Abstract
Cyclic AMP affects microvascular smooth muscle contraction and growth. Therefore, it is important to elucidate mechanisms regulating cyclic AMP production in microvascular smooth muscle. In this study, we determined whether several signal transduction pathways regulate receptor-induced cyclic AMP in isolated preglomerular microvessels and microvascular smooth muscle cells. Preglomerular microvessels were incubated with isoproterenol (β-adrenoceptor agonist) and with and without U73122 (phospholipase C inhibitor), GF109203X (protein kinase C inhibitor), 1-butanol (phospholipase D inhibitor), CGP77675 (c-src inhibitor), HA1077 (Rho kinase inhibitor), Y27632 (Rho kinase inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), dipenyleneiodonium (NADPH oxidase inhibitor), or Tempol (superoxide dismutase mimetic). Cultured preglomerular microvascular smooth muscle cells were incubated with isoproterenol or forskolin (direct activator of adenylyl cyclase) and with or without U73122, C2-ceramide (phospholipase D inhibitor), or PP1 [src family inhibitor, 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine]. All studies were conducted with 3-isobutyl-1-methylxanthine (broad-spectrum phosphodiesterase inhibitor) to eliminate changes in cyclic AMP degradation. In microvessels isoproterenol-induced cyclic AMP was not affected by Y27632, HA1007, LY294002, dipenylene-iodonium, or Tempol; was increased by U73122 and GF109203X; and was decreased by 1-butanol and CGP77675. In cells, U73122 increased and C2-ceramide and PP1 decreased isoproterenol-induced cyclic AMP. Forskolin-induced cyclic AMP was not altered. These results indicate that receptor-mediated activation of adenylyl cyclase is 1) not modulated by Rho kinase, phosphatidylinositol-3-kinase, NADPH oxidase, or superoxide; 2) is attenuated by phospholipase C and protein kinase C; and 3) is augmented by phospholipase D and src. Phospholipase C, phospholipase D, and src modulate receptor-induced cyclic AMP by affecting β-adrenoreceptor/G protein/adenylyl cyclase coupling rather than by directly affecting adenylyl cyclase activity.
Cyclic AMP is a critical second messenger in vascular smooth muscle cells. In this regard, in intact blood vessels increases in cyclic AMP are associated with diminished vascular resistance (Lincoln and Cornwell, 1991), and in vascular smooth muscle cells in culture cyclic AMP inhibits cellular migration (Mooradian et al., 1995; Newman et al., 2003) and proliferation (Kronemann et al., 1999; Ii et al., 2001). Moreover, abnormalities in cyclic AMP production may participate in the pathophysiology of vascular diseases such as hypertension (Chatziantoniou et al., 1995; Vyas et al., 1996) and arteriosclerosis (Tintut et al., 2000). Clearly, it is important to have a complete understanding of the mechanisms that determine the level of cyclic AMP in vascular smooth muscle cells, particularly in the microcirculation where vascular resistance is a critical determinant of blood pressure and tissue perfusion.
The levels of intracellular messengers are regulated by a balance between production rate and elimination rate, and cyclic AMP is no exception to this general rule. There are multiple isoforms of adenylyl cyclases (Sunahara et al., 1996; Schwartz, 2001; Watts, 2002) and phosphodiesterases (Soderling and Beavo, 2000) that catalyze the production and metabolism, respectively, of cyclic AMP. In mammals, the complexity and diversity of adenylyl cyclases is particularly striking (Schwartz, 2001; Watts, 2002), with at least nine different isoforms that are differentially regulated by G protein α subunits, G protein βγ subunits, protein kinase C, calcium, and calmodulin (Schwartz, 2001; Watts, 2002).
Although investigators have elucidated some of the factors that regulate individual isoforms of adenylyl cyclase in isolation or in cells manipulated so as to overexpress isoforms of adenylyl cyclase and/or specific signal transduction molecules, it remains unclear what signal transduction pathways in intact, normal cells influence the ability of receptors to stimulate adenylyl cyclase activity and whether the integrated, net effects of these signal transduction pathways on cyclic AMP production are positive or negative. Indeed, the role of signal transduction pathways in this regard may vary depending on the relative expression of different adenylyl cyclase isoforms in a given cell and the relative activity of various signal transduction pathways in the cell (Ishikawa and Homcy, 1997).
Because our interest is in preglomerular microvascular smooth muscle and because the role of signal transduction systems to modify receptor-mediated activation of adenylyl cyclase may well be tissue- and cell-specific, we decided to examine the participation of key signal transduction processes on β-adrenoceptor-induced cyclic AMP in renal preglomerular microvascular smooth muscle. In this regard, we first conducted studies in freshly isolated preglomerular microvessels so as to avoid changes in signal transduction mechanisms that may occur in vascular smooth muscle cells in culture. To achieve this objective, we developed a preglomerular microvascular preparation that is highly response to β-adrenoceptor activation and provides a robust and reproducible response. We then investigated the response to β-adrenoceptor activation, using the β-adrenoceptor agonist isoproterenol, in the absence and presence of well established inhibitors of signal transduction systems known to importantly regulate microvascular resistance and vascular smooth muscle cell growth. To confirm that observed changes were occurring in the vascular smooth muscle cells, rather than in other vascular elements, additional experiments were conducted in cultured preglomerular microvascular smooth muscle cells (PGSMCs). In these studies, we focused our attention on the signal transduction pathways that, according to our results in freshly isolated preglomerular microvessels, were the most important with regard to regulating receptor-induced cyclic AMP production. In our studies in PGSMCs, we also took the opportunity to use some different signal transduction inhibitors so as to increase the overall confidence in our conclusions. Finally, in the PGSMC studies we compared the effects of the selected inhibitors on both receptor-mediated (isoproterenol) as well as forskolin-induced cyclic AMP. Because forskolin, unlike isoproterenol, directly activates adenlylyl cyclase, these experiments permitted inferences regarding the mechanism by which the investigated signal transduction systems altered receptor-induced cyclic AMP production. Here, we show for the first time that in preglomerular microvascular smooth muscle, phospholipases C and D and src importantly modulate receptor-induced activation of adenylyl cyclase by affecting receptor/G protein/adenylyl cyclase coupling efficiency rather than by directly affecting adenylyl cyclase activity.
Materials and Methods
Animals. In a previous study (Mokkapatti et al., 1998), we found that preglomerular vascular smooth muscle cells in long-term culture produced more cyclic AMP in response to isoproterenol when the cells were obtained from spontaneously hypertensive rats compared with normotensive rats. Therefore, to maximize the cyclic AMP signal, in the present study we used adult (approximately 16-week-old) male spontaneously hypertensive rats purchased from Taconic Farms (Germantown, NY). Rats were maintained in the University of Pittsburgh Animal Care Facility and were provided free access to Prolab Isopro RMH 3000 rodent diet (PMI Nutrition International, Richmond, IN) and tap water.
Isolation of Preglomerular Microvessels. We previously reported a method for isolating preglomerular microvessels that results in a preparation responsive to isoproterenol with respect to activation of cyclic AMP production via β-adrenoceptors coupled to adenylyl cyclase (Jackson and Mi, 2000). In our previous study, we found that the preparations were much more responsive to isoproterenol if the microvessels were allowed to recover from the isolation procedure for approximately 1 d. In our original protocol we used Tyrode's solution for all steps of the procedure, including the 1-day incubations for recovery. Subsequently, Bolz et al. (2000) reported a culture protocol that preserves for 48 h vascular integrity and function in small skeletal arteries from golden Syrian hamsters using Leibovitz (L15) medium containing 15% fetal calf serum and antibiotics (Bolz et al., 2000). After reading this article, we decided to modify our original method for isolating preglomerular microvessels and substitute L15 (Sigma-Aldrich, St. Louis, MO) for Tyrode's solution. We found that this revised method gave a 5-fold increase in the amount of cyclic AMP that accumulates in response to isoproterenol. We also found that variability in cyclic AMP production induced by isoproterenol was markedly reduced using L15 versus Tyrode's solution. In the present study, we use this modified technique as described below.
Rats were anesthetized with Inactin (100 mg/kg i.p.), and the aorta below the renal artery was cannulated with polyethylene-190 tubing. The proximal aorta, mesentery artery, and small side branches of the aorta were ligated, and the kidneys were flushed (10 ml) with oxygenated L15 medium at room temperature (RT). A 1% suspension of iron oxide (Aldrich Chemical Co., Milwaukee, WI) in oxygenated L15 medium (10 ml; RT) was flushed into the kidneys. The kidneys were harvested, placed in oxygenated, ice-cold L15 medium, and dissected by removing the renal medulla and interlobar arteries. The cortex was sliced into small pieces, suspended in oxygenated, ice-cold L15 medium, and dispersed by pushing the cortical material through a series of increasingly small needle hubs (16, 18, 21, and then 23 gauge). The dispersed cortical material was suspended in ice-cold, oxygenated L15 medium, and a magnet was applied to the tube to retrieve the iron oxide-laden microvessels while the unwanted material was decanted. The glomeruli were removed from the microvessels by filtering the microvessel suspension through a 149-μm nylon mesh. The microvessels were retrieved from the nylon mesh and distributed into the wells of a 24-well culture plate. A sample of the microvessels was examined by phase contrast microscopy to confirm that the preparation consisted of interlobular, accurate, and afferent arterioles without contaminating glomeruli or tubules. To each well was added 1 ml of oxygenated L15 medium containing streptomycin (100 μg/ml), penicillin (100 U/ml), amphotericin B (25 μg/ml), and 15% fetal calf serum. Microvessels were incubated for 24 h (RT).
Protocol in Preglomerular Microvessels. After incubation for 24 h at RT, microvessels were washed three times with 1 ml of MOPS-buffered salt solution (145 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl2, 1.17 mM MgSO4, 1.2 mM NaH2PO4, 2.0 mM pyruvate, 0.02 mM EDTA, 3.0 mM MOPS, and 5.0 mM glucose). Next, the microvessels were incubated in MOPS-buffered salt solution for 30 min at 37°C. The MOPS solution was then replaced with 1 ml of fresh MOPS solution containing 0.3 mM 3-isobutyl-1-methylxanthine (broad-spectrum phosphodiesterase inhibitor) and the various treatments: 1) no isoproterenol and no inhibitor; 2) isoproterenol (1 μmol/l; Sigma-Aldrich) without inhibitor; 3) inhibitor (1 μmol/l for CGP77675, 1 mmol/l for Tempol, 10 mmol/l for 1-butanol and 10 μmol/l for all other inhibitors), but no isoproterenol; or 4) isoproterenol plus inhibitor. After incubation for 30 min at 37°C, the medium and microvessels were collected separately. Tissue cyclic AMP was extracted with 1 ml of 1-propanol at 4°C for 30 min with continuous shaking. The 1-propanol was evaporated, and samples were resuspended in phosphate buffer for assay of cyclic AMP.
Culture of PGSMCs. Primary PGSMCs were cultured by explant from freshly isolated microvessels using our previously described method (Mokkapatti et al., 1998). PGSMCs were maintained under standard cell culture conditions, and studies were conducted in PGSMCs with low passage number.
Protocol in PGSMCs. PGSMCs were washed twice with 1 ml of phosphate-buffered saline, placed in phosphate-buffered saline containing 0.3 mM 3-isobutyl-1-methylxanthine and treated with 1) no isoproterenol, no forskolin, and no inhibitor; 2) isoproterenol (1 μmol/l) without inhibitor; 3) forskolin (10 μmol/l) without inhibitor; 4) isoproterenol with U73122 (1 and 3 μmol/l); 5) forskolin with U73122 (3 μmol/l); 6) isoproterenol with C2-ceramide (1, 3, and 10 μmol/l); 7) forskolin with C2-ceramide (10 μmol/l); 8) isoproterenol with PP1 [0.03 and 0.1 μmol/l, 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine]; or 9) forskolin with PP1 (1 μmol/l). After a 30-min incubation with the aforementioned treatments, the medium was removed and cyclic AMP was extracted from cells with 1-propanol as described for the microvessels.
Assay for Cyclic AMP. In the microvessel experiments, cyclic AMP in both the medium (extracellular compartment) and microvessels (intracellular compartment) were measured. In the PGSMC experiments, only cyclic AMP in the cells (intracellular compartment) was measured. In both experiments, cyclic AMP was determined using a previously described high-performance liquid chromatography-fluorometric assay (Mokkapatti et al., 1998). Protein content in microvessels and PGSMCs was determined by dissolving microvessels and PGSMCs in 0.1% SDS and 0.1 N sodium hydroxide and measuring protein levels using the bicinchoninic acid method. Results are expressed as picomoles of cyclic AMP per milligram of protein.
Drugs.Table 1 lists each signal transduction inhibitor used in the present study along with its source. For reference, Table 1 also includes potency values made available by the manufacturer. However, these potencies were obtained mostly from reconstitution studies, and there relevance to studies in intact tissues/cells is uncertain.
Statistical Methods. For the microvessels experiments, data were analyzed by two-factor analysis of variance (ANOVA) in which one factor was level of isoproterenol and the second factor was level of inhibitor. For the PGSMC experiments, data were analyzed by one-factor analysis of variance. A Fisher's least significant difference (LSD) test was used for post hoc analyses. The criterion of significance was P < 0.05. All data are presented as means ± S.E.M.
Results
Studies in Freshly Isolated Preglomerular Microvessels. In control microvessels, isoproterenol (β-adrenoceptor agonist) significantly increased extracellular, intracellular, and total cyclic AMP (Fig. 1) with little variability. Figure 1 illustrates the effects of U73122 (phospholipase C inhibitor) on extracellular (top), intracellular (middle), and total (bottom) cyclic AMP in preglomerular microvessels. Although U73122 did not augment isoproterenol-induced extracellular cyclic AMP, U73122 did significantly (P < 0.0001) enhance isoproterenol-induced intracellular and total cyclic AMP. In this regard, the levels of intracellular and total cyclic AMP in the presence of isoproterenol were 89 and 67% greater, respectively, in U73122-treated microvessels compared with control microvessels.
Figure 2 illustrates the effects of GF109203X (protein kinase C inhibitor) on extracellular (top), intracellular (middle), and total (bottom) cyclic AMP in preglomerular microvessels. Similar to U73122, GF109203X did not augment isoproterenol-induced extracellular cyclic AMP, but it did significantly (P = 0.0478) enhance isoproterenol-induced total cyclic AMP. GF109203X tended to enhance intracellular cyclic AMP, but this effect did not reach statistical significance (P = 0.0582). The levels of intracellular and total cyclic AMP in the presence of isoproterenol were 35 and 29% greater, respectively, in GF109203X-treated microvessels compared with control microvessels.
Figure 3 illustrates the effects of 1-butanol (phospholipase D inhibitor) on extracellular (top), intracellular (middle), and total (bottom) cyclic AMP in preglomerular microvessels. 1-Butanol significantly inhibited isoproterenol-induced extracellular (P = 0.0006), intracellular (P < 0.0001), and total (P < 0.0001) cyclic AMP. In 1-butanol-treated microvessels, the levels of extracellular, intracellular, and total cyclic AMP in the presence of isoproterenol were 80, 5, and 23%, respectively, of the amounts in control microvessels.
Figure 4 illustrates the effects of CGP77675 (c-src inhibitor) on extracellular (top), intracellular (middle), and total (bottom) cyclic AMP in preglomerular microvessels. CGP77675 did not affect isoproterenol-induced extracellular cyclic AMP, but it significantly inhibited isoproterenol-induced intracellular (P = 0.0168) and total (P < 0.0001) cyclic AMP. In CGP77675-treated microvessels, the levels of intracellular and total cyclic AMP in the presence of isoproterenol were 52 and 61%, respectively, of the amounts in control microvessels.
Tables 2, 3, 4 summarize the effects of HA1077 (Rho kinase inhibitor), Y27632 (Rho kinase inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), dipenylene-iodonium (NADPH oxidase inhibitor), and Tempol (superoxide dismutase mimetic) on extracellular (Table 2), intracellular (Table 3), and total (Table 4) cyclic AMP. These inhibitors did not significantly alter isoproterenol-induced extracellular, intracellular, or total cyclic AMP.
Studies in Cultured PGSMCs. In all studies in cultured PGSMCs, we measured intracellular, but not extracellular, cyclic AMP because our experiments in freshly isolated microvessels indicated that intracellular cyclic AMP and total cyclic AMP afforded similar results. As shown in Fig. 5, 1 and 3 μmol/l U73122 increased isoproterenol-induced cyclic AMP by 5- and 6-fold, respectively; however, 3 μmol/l U73122 did not affect forskolin (direct activator of adenylyl cyclase)-induced cyclic AMP (Fig. 6). As shown in Fig. 7, 1, 3, and 10 μmol/l C2-ceraminde (phospholipase D inhibitor) decreased isoproterenol-induced cyclic AMP by 32, 78, and 82%, respectively; yet 10 μmol/l C2-ceraminde did not affect forskolin-induced cyclic AMP (Fig. 8). Both 0.03 and 0.1 μmol/l PP1 (src family inhibitor) reduced isoproterenol-induced cyclic AMP by 60 and 68% (Fig. 9), respectively; however, even a much higher concentration of PP1 (1 μmol/l) did not influence forskolin-induced cyclic AMP (Fig. 10).
Discussion
An important aspect of the present study is the development of a robust and reproducible model system for examining signal transduction processes in freshly isolated preglomerular microvessels. The present study demonstrates that the preglomerular microvessel preparation is an excellent model system for such studies. This model system, as described in the present study, was developed by combining our previous experience with isolation of preglomerular microvessels (Jackson and Mi, 2000) with the excellent method described by Bolz et al. (2000) for maintaining skeletal muscle microvessels in organ culture. The advantage of the present method is that signal transduction processes can be investigated in intact microvessels, which are more physiological compared with cell culture model systems in which signal transduction mechanisms may be perturbed. However, the drawbacks of the preglomerular microvessel preparation are that it is a labor-intensive method and cell types other than vascular smooth muscle cells (for example, endothelial cells and fibroblasts) are also present in the preparation, albeit in much lower mass compared with smooth muscle cells. Therefore, we used freshly isolated microvessels to identify signal transduction systems that participate in the physiological regulation of receptor-induced cyclic AMP production in intact blood vessels and then used cultured PGSMCs to confirm the involvement of vascular smooth muscle cells and to conduct more detailed studies with regard to concentration response and mechanism.
U73122 is the most widely used pharmacological inhibitor to probe the role of phospholipase C in signal transduction processes (Bleasdale et al., 1990). In freshly isolated preglomerular microvessels, U73122, at a concentration several times the IC50 value for inhibition of phospholipase C, markedly potentiated isoproterenol-induced intracellular and total cyclic AMP. These results strongly suggest that phospholipase C normally restrains receptor-induced adenylyl cyclase activation in intact preglomerular microvessels.
Most likely, U73122-induced enhancement of receptor-mediated cyclic AMP in preglomerular microvessels occurs in vascular smooth muscle cells, the dominant cell type in this preparation. However, to specifically test this hypothesis, we examined the ability of U73122 to enhance receptor-induced cyclic AMP in cultured PGSMCs. In these studies, we used lower concentrations of U73122 to avoid nonspecific effects and included experiments with forskolin to investigate the mechanism of U73122-induced enhancement of receptor-induced cyclic AMP. Importantly, even 10-fold lower concentrations of U73122 enhanced isoproterenol-induced cyclic AMP in cultured PGSMCs. Moreover, a concentration of U73122 that caused a 6-fold increase in the response to isoproterenol did not alter the response to forskolin. These findings indicate that phospholipase C modulates receptor-induced cyclic AMP in vascular smooth muscle cells by regulating the efficiency of receptor/G protein/adenylyl cyclase coupling, rather than by directly increasing the activity of adenylyl cyclase.
Because activation of phospholipase C increases the activity of protein kinase C (Rana and Hokin, 1990; Majerus, 1992), it is possible that protein kinase C mediates all or part of the effects of phospholipase C on receptor-induced adenylyl cyclase. Although GF109203X, a well characterized protein kinase C inhibitor (Ku et al., 1997; Hers et al., 1999), augmented isoproterenol-induced cyclic AMP in preglomerular microvessels, the enhancement was very modest (only approximately 30%). These results indicate that the contribution of protein kinase C to the regulation of receptor-induced cyclic AMP by phospholipase C is small and of questionable biological significance. Accordingly, we did not pursue additional studies with GF109203X in cultured PGSMCs.
Perhaps the most provocative finding in the present study is that concentrations of 1-butanol that inhibit phospholipase D activity (Kotter and Klein, 1999) nearly completely block isoproterenol-induced cyclic AMP in preglomerular microvessels. These results suggest that phospholipase D may importantly regulate adenylyl cyclase activity and in a positive manner. Consistent with this hypothesis are our observations that in cultured vascular smooth muscle cells obtained from preglomerular microvessels, angiotensin II activates phospholipase D more potently (Andresen et al., 2001) and augments isoproterenol-induced cyclic AMP more potently (Mokkapatti et al., 1998) in cells obtained from spontaneously hypertensive rats compared with cells obtained from normotensive rats.
An obvious caveat to the hypothesis that phospholipase D regulates adenylyl cyclase activity is that 1-butanol may elicit nonspecific effects. Although concentrations of 1-butanol much higher than used in the present study have been used in numerous investigations to probe the physiological role of phospholipase D (Chen et al., 1997; Lee et al., 2000), nonspecific effects of 1-butanol remain a concern. Therefore, we conducted additional experiments in cultured PGSMCs with both a lower concentration of 1-butanol (data not shown) and with the alternative phospholipase D inhibitor C2-ceramide (Venable et al., 1996). A lower concentrations of 1-butanol (3 mM) also inhibited isoproterenol-induced cyclic AMP (data not shown), a finding consistent with the results in preglomerular microvessels. More convincing, however, are the results with C2-ceramide. In this regard, C2-ceramide caused a marked concentration-related inhibition of isoproterenol-induced cyclic AMP in cultured PGSMCs. In marked contrast, the highest concentration of C2-ceramide used to augment isoproterenol-induced cyclic AMP had no effect on forskolin-stimulated cyclic AMP. These data support the hypothesis that phospholipase D augments receptor-mediated cyclic AMP in vascular smooth muscle cells by regulating the efficiency of receptor/G protein/adenylyl cyclase coupling, rather than by directly increasing the activity of adenylyl cyclase. Thus, phospholipase D and phospholipase C seem to have opposite roles in modulating receptor-induced cyclic AMP in smooth muscle cells in the renal microcirculation.
Another key finding of the present study is that apparently src importantly regulates receptor-mediated cyclic AMP production in preglomerular microvessels. In preglomerular microvessels, the selective c-src inhibitor CGP77675 attenuated receptor-induced cyclic AMP. To further address the hypothesis that src regulates receptor-mediated cyclic AMP in vascular smooth muscle, we conducted additional experiments in cultured PGSMCs using an alternative and widely used src family inhibitor PP1 (Hanke et al., 1996). PP1 caused a concentration-dependent inhibition of isoproterenol-induced cyclic AMP in PGSMCs, yet even a 10-fold higher concentration of PP1 did not affect forskolin-induced cyclic AMP. These results are entirely consistent with the observation in murine fibroblasts that overexpression of c-src markedly enhances β-adrenoceptor-induced cyclic AMP (Bushman et al., 1990) by a mechanism involving increased coupling of stimulatory G proteins to adenylyl cyclase (Luttrell et al., 1992). Thus, the current findings extend the observation in experimentally manipulated fibroblasts to physiologically intact preglomerular microvessels and microvascular smooth muscle cells.
The RhoA/Rho kinase pathway is one of the most important mechanisms regulating vascular smooth muscle cell contractility (Wettschureck and Offermanns, 2002). Therefore, it would come at no surprise if this pivotal pathway also influenced the regulation of adenylyl cyclase. To test this hypothesis, we first used HA1077, a potent and selective inhibitor of Rho kinase (Amano et al., 1999). With regard to total cyclic AMP, HA1077 tended to attenuated isoproterenol-induced responses. However, this effect did not reach statistical significance. Therefore, we decided to repeat the study with an alternative inhibitor, Y27632. Y27632 is both a highly potent and selective inhibitor of Rho kinase (Uehata et al., 1997). Importantly, the results with Y27632 were not significant, and indeed isoproterenol-induced cyclic AMP tended to be higher in Y27632-treated microvessels. Together, our data eliminate the possibility that Rho kinase regulates, either positively or negatively, receptor-mediated stimulation of cyclic AMP production in preglomerular microvessels. Because of these negative findings, we did not pursue additional studies in cultured PGSMCs.
Another key signal transduction pathway in vascular smooth muscle is the phosphatidylinositol-3-kinase mechanism. This pathway is critically involved in vascular smooth muscle cell contraction (Komalavilas et al., 2001) and growth (Lipskaia et al., 2003; Sedding et al., 2003), and so it too is a likely candidate for modulating receptor-induced activation of adenylyl cyclase in vascular smooth muscle. However, we found that LY294002, a potent and selective inhibitor of phosphatidylinositol-3-kinase (Vlahos et al., 1994), had no observable effect on isoproterenol-induced cyclic AMP. Therefore, our data rule out any role of phosphatidylinositol-3-kinase as a modulator of receptor-mediated stimulation of cyclic AMP production in preglomerular microvessels. Because of these negative findings, we did not conduct additional studies in PGSMCs.
Yet another extremely important signal transduction pathway that is involved in vascular smooth muscle cell contraction (Hanna et al., 2002) and growth (Viedt et al., 2000) is the NADPH oxidase mechanism that generates superoxide anion. In the present study, we observed a near significant ability of diphenylene-iodonium, a standard inhibitor of NADPH oxidase (Inoguchi et al., 2000), to augment receptor-mediated stimulation of total cyclic AMP production. To ascertain whether this near significant effect was real, we repeated the experiment in the presence of a high concentration of Tempol, a widely used superoxide dismutase mimetic (Iannone et al., 1989; Nilsson et al., 1989). Importantly, we found no evidence that Tempol alters receptor-mediated stimulation of cyclic AMP production. Therefore, our data eliminate any role for NADPH oxide as a modulator of receptor-mediated stimulation of cyclic AMP production in preglomerular microvessels. Accordingly, additional studies in PGSMCs were not performed.
The results of the present study strongly support the conclusion that the phospholipase C, phospholipase D, and src pathways importantly regulate receptor-mediated stimulation of cyclic AMP production in preglomerular microvessels and PGSMCs. It is important to note, however, that in the present discussion the terms phospholipase C, phospholipase D, and src refer to families of enzymes, rather than to homogenous populations of proteins. In this regard, there are several members of the phospholipase C, phospholipase D, and src families, and the currently available pharmacological inhibitors are not sufficiently selective to differentiate reliably among the various family members of these classes of enzymes. Which specific isoforms/family members of phospholipase C, phospholipase D, and src regulate receptor-induced cyclic AMP must be resolved using molecular biological approaches.
In summary, the current study examined whether key signal transduction mechanisms modulate receptor-mediated stimulation of cyclic AMP production in preglomerular microvessels and PGSMCs. Our results rule out the participation of the RhoA/Rho kinase, phosphatidylinositol-3-kinase, and the NADPH oxidase pathways in this regard. However, our results strongly suggest that the phospholipase C, phospholipase D, and src pathways importantly regulate receptor-mediated stimulation of cyclic AMP production in preglomerular microvessels and PGSMCs. Phospholipase C, phospholipase D, and src modulate receptor-induced cyclic AMP not by directly affecting adenylyl cyclase activity but by altering the efficiency of receptor/G protein/adenylyl cyclase coupling.
Footnotes
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This work was supported by National Institutes of Health Grant HL069846.
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DOI: 10.1124/jpet.103.063081.
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ABBREVIATIONS: PGSMC, preglomerular microvascular smooth muscle cell; RT, room temperature; MOPS, 3-(N-morpholino)propanesulfonic acid; PBS, phosphate-buffered saline; PPI, 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; ANOVA, analysis of variance; LSD, least significant difference; U73122, 1-(6-((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide; CGP77675, 1-(2-{4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenyl}ethyl)piperidin-4-ol; HA1077, 1-(5-isoquinolinesulfonyl)homopiperazine; Y27632, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one.
- Received November 17, 2003.
- Accepted April 12, 2004.
- The American Society for Pharmacology and Experimental Therapeutics