Activation of the β-adrenergic receptor (β-AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release from erythrocytes. cAMP levels depend on a balance between synthesis via adenylyl cyclase and hydrolysis by phosphodiesterases (PDEs). Previously, we reported that cAMP increases associated with activation of the β-AR and IPR in rabbit and human erythrocytes are tightly regulated by distinct PDEs (1). Importantly, inhibitors of these PDEs potentiated both increases in cAMP and ATP release. It has been shown that increases in protein kinase (PK) activity can activate PDE3 and PDE4. Both PKA and PKC are present in the erythrocyte and can phosphorylate and activate these PDEs. Here we investigate the hypothesis that PKA regulates PDE activity associated with the β-AR and both PKA and PKC regulate the PDE activity associated with the IPR in rabbit erythrocytes. Pretreatment of erythrocytes with the PKA inhibitor, H89 (10 μM), in the presence of the PDE4 inhibitor, rolipram (10 μM), augmented isoproterenol (1 μM)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 μM), pretreatment of erythrocytes with either H89 (1 μM) or two chemically dissimilar inhibitors of PKC, calphostin C (1 μM) or GFX109203X (1 μM), potentiated iloprost (1 μM)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFX109203X in the presence of cilostazol augmented the iloprost-induced increases in cAMP to a greater extent than either PK inhibitor individually. These results support the hypothesis that PDEs associated with receptor-mediated increases in cAMP in rabbit erythrocytes are regulated by kinases specific to the receptor's signaling pathway.
in mammalian erythrocytes, receptor-mediated activation of β-adrenergic receptors (β-AR) and prostacyclin receptors (IPR) results in stimulation of signaling pathways that culminate in ATP release (32, 42). Although β-AR and IPR agonists can directly stimulate vascular smooth muscle relaxation, both isoproterenol (ISO) and prostacyclin (PGI2) analogs have also been shown to produce receptor-mediated ATP release from erythrocytes (41–43). This release of ATP from erythrocytes can then stimulate the local synthesis and release of endothelium-derived relaxing factors, permitting these cells to participate in the local control of vascular caliber (12, 43).
Components of the signaling pathways for ATP release that are associated with the β-AR and IPR include the heterotrimeric G protein, Gs, and adenylyl cyclase (AC) (41, 44). Activation of each receptor results in increases in the second messenger, cAMP, a requisite for ATP release (41, 42). cAMP is an important second messenger in most cell types and influences a vast array of cellular events vital for the cell's response and control of signaling pathways. Clearly, within the erythrocyte, regulation of intracellular cAMP concentrations and their localization to a specific signaling pathway is critical to ensure discrete responses to β-AR agonists and prostacyclin analogs.
The local concentration of cAMP within erythrocytes is tightly regulated by a balance of its synthesis by ACs and hydrolysis by phosphodiesterases (PDEs) (5). PDEs are a family of enzymes consisting of 11 isoforms, which are the only known physiological means of inactivation of cyclic nucleotides. Most cells contain representatives of more than one PDE gene family, but in different proportions and locations. It has become increasingly clear that PDEs serve not only to regulate local intracellular levels of cAMP, but also to limit diffusion of cAMP from its site of synthesis, permitting the activation of surface receptors to produce discrete signaling events within cells (2, 30). Failure to maintain a confined pool of cAMP could lead to aberrant phosphorylation of proteins and an improper signal outcome or nonspecific effects (18, 35).
Previously, we reported that PDE2, -3, and -4 are present in rabbit and human erythrocytes. More importantly, we established that PDE3 regulates increases in cAMP produced by activation of the IPR, while PDE2 and -4 hydrolyze cAMP produced in response to activation of the β-AR (1). Before these reports, segregation of PDEs to any signaling pathway in erythrocytes had not been established. Although it is clear that PDE activity is present in erythrocytes and that distinct PDEs are associated with regulation of increases in cAMP produced by activation of the β-AR and IPR signaling pathways, there are no reports addressing the mechanisms responsible for the stimulation of PDE activity in either pathway in these cells.
It is well recognized that, in nonerythroid cells, PDE3 and -4 can be regulated by specific protein kinases (PKs). Mammalian erythrocytes have been shown to possess the cAMP-dependent PKA and cAMP-independent PKC activity (11, 19, 33). An increase in PK activity can be stimulated by receptor-mediated activation of Gs and Gq, G proteins that couple to the β2-AR and IPR, respectively (40, 49). An increase in PKA activity has been reported to phosphorylate (activate) both isoforms of PDE3, PDE3A and PDE3B (9). In addition to phosphorylation of PDE3A by PKA, PDE3A can be also be phosphorylated by PKC (9, 34). Like PDE3, there are PDE4 isoforms that require phosphorylation by PKA for their activation (7, 8). Thus, although erythrocytes possess both PDEs and the PKs that regulate their activity, the interaction among these proteins in specific G protein-coupled receptor (GPCR) signaling pathways in mammalian erythrocytes has not been examined previously.
Here, we investigated the contribution of PKA and PKC to the regulation of PDE activity in erythrocytes in response to activation of two distinct GPCRs, the β-AR and the IPR. We hypothesized that, in erythrocytes, PKA regulates the activity of both PDE3 and PDE4 following stimulation of either receptor, whereas PKC alone regulates the activity of PDE3 associated with activation of the IPR.
Isolation of erythrocytes.
Male New Zealand White rabbits were anesthetized with ketamine (12.5 mg/kg) and xylazine (1.5 mg/kg) intramuscularly, followed by pentobarbital sodium (10 mg/kg) administered via a cannula placed in an ear vein. A catheter was subsequently placed in a carotid artery, and heparin (500 units) was administered. After 10 min, the animals were exsanguinated. Immediately after collection of blood, erythrocytes were isolated by centrifugation at 500 g at 4°C for 10 min with the supernatant, and buffy coat was removed by aspiration. Packed erythrocytes were resuspended and washed three times in a physiological salt solution containing the following, in mM: 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 140.5 NaCl, 21.0 Tris-base, and 5.5 dextrose with 0.5% bovine serum albumin, pH adjusted to 7.4. Erythrocytes were prepared on the day of use. The protocol for blood collection was approved by the Institutional Animal Care and Use Committee of St. Louis University.
Incubation of erythrocytes with pharmacological agents.
Washed erythrocytes were diluted to a 50% hematocrit (1 ml) and were preincubated with a PDE inhibitor, a kinase inhibitor, or their respective vehicles for 30 min. The PDE inhibitors used were rolipram (ROL), a selective PDE4 inhibitor (Tocris) (48), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (28), a selective PDE2 inhibitor (Biomol), and cilostazol (CILO), a selective PDE3 inhibitor (Sigma-Aldrich) (6). The kinase inhibitors used were H89, a PKA inhibitor (Biomol) (29), calphostin C (CALC), a selective PKC inhibitor (Biomol) (37), and GFX109203X (GFX), a chemically dissimilar selective PKC inhibitor (Biomol) (47). The concentrations of inhibitors were chosen based on the IC50 values of each inhibitor in other cell types. Importantly, these concentrations had no effect on baseline cAMP levels. The vehicle for ROL, CILO, H89, CALC, and GFX was N′,N-dimethylformamide (Sigma-Aldrich), and the vehicle for EHNA, ISO (Sigma-Aldrich), and iloprost (ILO, Cayman) was saline. Following incubation with inhibitors or vehicles, erythrocytes were incubated with either ISO (1 μM, 30 min) or ILO (1 μM, 15 min). Reactions were stopped with the addition of 4 ml ice-cold acidified ethanol containing 1 mM HCl.
Measurement of cAMP.
The erythrocyte-ethanol mixture was centrifuged at 14,000 g for 10 min at 4°C, to remove precipitated proteins. The supernatant was removed and stored overnight at −20°C. Samples were centrifuged a second time at 3,700 g for 10 min at 4°C, to remove cryoprecipitates. The supernatant was again removed and dried under vacuum centrifugation. Concentrations of cAMP were determined by EIA (GE Healthcare), according to the manufacturer's instructions.
Statistical significance was determined using an ANOVA. In the event that the F-ratio indicated a change had occurred, a Fisher's least significant difference test was performed to identify individual differences. Results are reported as means ± SE.
Effect of an inhibitor of PKA, H89, on ISO-induced increases in cAMP.
In rabbit and human erythrocytes, increases in cAMP produced by receptor-mediated activation of the β-AR are regulated by the activity of both PDE2 and PDE4 (1). The isoform of PDE4 demonstrated to be associated with the β-AR is activated via phosphorylation by PKA (38). Therefore, to determine whether the PDE4 isoform associated with the β-AR in rabbit erythrocytes can be regulated by PKA, erythrocytes were incubated with the PKA inhibitor, H89 (10 μM), in the absence and presence of ROL (10 μM), a selective inhibitor of PDE4, before stimulation with ISO (1 μM). H89 alone had no effect on baseline cAMP levels (data not shown) or on ISO-induced increases in cAMP (Fig. 1). Importantly, we have reported previously that, at this concentration, ROL has no effect on baseline or ISO-induced increases in cAMP (1). However, in the presence of ROL, H89 potentiated the increases in cAMP produced by ISO (Fig. 1).
Effect of an inhibitor of PKC, CALC, on ISO-induced increases in cAMP.
PKC has been reported to activate PDE2 (14) and PDE4 (46). We have shown that both PDEs are associated with the regulation of cAMP levels, resulting from receptor-mediated activation of the β-AR in rabbit and human erythrocytes (1). Therefore, to determine whether the increases in cAMP seen upon inhibition of PKC in the IPR signaling pathway were specific to this receptor, rabbit erythrocytes were incubated with CALC (1 μM) in the presence of inhibitors of either PDE2 (EHNA, 10 μM) or PDE4 (ROL, 10 μM), concentrations of PDE inhibitors that have no effect on ISO-induced increases in cAMP (1). CALC had no effect on the ISO-induced increases in cAMP alone (Table 1) or in the presence of either EHNA or ROL (Fig. 2).
Effect of H89 on ILO-induced increases in cAMP.
Incubation of rabbit and human erythrocytes with a selective inhibitor of PDE3, CILO, was shown to potentiate ILO-induced increases in cAMP (1, 16). PKA was reported to phosphorylate and activate an isoform of PDE3, PDE3B, which is present in rabbit and human erythrocyte membranes (9, 16). Therefore, to determine if PKA regulates ILO-induced increases in cAMP, rabbit erythrocytes were incubated with H89 (1 μM) in the absence and presence of CILO (10 μM). H89 alone had no effect on either baseline (data not shown) or ILO (1 μM)-induced increase in cAMP (Fig. 3A). However, H89 augmented the increase in cAMP produced by CILO (Fig. 3B).
Effect of inhibitors of PKC, CALC, and GFX on ILO-induced increases in cAMP.
In addition to PKA, PKC was also demonstrated to phosphorylate and activate the PDE3 isoform, PDE3A (20). Therefore, to determine whether PKC is involved in regulating erythrocyte cAMP levels associated with activation of the IPR, rabbit erythrocytes were preincubated with two chemically dissimilar inhibitors of PKC, GFX (1 μM) or CALC (1 μM), before treatment with ILO (1 μM). Importantly, incubation of erythrocytes with either inhibitor alone had no effect on baseline cAMP levels (data not shown) or ILO-induced increases in cAMP (Table 1). However, preincubation of these cells with either GFX (Fig. 4A) or CALC (Fig. 4B) in the presence of CILO (10 μM) augmented the ILO-induced increases in cAMP produced by CILO.
Effect of the combination of H89 and GFX on ILO-induced increases in cAMP.
It was reported that both PKA and PKC can phosphorylate and activate PDE3A on different residues (20). Therefore, to establish that IPR signaling and regulation of cAMP in the erythrocyte involves the activity of both PKA and PKC, rabbit erythrocytes were pretreated with CILO (10 μM), an inhibitor of PDE3, and either GFX (1 μM), an inhibitor of PKC, or H89 (1 μM), an inhibitor of PKA, alone or in combination, before stimulation with ILO (1 μM). As demonstrated above, both kinase inhibitors alone enhance ILO-induced increases in cAMP in the presence of CILO. However, when the kinase inhibitors were administered in combination with CILO, their effect on cAMP accumulation was greater than that of either inhibitor alone (Fig. 5).
Kinases are integral parts of many signal transduction pathways that mediate specific functions in cells. PKs are the largest group of kinases that modify the activity of specific target proteins via phosphorylation of specific amino acid residues. The ability of PKs to determine the activity, localization, and overall function of proteins results in the orchestration of most cellular processes. Two serine/threonine kinases have been identified in the erythrocyte, PKA and PKC (11, 19, 33). PKA is activated by cAMP and is the main effector protein of that cyclic nucleotide. In contrast, PKC is not activated by cAMP, but rather by increases in intracellular Ca2+ and/or diacylglycerol. Moreover, depending on the isoform present, some isoforms of PKC require neither for their activation (31).
Recently, we reported that activation of either the β-AR or IPR in rabbit and human erythrocytes results in receptor-mediated increases in cAMP that are regulated by distinct PDEs (1). In the case of the β-AR, the PDEs that regulate cAMP levels were determined to be PDE2 and -4. The PDE associated with the IPR signaling pathway in these cells is PDE3. Although the activation of these PDEs is regulated by phosphorylation via PKs in other cell types (20, 34, 38), in erythrocytes this relationship has not been evaluated previously. Here we demonstrate pharmacologically that PKA regulates the increases in cAMP produced by activation of the β-AR and that both PKA and PKC are involved in the regulation of the increases in cAMP produced by activation of the IPR in rabbit erythrocytes.
PDEs provide the sole physiological means of inactivating cyclic nucleotides in cells. In the erythrocyte, they play a pivotal role in mediating the release of ATP, since increases in cAMP are necessary for that release (1, 44). The increase in cAMP produced by activation of the β-AR has been shown to stimulate the activity of PKA (22, 51). This increase in PKA activity can phosphorylate isoforms of PDE4, increasing its activity (4, 50). We and others have demonstrated the importance of PDE4 in regulating the increases in cAMP associated with activation of the β-AR (1, 48, 50). Since the PDE4 isoforms associated with this receptor in nonerythroid cells are phosphorylated by PKA, we hypothesized that, in rabbit erythrocytes, increases in cAMP produced by activation of the β-AR are regulated by PKA-stimulated PDE activity. Here we report that in rabbit erythrocytes, an inhibitor of PKA, H89, when administered alone, had no effect on ISO-induced increases in cAMP (Fig. 1). However, the combination of inhibitors of both PKA and PDE4, at concentrations that had no effect alone, potentiated the increases in cAMP produced by ISO (Fig. 1). These results suggest that within this signaling pathway, a local increase in cAMP results in activation of PKA which, in turn, phosphorylates and activates PDE4.
In addition to activation of PDE4, PKA can also activate both isoforms of PDE3, PDE3A and PDE3B (9). PDE3A is predominantly cytosolic; however, splice variants of this isoform can associate with the membrane (39). On the other hand, PDE3B is membrane associated (27). We identified PDE3B in rabbit and human erythrocyte membranes and showed that PDE3 inhibitors potentiated increases in cAMP produced by activation of the IPR (16). Similar to the β-AR, activation of the IPR increases the activity of PKA (13, 49). Phosphorylation of PDE3B by PKA has been demonstrated in adipocytes (9), and activation of PDE3A has been reported in platelets and vascular smooth muscle cells (20, 26). Therefore, the possibility exists that in addition to activation of PDE4, PKA also activates PDE3 in erythrocytes. Here we demonstrate that the PKA inhibitor, H89, alone had no effect on ILO-induced increases in cAMP (Fig. 3A). However, H89 potentiated ILO-induced increases in cAMP in erythrocytes in the presence of the PDE3 inhibitor CILO (Fig. 3B). These results are consistent with the hypothesis that activation of IPR increases PKA activity that increases the activity of PDE3 in this pathway.
The finding that PKA is involved in two different signaling pathways has been demonstrated in other cell types (10, 17). There are two isoforms of PKA, a membrane-associated isoform and a cytoplasmic isoform (25, 45). It has been proposed that these distinct isoforms are associated with different signaling pathways. For example, in cardiac myocytes, activation of different GPCRs has been shown to stimulate different PKA isoforms (10). In these cells, β2-AR stimulation was reported to give rise to a discrete pool of cAMP which activates the membrane-associated PKA, whereas activation of the IPR results in activation of the cytoplasmic PKA (10). Subsequently, it was confirmed that these two isoforms of PKA reside in physically segregated and distinct compartments in myocytes (10). Although we cannot define discrete pools of cAMP in erythrocytes, it is attractive to speculate that a similar association of PKA isoforms with the β-AR and IPR exists in rabbit erythrocytes.
In addition to increasing PKA activity, activation of the IPR was also shown to increase the activity of PKC via activation of the G protein Gq. Receptor-mediated activation of Gq results in stimulation of phospholipase C and increases in intracellular second messengers, such as inositol triphosphate and diacylglycerol, the latter a direct activator of PKC (36). Here we demonstrate that two chemically dissimilar inhibitors of PKC, GFX (Fig. 4A) and CALC (Fig. 4B), potentiate CILO-induced increases in cAMP produced by activation of the IPR. These results suggest that, in addition to PKA (Fig. 2B), PKC also regulates the PDE3 activity associated with the IPR. The involvement of both PKA and PKC in the regulation of PDE3 activity associated with activation of the IPR could be attributed to the association of two distinct PDE3 isoforms, PDE3A and PDE3B, with the receptor. Although PDE3B has been identified in rabbit and human erythrocyte membranes (16), there are no reports of PDE3A. It must be noted that available selective PDE3 inhibitors, including CILO, inhibit both isoforms of PDE3. Thus, although it is attractive to hypothesize that PKA and PKC regulate the activity of distinct isoforms of PDE3 in the IPR signaling pathway in rabbit erythrocytes, this cannot be resolved with currently available inhibitors in the mature erythrocyte, a cell in which alterations in protein synthesis cannot be stimulated. Notwithstanding, studies using inhibitors of both PKA and PKC in combination suggest that both kinases are associated with the IPR receptor, and together they regulate the PDE activity that hydrolyzes IPR-induced increases in cAMP. This conclusion is based on the findings that the effect on cAMP levels in the presence of inhibitors of both kinases together was greater than when either inhibitor was administered alone (Fig. 5).
In addition to effects on PDEs, PKs have been shown to alter the activity of other components of signaling pathways. PKC is known to affect the activity of AC, either positively or negatively, depending on the isoform of PKC or AC present (21, 52). Importantly, neither of the two chemically distinct PKC inhibitors used in these studies, GFX or CALC, had an effect on either baseline or stimulated levels of cAMP. Thus, in rabbit erythrocytes, the PKC isoform(s) present does not appear to directly modulate AC activity. In addition to effects on the synthesis of cAMP, PKA and PKC have also been reported to desensitize and resensitize receptors (15, 24). However, the PK inhibitors used in these studies had no effect on receptor-mediated increases in cAMP (Table 1). The latter finding suggests that PKA and PKC do not directly affect the receptors studied. Therefore, the data presented here support the hypothesis that the ability of the kinase inhibitors to increase receptor-mediated increases in cAMP in rabbit erythrocytes reflects their effects on PDE activity.
It is now generally accepted that much of the specificity of cAMP signaling is accomplished by spatio-temporal compartmentalization of cAMP transients, as well as by physical coupling of cAMP with relevant downstream signaling molecules close to their sites of action (2, 3). Compartmentalization of PDEs is also crucial for controlling the duration and magnitude of cAMP-dependent events. Intracellular pools of cAMP are not uniform, and these distinct pools allow localized cAMP-mediated effects (23, 30). Thus the localization of individual PDEs and the PKs that regulate them within individual signaling pathways is critical. For example, the tethering of PKA to selected intracellular sites via scaffold proteins is acknowledged to allow compartmentalization of PKA-dependent effects (3). Like PKA, PKC and PDEs can also be anchored to specific intracellular sites and complexes via scaffold or association proteins (2, 24). However, most of the compartmentalization of cAMP is thought to be due to the PDEs in the signaling pathways which keep the cAMP from diffusing throughout the cell (53). In the erythrocyte, PKA and PKC fulfill unique functions, and disruption of their activity would be expected to impact cAMP-regulated functions in that cell. In the present study, as depicted in Fig. 6, we demonstrate that both PKA and PKC are involved in regulating increases in cAMP produced by activation of the β2-AR and IPR. The effects of these PKs are proposed to result from the kinase-dependent activation of the specific PDEs associated with each pathway.
This work is supported by National Heart, Lung, and Blood Institute Grants HL-64180 and HL-89094 and American Diabetes Association Grant RA-133.
I am not aware of financial conflict(s) with the subject matter or materials discussed in this manuscript with any of the authors, or any of the authors– academic institutions or employers.
The authors thank J. L. Sprague for inspiration.
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