH7.4. The membranes were then immunoblotted with one of four rabbit polyclonal antibodies specific for heterotrimeric G protein ß subunits 1 through 4 (anti-ß1,2 and 3 antibodies were obtained from Calbiochem, La Jolla, CA and anti-ß 4 antibody was obtained from Sant Cruz Biotechnology, Sant Cruz, CA) in a buffer solution containing 1% non-fat dry milk, 10 mM NaPO4, 150 mM NaCI and 0.1% Tween-20 at pH 7.4. The specificity of the antibodies is as follows: anti-ß1 does not recognize ß2, ß3 or ß5 subunits; anti-ß2 does not recognize ß1 or ß3 subunits and reacts only weakly with ß5; anti-ß3 does not recognize ß1, ß2 or ß5 subunits and anti-ß4 is stated to be specific for that subunit. Importantly, an authentic standard for the ß3 subunit (Calbiochem, La Jolla, CA) and a blocking peptide that specifically inhibits the binding of the anti-ß4 antibody to the ß4 protein (Sant Cruz Biotechnology, Sant Cruz, CA) were used to verify the selectivity of the antibodies directed at these ß subunits. Antibody-bound proteins were visualized by enhanced chemiluminescence after incubation for 1 hr with 3 µl donkey anti-rabbit IgG peroxide-linked antibody (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) in 15 ml of phosphate buffered saline containing 1% milk.

Measurement of ATP and Hemoglobin

ATP was measured by the luciferin-luciferase technique (1-4) which utilizes the ATP concentration-dependence of light generated by the reaction of ATP with firefly tail extract. In brief, a 200 µl sample of the RBC suspension was injected into a cuvette containing 100 µ1 crude firefly tail extract (5 mg/5 ml distilled water, FLE-50, Sigma, St. Louis, MO) and 100 µl of synthetic D- luciferin (50 mg/100 ml distilled water, Sigma, St. Louis, MO). The light emitted was detected using a luminometer (Turner-Designs, TD-20/20, Sunnyvale, CA). A standard curve was obtained on the day of each experiment. To exclude the presence of significant hemolysis, after ATP determinations, samples were centrifuged at 1000 x g at 4°C for 10 min. The presence of hemoglobin in the supernatant was then determined by light absorption at a wavelength of 405 nm. In response to the application of mastoparan, the ATP signal increased in the absence of any consistent increase in hemoglobin. All data from experiments in which increases in hemoglobin were detected were excluded. To ensure that the results of the ATP assay were not altered by interference from mastoparan, the effects ofmastoparin on ATP measurement was determined. At the concentrations used in this study, mastoparan did not alter the sensitivity of the assay for authentic ATP standard solutions. Finally, in all experiments, ATP content oferythrocytes was determined by measurement of ATP in solution following lysis of a known number of RBCs in distilled water.

Incubation of Erythrocytes with Mastoparan or its Vehicle

Erythrocytes (hematocrit 10%), were incubated at 37 °C with mastoparan (MAS, 10 µM), an agent that stimulates the activity ofheterotrimeric G proteins of the Gi/o subclass (15) (Biomol Research Labs Inc., Plymouth Meeting, PA) or its vehicle (saline).

Statistical Methods

Statistical significance between experimental periods was determined with a Student's T-test. A P value of 0.05 or less was considered statistically significant. Results are reported as means ± SEM.


RESULTS
Effect of Mastoparan on ATP Release from Erythrocytes

Incubation of human erythrocytes with mastoparan resulted in a 307 ± 61% increase in ATP concentration (P<0.01, n=5, Fig. 1). Maximal ATP release in response to mastoparan administration occurred at 5 min after exposure in three studies and after 10 and 15 min in two additional experiments. The concentration of ATP in the erythrocytes was 1.80 ± 0.41 mM.

Fig. 1. Effect of incubation of human erythrocytes (20% hematocrit, n=5) with mastoparin (10 µM) or its vehicle (saline) on ATP release (per 2 x 105 RBCs/mm3 ). *, different from vehicle value (P<0.01).

Identification of the Heterotrimeric G Protein ? Subunits in Human Erythrocyte Membranes

Immunoblots oferythrocyte membranes were probed with antibodies directed against either ß1, ß2, ß3 or ß4 subunits. Human erythrocyte membranes stained positive for the ß1 and ß2 subunits with apparent molecular weights of 36 and 35 kDa, respectively (Fig. 2, A, B). The membranes also stained positive for the ß3 subunit (Fig. 2, C). The presence of ß3 was confirmed with an authentic ß3 standard (MW: 39.9 kDa) (Fig. 2, C). Finally, the ß4 subunit was identified in human erythrocyte membranes with an apparent molecular weight of approximately 35 kDa (Fig. 2, D).

Fig. 2. Identification of heterotrimeric G protein ß subunits in human erythrocyte membranes. Membranes were prepared as described and the protein resolved using a 10% SDS-PAGE gel, transferred to PVDF membrane and incubated with rabbit polyclonal antibodies directed against ß subunits 1, 2, 3 and 4. Panel A; erythrocyte membranes (100 µg of protein) probed with anti-ß1 antibody (n=3). Panel B; erythrocyte membranes (75 µg of protein) probed with anti-ß2 antibody (n=5). Panel C; authentic ß3 standard (1; 125 ng of protein) and erythrocyte membranes (2; 100 µg of protein) probed with anti-ß3 antibody (n=3). Panel D; erythrocyte membranes (100 µg of protein) probed with anti-ß4 antibody (n=5).

In the case of the ß4, immunoreactivity was attenuated by preincubation of the antibody with a blocking peptide.


DISCUSSION
Heterotrimeric G proteins, composed of alpha, ß, and subunits, are components of a vast array of signal-transduction pathways in mammalian cells. Nomenclature for the specific heterotrimeric G proteins is based on the identification and activity of the a subunit component of the trimer. Recently, it has become increasingly clear that, in addition to the alpha subunit, ß subunits of G proteins have the capacity to participate in signal-transduction pathways. Indeed it was reported that ß subunits participate in the regulation of the activity of adenylyl cyclase (11-14), muscarinic K+ channels (16-17) and the ß2 isoform of phospholipase C (18-19). Moreover, it has been reported that for an individual heterotrimeric G protein, the a subunit does not necessarily activate the same effectors as does the associated ß dimer (20).

The finding that ß subunits of heterotrimeric G proteins can activate some types of adenylyl cyclase is of particular interest with respect to ATP release from erythrocytes. We have reported that the activation of adenylyl cyclase is a required component of a signal-transduction pathway that relates mechanical deformation of erythrocyte to ATP release (6). Thus, incubation of human erythrocytes with forskolin, an agent that stimulates adenylyl cyclase activity, resulted in both increases in intracellular cAMP and ATP release (6). These studies establish that adenylyl cyclase is a component of a signal-transduction pathway for ATP release from erythrocytes.

Here, we demonstrate that incubation of human erythrocytes with mastoparan, an agent that directly activates heterotrimeric G proteins of the Gi/o subclass (15) results in activation of a signal-transduction pathway for ATP release. It was reported previously that human erythrocyte membranes contain the heterotrimeric G protein Gi, but not Go (8). These reports are consistent with the hypothesis that the release of ATP from human erythrocytes in response to incubation with mastoparin resulted from the activation of Gi.

The finding that activation of the heterotrimeric G protein, Gi, results in ATP release suggests that this effect is not mediated via the activity of the a subunit. Indeed, the a subunit of Gi inhibits the activity of adenylyl cyclase types V and VI, but does not alter the activity of other adenylyl cyclase types (10, 20). In contrast, heterotrimeric G protein ß subunit types 1, 2, 3 and 4, in association with a common g subunit, have been shown to activate adenylyl cyclase types II and IV (11-14). The latter findings provide support for the hypothesis that one or more of these subunits could, via stimulation of the activity of adenylyl cyclase, be a component of a signal-transduction pathway for ATP release from erythrocytes.

To address this important issue, we looked for the presence of b subunits of the 1, 2, 3 and 4 subtypes in the membrane of human erythrocytes. As shown in figure 2, all four of the ß subunits that have bee reported to stimulate adenylyl cyclase types II and IV are present in human erythrocyte membranes. Although these studies do not establish which of these ß subunits are associated with the a subunit of Gi in the erythrocyte membrane, the data demonstrate that exposure of human erythrocytes to mastoparan results in activation of heterotrimeric G proteins of the Gi subclass and that this is associated with the stimulation of a signal-transduction pathway for ATP release. Since ATP release from erythrocytes requires the activation ofadenylyl cyclase, the data provides strong support for the hypothesis that this effect of activated Gi is mediated via the activity of bg subunits associated with that heterotrimeric G protein in the erythrocyte membrane.

In summary, we have demonstrated that incubation of human erythrocytes with mastoparan, an activator of the heterotrimeric G protein, Gi, results in the release of ATP. In addition, we have characterized the types of G protein ß subunits that are present in the human erythrocyte membrane as those capable of stimulating some types of adenylyl cyclase (11-14), a known component of the signal-transduction pathway for ATP release from human and rabbit erythrocytes (6). These findings provide strong support for the hypothesis that heterotrimeric G proteins of the Gi subclass are components of a signal-transduction pathway that relates deformation of erythrocytes to ATP release and that this activity likely resides with the activity of the ß subunit.

Acknowledgments: The authors thank Dr. J. Baldassare for technical assistance and advice and J. L. Sprague for inspiration. This work is supported by National Heart Lung and Blood Institute Grants HL-51298, HL-52675 and HL-39226.


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