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NO inhibits signal transduction pathway for ATP release from erythrocytes via its action on heterotrimeric G protein G_i

Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri 63104

Submitted 17 February 2004 ; accepted in final form 30 March 2004

	ABSTRACT

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The release of ATP from erythrocytes involves a signal transduction pathway of which cystic fibrosis transmembrane conductance regulator, PKA, adenylyl cyclase, and the heterotrimeric G proteins G_s and G_i are components. In the pulmonary circulation, ATP released from the erythrocyte stimulates nitric oxide (NO) synthesis, thereby regulating vascular resistance. We reported that NO liberated from an NO donor inhibited ATP release from erythrocytes in response to decreased PO₂ or mechanical deformation. Here, we investigated the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of G_i. Washed rabbit erythrocytes were incubated in the presence or absence of the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate; 100 nM, 20 min), followed by treatment with agents that activate specific components of the signal transduction pathway promoting ATP release. Neither ATP release nor cAMP accumulation induced by either forskolin (100 µM, n = 7) or iloprost (100 nM, n = 6) was inhibited by spermine NONOate. These experiments suggest that the inhibitory action of NO is not the result of inactivation of adenylyl cyclase or G_s, respectively. However, spermine NONOate completely inhibited ATP release in response to mastoparan (10 µm, P < 0.05, n = 5), a specific activator of G_i. Spermine (100 nM, 20 min), the polyamine remaining after liberation of NO from spermine NONOate, had no affect on mastoparan-induced ATP release (n = 4). These results support the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein G_i.

red blood cell; vascular control; adenine nucleotides; pulmonary circulation

ATP release from erythrocytes has been reported to require the activation of a signal transduction pathway. Components of this signal transduction pathway that have been identified include the heterotrimeric G proteins, G_s and G_i, adenylyl cyclase, PKA, and the cystic fibrosis transmembrane conductance regulator (CFTR) (22, 23, 25, 27). Within this signal transduction pathway, the activation of G_s or G_i via a receptor-dependent or independent mechanism results in the dissociation of the -subunit from the -subunit complex. Both the -subunit of G_s and the -subunit complex of G_i can stimulate the catalytic activity of adenylyl cyclase, resulting in increased intracellular concentrations of cAMP (7, 14, 20, 30). Within the proposed signal transduction pathway for ATP release from erythrocytes, an increase in intracellular cAMP would result in the activation of PKA that in turn stimulates CFTR activity. Once activated, CFTR could regulate the activity of an as-yet-unidentified conduit for the release of ATP from erythrocytes. We hypothesize that the previously described inhibitory action of NO on ATP release from erythrocytes is the result of inactivation of one or more components of this signal transduction pathway. NO has been reported to reversibly inhibit the catalytic activity of certain isoforms of adenylyl cyclase by S-nitrosylation (13, 16, 32). In addition, NO has been reported to indirectly inhibit the activity of G_i by stimulating the activity of an endogenous mono-ADP-ribosyltransferase (4, 24). Once activated, the endogenous mono-ADP-ribosyltransferase, like pertussis toxin, catalyzes the addition of an ADP-ribose moiety to the -subunit of G_i, preventing the dissociation of the -subunit from the -subunit complex (18, 19).

In the present studies, we investigated the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein G_i and not other components of this signal transduction pathway. We examined the effects of NO liberated from the NO donor spermine NONOate on ATP release and cAMP accumulation in rabbit erythrocytes in response to pharmacological agents that specifically activate components of the signal transduction pathway for ATP release from erythrocytes. Specifically, we investigated the effects of NO on mastoparan, which activates the heterotrimeric G protein G_i, iloprost, which activates the heterotrimeric G protein G_s, and forskolin, which stimulates the catalytic activity of adenylyl cyclase. In addition, we examined the effects of pertussis toxin on ATP release from rabbit erythrocytes in response to decreased PO₂.

	MATERIALS AND METHODS

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Isolation of rabbit erythrocytes.

Rabbit erythrocytes were obtained from New Zealand White rabbits (random sex; 2–3 kg body wt). The animals were anesthetized with ketamine (12.5 ml/kg im) and xylazine (1 mg/kg im), followed by pentobarbital sodium (10 mg/kg iv). After tracheal intubation, the animals were mechanically ventilated with room air (tidal volume 10 ml/kg, rate 20–25 breaths/min). A catheter was placed into the carotid artery for administration of heparin (50 units) and for phlebotomy. Ten minutes after the administration of heparin, the animals were exsanguinated. The protocols used to obtain rabbit blood were approved by the Institutional Animal Care and Use Committee of St. Louis University.

Rabbit 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 containing 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 albumin, fraction V (ICN Biomedicals; Aurora, OH), final pH 7.4. The hematocrit of the washed erythrocytes was determined using an Autocrit Ultra 3 centrifuge (Becton Dickinson; Bedford, MA).

Measurement of ATP.

Washed rabbit erythrocytes were brought to a 20% hematocrit and incubated with either spermine NONOate (100 nM) (21) or spermine (100 nM) (21) for 20 min, followed by incubation with either 100 µM forskolin (27), 100 nM iloprost (23), or 10 µM mastoparan (22) for 10 min. ATP was measured using the luciferin-luciferase technique (29). A sample (200 µl) of an erythrocyte suspension was injected into a cuvette containing 100 µl of 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 light emitted from the reaction of ATP with the crude firefly extract/D-luciferin was measured using a luminometer at 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 pharmacological agents with which the erythrocytes were incubated did not alter the results of the ATP assay, the effects of forskolin, iloprost, and mastoparan on the measurements of authentic ATP were determined. These agents, at the concentrations used in these studies, did not have a direct effect on the measurement of ATP.

Measurement of erythrocyte lysis.

To exclude the possibility of erythrocyte hemolysis, after ATP was measured 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 (34).

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 diluted serially. The concentration of ATP and the absorbance at 405 nm were measured at each dilution. In dilutions where ATP was detected, an absorbance at 405 nm was observed, 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 containing 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 albumin (PSS). The erythrocyte suspension was preincubated with 100 nM spermine NONOate for 20 min, followed by incubation with either 100 µM forskolin (27), 100 nM iloprost (23), or vehicle (PSS) for 10 min in the presence of 10 µM IBMX (27). After incubation, the reaction was stopped by the addition of 1 ml of the erythrocyte suspension to 4 ml of ice-cold ethanol containing 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 Biosciences; Piscataway, NJ).

Decreased PO₂ induced ATP release.

Washed rabbit or human erythrocytes were brought to a 10% hematocrit with a buffered solution (140.5 mM NaCl, 21.0 mM Tris·HCl, 4.7 mM KCl, 2.0 mM CaCl₂, 1.2 mM MgSO₄, and 0.5% dextrose, pH 7.4) and incubated in the presence or absence of the pertussis toxin holotoxin (100 ng/ml) for 2 h. The erythrocytes were then diluted to a 0.1% hematocrit with buffered solution in a tissue chamber set at 37°C. The buffered solution in each tissue chamber was equilibrated with either 15% O₂-6% CO₂-79% N₂ (PO₂ = 112.4 ± 0.3 mmHg, PCO₂ = 30.9 ± 0.2 mmHg) or 0% O₂-6% CO₂-94% N₂ (PO₂ = 36.8 ± 0.3 mmHg, PCO₂ = 30.8 ± 0.2 mmHg). Immediately after the erythrocytes were added to the tissue chamber, the chamber was covered and the gas was turned off. ATP released into the buffered solution was measured 1 min after the addition of the erythrocytes to the chamber. The pH and gas tensions were determined immediately after the measurement of ATP with the use of a blood gas analyzer (Radiometer; Copenhagen, Denmark).

Statistical methods.

The statistical significance between experimental periods was determined by ANOVA. In the event that the associated F ratio indicated that changes had occurred, a least-significant difference test was used to identify individual differences. P 0.05 was considered statistically significant. Results are reported as means ± SE.

	RESULTS

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Effect of spermine NONOate on mastoparan-induced ATP release from rabbit erythrocytes.

Our laboratory has reported that NO inhibits ATP release from erythrocytes in response to decreased PO₂ or mechanical deformation (21). In addition, we have identified a signal transduction pathway for ATP release from erythrocytes involving the heterotrimeric G protein G_i (22). Within this signal transduction pathway, activation of G_i resulted in an increase in ATP release from washed rabbit erythrocytes. To determine whether NO inhibits ATP release from erythrocytes via its action on the heterotrimeric G protein G_i, washed rabbit erythrocytes were incubated with spermine NONOate (100 nM, 20 min), followed by treatment with mastoparan (10 µM, 10 min). Mastoparan is a tetradecapeptide derived from wasp venom that stimulates the dissociation of the -subunit from the -subunit complex of G_i. As shown in Fig. 1A, mastoparan-induced ATP release from rabbit erythrocytes was inhibited by spermine NONOate. Importantly, incubating rabbit erythrocytes with mastoparan did not alter intracellular concentrations of ATP or result in measurable increases in free hemoglobin, an indicator of erythrocyte lysis.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Effects of N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine and spermine (spermine NONOate; SpNO) on mastoparan-induced ATP release from rabbit erythrocytes. A: washed rabbit erythrocytes were incubated for 20 min with 100 nM spermine NONOate, followed by treatment for 10 min with 10 µM mastoparan (n = 6) and the measurement of ATP release. *P < 0.01, different from baseline. B: washed rabbit erythrocytes were incubated for 20 min with 100 nM spermine, followed by treatment for 10 min with 10 µM mastoparan (n = 6). *P < 0.01, different from baseline.

NO is a highly reactive free radical that has been reported to directly inhibit the activity of proteins via S-nitrosylation (15, 28). S-nitrosylation is the nonenzymatic addition of NO to protein cysteine residues (18). Mastoparan is a peptide consisting of several amino acids that could be S-nitrosylated by NO. To ensure that the inhibition in mastoparan-induced ATP release was not the result of inactivation of mastoparan by NO, washed rabbit erythrocytes were incubated with mastoparan that was previously incubated with the NO donor 6-(2-hydroxyl-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (NOC-9; 100 nM). NOC-9 is a NO donor with a half-life of 3 min at 22°C. Therefore, incubating mastoparan with NOC-9 for 10 min would allow for the complete liberation of NO. Incubation of washed rabbit erythrocytes with mastoparan resulted in a 65.0 ± 22.5% increase in ATP release compared with untreated erythrocytes (17.0 ± 3.6 nM per 2 x 10⁴ erythrocytes/mm³). Incubation of washed rabbit erythrocytes with mastoparan that had been incubated with NOC-9 (100 nM, 10 min) resulted in a 78.2 ± 14.9% increase in ATP release. In these experiments, exposure to NO did not alter the ability of mastoparan to stimulate ATP release. Taken together, these experiments provide support or the hypothesis that the inhibition of mastoparan-induced ATP release from erythrocytes by spermine NONOate is not the result of direct inactivation of mastoparan by NO, but rather it is the result of an effect of NO on G_i.

Effects of spermine NONOate on iloprost-induced ATP release from and cAMP accumulation in rabbit erythrocytes.

The proposed signal transduction pathway for ATP release from erythrocytes involves the heterotrimeric G protein G_s in addition to G_i. To determine whether inhibition of ATP release by NO was the result of an action on the heterotrimeric G protein G_s, washed rabbit erythrocytes were incubated with spermine NONOate (100 nM, 20 min) before treatment with iloprost (100 nM) for 10 min. Iloprost is a prostacyclin analog that stimulates the activity of G_s via its action on the prostaglandin I receptor (33). As shown in Fig. 2A, iloprost-induced ATP release from rabbit erythrocytes was not inhibited by spermine NONOate. In these experiments, there were no differences in total intracellular concentrations of ATP or measurable increases in free hemoglobin in any of the samples, an indicator of erythrocyte lysis. In separate experiments, washed rabbit erythrocytes were incubated with spermine NONOate before treatment with iloprost in the presence of IBMX (10 µM) to measure the effects of G_s activation on intracellular concentrations of cAMP. Iloprost stimulated increases in intracellular concentrations of cAMP that were not inhibited by spermine NONOate (Fig. 2B). Taken together, these experiments provide support for the hypothesis that NO does not inhibit ATP release from erythrocytes via its action on the heterotrimeric G protein G_s.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effect of spermine NONOate on iloprost-induced ATP release and cAMP accumulation. A: washed rabbit erythrocytes were incubated for 20 min with 100 nM spermine NONOate, followed by treatment for 10 min with 100 nM iloprost (n = 6) and the measurement of ATP release. *P < 0.01, different from baseline. B: washed rabbit erythrocytes were incubated for 20 min with 100 nM spermine NONOate, followed by treatment for 10 min with 100 nM iloprost in the presence of 10 µM IBMX (n = 6), and total concentrations of cAMP measured. *P < 0.05, different from baseline.

The heterotrimeric G proteins G_i and G_s, when activated, regulate the activity of adenylyl cyclase (11). Adenylyl cyclase has also been identified as a component of a signal transduction pathway for the release of ATP from erythrocytes (27); however, the specific types of adenylyl cyclase that are expressed in the mature circulating erythrocyte are not known. It was reported that NO, via S-nitrosylation, inhibits the catalytic activity of adenylyl cyclase types V and VI, but not other types of adenylyl cyclase (13, 16, 32). To determine whether NO inhibits ATP release via an action on adenylyl cyclase, washed rabbit erythrocytes were incubated with spermine NONOate (100 nM, 20 min) and then treated with forskolin (100 µM, 10 min) in the presence of IBMX (10 µM). As shown in Fig. 3A, forskolin-induced ATP release was not inhibited by spermine NONOate. Incubation of erythrocytes with forskolin did not alter intracellular concentration of ATP. In addition, increases in ATP release were independent of erythrocyte lysis, as increases in free hemoglobin were not measured. In separate experiments, washed rabbit erythrocytes were incubated with spermine NONOate (100 nM, 20 min) and then treated with forskolin (100 µM) in the presence of IBMX (10 µM), and measurements of intracellular concentrations of cAMP were made. Treatment of rabbit erythrocytes with forskolin stimulated increases in intracellular concentrations of cAMP that were not inhibited by spermine NONOate (Fig. 3B). Taken together, these data demonstrate that the inhibition of ATP release from erythrocytes by spermine NONOate is not the result of the action of NO on adenylyl cyclase.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effect of spermine NONOate on forskolin-induced ATP release and cAMP accumulation. A: washed rabbit erythrocytes were incubated for 20 min with 100 nM spermine NONOate, followed by treatment for 10 min with 100 µM forskolin (n = 7) and the measurement of ATP release. *P < 0.05, different from baseline. B: washed rabbit erythrocytes were incubated for 20 min with 100 nM spermine NONOate, followed by treatment for 10 min with 100 µM forskolin in the presence of 10 µM IBMX (n = 7), and total concentrations of cAMP measured. *P < 0.05, different from baseline.

We have provided support for the hypothesis that NO inhibits ATP release from erythrocytes via its action on the heterotrimeric G protein G_i; however, the mechanism by which NO acts is unknown. It was reported that NO stimulates the activity of a mono-ADP-ribosyltransferase that has been identified in human erythrocytes (4, 31). Once activated, this protein catalyzes the addition of an ADP-ribose moiety from NADH to the -subunit of G_i (4). Interestingly, the endogenous mono-ADP-ribosyltrasferase mono-ADP-ribosylates the same cysteine residue of the -subunit of G_i as does pertussis toxin (18, 19). Therefore, under our hypothesis, incubation of erythrocytes with pertussis toxin would be expected to inhibit ATP release. Previously, we reported that pertussis toxin inhibits mechanical deformation-induced ATP release from erythrocytes (22). Therefore, if mechanical deformation and decreased PO₂ stimulate a common signal transduction pathway for ATP release from erythrocytes, pertussis toxin would be anticipated to inhibit ATP release in response to decreased PO₂ as well. To investigate this possibility, washed rabbit erythrocytes were incubated with pertussis toxin holotoxin (100 ng/ml, 2 h) and then exposed to either normal or decreased PO₂. As depicted in Fig. 4, pertussis toxin inhibited ATP release in response to decreased PO₂. In these experiments, there were no measurable differences in the total intracellular concentrations of ATP between erythrocytes treated with pertussis toxin and those that were untreated. Importantly, increases in ATP release were not the result of erythrocyte lysis because no change in free hemoglobin was measured in any of the samples.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effect of pertussis toxin (PTX) on ATP release from rabbit erythrocytes in response to decreased PO2. Washed rabbit erythrocytes were incubated for 2 h with PTX (100 ng/ml) and then exposed to either a normal or a decreased PO2 (n = 5). The amount of ATP released into the buffer was measured using an ATP assay. *P < 0.01, different from baseline.

	DISCUSSION

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Preincubating washed rabbit erythrocytes with the NO donor spermine NONOate inhibited mastoparan-induced ATP release (see Fig. 1A). Mastoparan activates G_i by stimulating the dissociation of the -subunit from its -subunit complex (12). In identical experiments, spermine, the polyamine remaining from liberation of NO from spermine NONOate, had no effect on mastoparan-induced ATP release (see Fig. 1B). Importantly, the inhibitory effect of NO on ATP release was not the result of direct inactivation of mastoparan as evidenced by the finding that when mastoparan was incubated with the NO donor NOC-9, there was no measurable decrease in the ability of mastoparan to stimulate the release of ATP from rabbit erythrocytes. Taken together, these results provide support for the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein G_i.

The erythrocyte contains millimolar concentrations of ATP that are maintained by an active glycolytic pathway (17). NO has been reported to stimulate the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, resulting in a loss of enzymatic activity (5). Therefore, it is possible that the inhibition in mastoparan-induced ATP release by spermine NONOate was the result of decreased ATP synthesis. However, in those experiments in which spermine NONOate inhibited mastoparan-induced ATP release, there was no measurable difference in total intracellular concentrations of ATP, arguing against this possibility. In addition, iloprost and forskolin stimulated ATP release from erythrocytes in the presence of NO.

To determine whether, in addition to the heterotrimeric G protein G_i, NO inhibits other components of the proposed signal transduction pathway for ATP release, washed rabbit erythrocytes were preincubated with spermine NONOate and then treated with iloprost, which selectively activates the heterotrimeric G protein G_s via stimulation of the prostaglandin I receptor. Spermine NONOate did not inhibit iloprost-induced ATP release or cAMP accumulation (see Fig. 2, A and B, respectively). These experiments suggest that NO does not inhibit ATP release via its action on the heterotrimeric G protein G_s or other downstream components of the proposed signal transduction pathway.

NO has been reported to directly and reversibly inhibit protein activity by S-nitrosylation (15, 28). S-nitrosylation is the nonenzymatic addition of NO to a protein thiol (18). It has been reported that some isoforms of adenylyl cyclase, a protein that is a component of the signal transduction pathway for ATP release from erythrocytes, can be inhibited by NO (13, 16, 32). To determine whether NO inhibits ATP release via S-nitrosylation of adenylyl cyclase, washed rabbit erythrocytes were preincubated with spermine NONOate and then treated with forskolin, which indiscriminately stimulates the catalytic activity of all isoforms of adenylyl cyclase. In these experiments, spermine NONOate did not inhibit forskolin-induced ATP release (see Fig. 3A). In addition, spermine NONOate did not inhibit forskolin/IBMX-induced cAMP accumulation (see Fig. 3B). These data suggest that NO does not inhibit ATP release from erythrocytes via its action on adenylyl cyclase.

Taken together, the results of these studies provide strong support for the hypothesis that NO inhibits ATP release from the erythrocyte via inactivation of G_i. However, the mechanism by which this occurs has not been defined. NO has been reported to inhibit the activity of G_i/o via stimulation of a mono-ADP-ribosyltransferase, an enzyme that catalyses the addition of the ADP-ribose moiety from nicotinamide adenine dinucleotide to the -subunit of G_i/o (4, 24). Importantly, a mono-ADP-ribosyltransferase, that is activated by NO and catalyses the mono-ADP-ribosylation of G_i, has been identified and purified from human erythrocytes. This NO-activated mono-ADP-ribosyltransferase ADP-ribosylates G_i on the same cysteine residue as does the well-characterized bacterial mono-ADP-ribosyltransferase, pertussis toxin (18, 19). Therefore, the effects of NO to inhibit ATP release from erythrocytes could be reproduced by incubation of erythrocytes with pertussis toxin. Previously, we demonstrated that incubation of rabbit erythrocytes with pertussis toxin inhibited mechanical deformation-induced ATP release (22). Here, we extended this observation and determined the effect of pertussis toxin on decreased PO₂-induced ATP release. Incubation of washed rabbit erythrocytes with pertussis toxin inhibited ATP release in response to decreased PO₂ (see Fig. 4). One interpretation of these results is that NO inhibits ATP release from erythrocytes indirectly through the activation of an endogenous mono-ADP-ribosyltransferase that ribosylates, thereby inactivating, G_i in the same manner that pertussis toxin does.

The results presented here provide support for the hypothesis that NO inhibits ATP release from erythrocytes via its action on the heterotrimeric G protein G_i, a component of a signal transduction pathway for the ATP release from erythrocytes. We have demonstrated that NO, liberated from the NO donor spermine NONOate, inhibited ATP release via its action of G_i, but not G_s or adenylyl cyclase. Importantly, the action of spermine NONOate cannot be attributed to any activity of spermine, the polyamine resulting from the liberation of NO from spermine NONOate. In addition, NO-induced inhibition of ATP release is not the result of direct inactivation of mastoparan and is not related to a general inhibition of ATP synthesis.

Within the proposed signaling paradigm ATP released into the vascular lumen from erythrocytes in response to decreased PO₂ or mechanical deformation stimulates the synthesis of NO in the vascular endothelium. NO released abluminally stimulates relaxation of vascular smooth muscle leading to vasorelaxation and an increase in vascular caliber. NO is also released into the vascular lumen, where it can interact with the erythrocyte in a negative feedback manner to inhibit ATP release. These results also support the hypothesis that this action of NO occurs via inhibition of the activity of the heterotrimeric G protein G_i. Taken together, these data identify an additional mechanism by which NO, through the inhibition of ATP release from erythrocytes, participates in the regulation of vascular resistance.

	GRANTS

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This work was supported by National Heart, Lung, and Blood Institute Grants HL-64180 and HL-51289, American Heart Association Grant 0215286Z, and the National Institute of General Medical Sciences Training Grant GM-08306-11.

	ACKNOWLEDGMENTS

 
The authors thank Elizabeth Bowles for technical expertise and J. L. Sprague for inspiration.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. J. Olearczyk, Dept. of Pharmacological and Physiological Science, St. Louis Univ. School of Medicine, M-208, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: olearcjj{at}slu.edu).

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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