Introduction

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Several studies demonstrate that Ang II enhances the formation of cAMP in response to a variety of agonists. Kubalak and Webb (1993) showed that Ang II increases the accumulation of cAMP in response to isoproterenol, adenosine and prostacyclin in rat aortic smooth muscle cells by a mechanism involving calcium-calmodulin. Similarly Zhang et al. (1997) confirmed that vasopressin, like Ang II, increases the cAMP response to isoproterenol and prostacyclin in rat aortic smooth muscle cells via a calcium-calmodulin mechanism. McCumbee et al. (1996) also demonstrated synergistic activation of adenylyl cyclase by Ang II and isoproterenol in rat aortic smooth muscle cells. Moreover, this effect appears to occur in intact blood vessels because Ang II enhances vasodilation of the rat aorta by agents that elevate cAMP (Brizzolara-Gourdie and Webb, 1997).

Ang II potentiation of cAMP formation has also been observed in other cell types. Baukal et al. (1994) reported that Ang II enhances ACTH-induced cAMP formation in bovine adrenal glomerulosa cells and in COS-7 cells transfected with the rat AT₁ receptor. However, unlike aortic vascular smooth muscle cells, in these cell types the cross-talk between Ang II and agonist-stimulated cAMP is mediated by the phosphatase calcineurin. Finally, a very recent study (Klingler et al., 1998) indicated that Ang II facilitates V₂ vasopressin receptor-mediated cAMP formation in CHO cells transfected with cDNA for both the AT₁ and V₂ receptors. However, in this case the interaction is mediated by protein kinase C.

Although Ang II enhances agonist-stimulated cAMP formation in smooth muscle cells from large conduit arteries, whether this occurs in smooth muscle cells from resistance vessels is unknown.

In this regard, in the perfused rat kidney, Ang II inhibits rather than stimulates isoproterenol-induced cAMP formation (Vyas et al., 1996), and in isolated glomeruli Ang II inhibits rather than stimulates adenylyl cyclase (Edwards and Stack, 1993). Thus, there is some evidence that Ang II might not enhance agonist-induced cAMP in vascular smooth muscle cells in the microcirculation.

Another important, yet open, question is whether Ang II modulation of agonist-induced cAMP is altered in genetic hypertension, particularly in the renal vascular bed. Several studies suggest that cAMP stimulating agents have a reduced ability to buffer Ang II-induced renal vasoconstriction in the SHR kidney compared with kidneys from normotensive WKY rats (Chatziantoniou and Arendshorst, 1992; Chatziantoniou et al., 1993, 1995; Jackson and Herzer, 1993, 1994). These latter finding suggest that in SHR kidneys, Ang II inhibits, rather than stimulates, agonist-induced cAMP production in the renal vasculature, an inference that has been verified directly in the perfused SHR kidney (Vyas et al., 1996).

A final question regarding the interaction between Ang II and cAMP stimulating agonists relates to the mechanism of the interaction. Thus far, three mechanisms have been proposed (i.e., calcineurin, calcium-calmodulin and protein kinase C). Which, if any, of these mechanisms apply in smooth muscle cells from the microcirculation remains to be elucidated.

The aforementioned considerations prompted the present study, which had three goals; 1) to determine whether Ang II enhances beta-adrenoceptor-induced cAMP in PMVSMCs; 2) to determine whether Ang II-induced enhancement of beta-adrenoceptor-induced cAMP differs in PMVSMCs from normotensive WKY rats vs. PMVSMCs from spontaneously hypertensive rats (SHR); and 3) to elucidate the mechanism of Ang II enhancement of beta-adrenoceptor-induced cAMP in both SHR and WKY PMVSMCs.

	Methods

Top Abstract Introduction Methods Results Discussion References

Materials. Rats were obtained from Taconic Farms (Germantown, NY). All cell culture reagents and supplies were purchased from Gibco Laboratories (Grand Island, NY), except for donor-defined FCS, which was obtained from Hyclone Laboratories (Miami, FL). Iron oxide and chloroacetaldehyde were from Aldrich Chemical (Milwaukee, WI); Pertussis toxin, forskolin, Ang II (acetate salt), (±)-isoproterenol, IBMX, W-7, calphostin C and chelerythrine chloride were from Sigma Chemical (St. Louis, MO); BAPTA-AM was from Molecular Probes (Eugene, OR); UK14,304 was from Research Biochemicals International (Natick, MA); A23187 and calmidazolium were from Calbiochem-Novabiochem (La Jolla, CA); FK506 was from Fujisawa USA (Deerfield, IL); 1-propanol was from J. T. Baker (Phillipsburg, NJ). Immunochemicals for cell characterization were obtained from sources described by Dubey and coworkers (1992). All other chemicals were of the highest grade available.

Animal care. Male SHR and WKY rats 12 to 14 weeks of age were housed at the University of Pittsburgh Animal Facility with controlled temperature, relative humidity and light cycle (22°C, 55% and 7 a.m. to 7 p.m., respectively). Animal care conformed with institutional guidelines. The animals were maintained on Wayne Rodent Blox 8604 (Madison, WI) and tap water ad libitum. Studies had prior approval of the Institutional Animal Care and Use Committee.

cAMP experiments in cultured preglomerular microvascular smooth muscle cells. Renal microvessels were isolated from 12- to 14-week-old male SHR and WKY rats using iron oxide infusion, mechanical separation and collagenase digestion coupled with the use of a magnet to retain the iron-laden vessels as described by Chaudhari et al. (1989) and modified by Dubey et al.(1992). Briefly, a 5% suspension of iron oxide particles in DMEM was infused into the aortas of pentobarbital-anesthetized rats. The kidneys were removed, decapsulated and sectioned longitudinally. Next, the medulla was removed and the cortical tissue was placed in supplemented DMEM (DMEM with antibiotics, fungicidals and HEPES). The cortical tissue was minced and dispersed using a wire mesh, effecting separation of microvessels from surrounding tissue. The preparation was then washed repeatedly in ice-cold supplemented DMEM while using a magnet to retain the iron-laden vessels. The samples were then digested with Type I collagenase at 37°C for 15 to 30 min after which they were passed through a 20-gauge hypodermic needle to shear off glomeruli. The arteriolar fraction retained after sieving through an 80 µm mesh was suspended in supplemented DMEM with 20% FCS, plated and incubated at 37°C in 5% CO₂, 95% air and 98% humidity. The medium was changed every day until cells attained confluence. The PMVSMCs were repeatedly subjected to selective plating (Aviv et al., 1983) to reduce fibroblast contamination. The following characteristics were used to verify that the cultured cells were PMVSMCs: a) characteristic cell morphology and hill-and-valley pattern of growth, b) contraction to Ang II and norepinephrine (>90% of cells contracted), c) positive immunofluorescence staining for smooth muscle-specific  and  isoactin (>98%), heavy chain myosin (>95%) and desmin (>90%), and d) absence of von Willebrand factor (Dubey et al., 1992). Experiments were conducted at confluence in the third to fifth passage. At this passage level, cells retain their phenotypic characteristics and grow exponentially.

HPLC. Preparation of samples for HPLC involved acidification to stabilize cAMP, addition of internal standard and derivatization of cAMP and the internal standard to allow quantification by HPLC with fluorometric detection. Samples were prepared by adding 10 µl of acetate buffer (0.5 mol/l sodium acetate, pH 4.35), 10 µl of a 1 µmol/l solution of internal standard (adenine-9-beta-D-arabinofuranoside) and 10 µl of 50% chloroacetaldehyde followed by incubation for 1 hr at 80°C. This affected derivatization of cAMP to N⁶-etheno-cAMP and of internal standard to N⁶-etheno-adenine-9--D-arabinofuranoside (Zhang et al., 1991). Derivatized samples were injected into an ISCO (Lincoln, NE) HPLC system (pump model 2350, gradient programmer model 2360, 4.6 × 250 mm C18 reverse-phase column with 5 µm particle size; ChemResearch Data Management System) (Jackson et al., 1996). Fluorometric detection was achieved at an excitation wavelength of 275 nm and emission wavelength of 420 nm with a Waters 470 fluorescence detector. The mobile phase was composed of 10 mmol/l citrate buffer with 3.5% acetonitrile and 0.5% tetrahydrofuran (pH 4.0) and was run isocratically at 1.2 ml/min. A standard curve for cAMP was constructed with the ratio of areas of cAMP with that of the internal standard. This method achieved a detection sensitivity of ~0.12 pmol/injection.

Calcium measurements in cultured preglomerular microvascular smooth muscle cells. Intracellular free Ca²⁺ was measured using a charge coupled device (CCD)-based imaging system. Cells were loaded via incubation with 5 µmol/l Fura-2 acetoxymethyl ester (Fura-2 AM, Molecular Probes, Eugene, OR) in buffer supplemented with 5 mg/ml bovine serum albumin for 45 min at 37°C. Following loading, coverslips were rinsed with buffer and mounted in a recording chamber. The imaging system used in these studies consisted of a Nikon Diaphot with 300 pixel resolution in combination with a Dage-MTI Gen II Sys image intensifier, a software package from Compix, Inc. (Cranberry, PA), and a 75 watt Xenon lamp-based monochromator illuminated with 345 nm and 375 nm light. Attenuation of incident light was achieved with neutral density filters (ND2, 1% transmittance). Emitted light passed through a 515 nm dichroic mirror and a 535 ± 12.5 nm band pass filter (Omega Optical, Brattleboro, VT). All recordings were made at room temperature (20-25°C) in Dulbecco's PBS buffered with HEPES and NaHCO₃. Determinations of [Ca²⁺]_i were made for 5 min under basal (unstimulated) conditions and then after the addition of Ang II (0.5 µmol/l). Background-subtracted fluorescence ratios were converted to [Ca²⁺]_i using a method originally described by Grynkiewicz et al. (1985). Relevant parameters were determined in vitro from calibration curves constructed from fluorescence ratios obtained from eighteen or more EGTA-buffered solutions containing the Fura-2 potassium salt (20 µmol/l) and known concentrations of Ca²⁺ (0-100 µmol/l).

Organization of experimental groups, calculation of isoproterenol-induced cAMP and calculation of the effects of Ang II on isoproterenol-induced cAMP. The basic treatment groups for PMVSMCs were as follows: control group (vehicles only), isoproterenol group (vehicle for Ang II followed by 0.1 µmol/l isoproterenol), Ang II group (Ang II 0.5 µmol/l followed by vehicle for isoproterenol), or Ang II and isoproterenol group (Ang II 0.5 µmol/l followed by 0.1 µmol/l isoproterenol). An experimental block consisted of 4 contiguous culture wells containing PMVSMCs from the same rat strain and subjected to the above modalities. Protein in PMVSMCs was assayed using the bicinchoninic acid assay (Pierce, Rockford, IL). For each experimental block, the isoproterenol-induced change in cAMP levels in the absence of Ang II was calculated by subtracting the cAMP levels in the control group and from the cAMP levels in the isoproterenol group. Similarly, the isoproterenol-induced change in cAMP levels in the presence of Ang II was calculated by subtracting the cAMP levels in the Ang II group from the cAMP levels in the Ang II and isoproterenol group. Finally, for each experimental block, the net effect of Ang II on the isoproterenol-induced cAMP response was calculated by subtracting the isoproterenol-induced cAMP response in the absence of Ang II from the isoproterenol-induced cAMP response in the presence of Ang II. Therefore, the net effect of Ang II on the isoproterenol-induced cAMP response was calculated as (Ang II and isoproterenol group - Ang II group) - (isoproterenol group - control group).

Data presentation and statistical analyses. In all experiments, the quantity of cAMP represents total cAMP (supernatant + cellular) expressed as pmol/mg protein, and all values in text and figures represent means ± SEM. The data in figures 1 and 5 were analyzed by unpaired Student's t tests. The data in figures 2 to 4 and 6 to 11 were analyzed using a two-factor analysis of variance (ANOVA), followed by a Bonferroni test for post-hoc comparisons if appropriate. In figures 3, 4 and 6 to 11, the control groups in each figure were the groups that were treated with the vehicle for the given pharmacological agent and were conducted in parallel with and at the same time as the experiments with the given pharmacological probe. Parallel controls were essential because the effects of isoproterenol on cAMP and the enhancement of these responses by Ang II varied from batch to batch of PMVSMCs. The data in figure 12 were analyzed with a one-factor analysis of variance followed by a Bonferroni test for post-hoc comparisons. Statistical analyses were performed using the Number Cruncher Statistical System (Kaysville, UT) and Excel spreadsheet.

	Results

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Production of cAMP in the presence and absence of isoproterenol and/or Ang II was studied in cultured PMVSMCs. All treatments were tested in four to nine experimental blocks per rat strain. Figure 1 illustrates the results of a typical experimental series. In this experimental series, in WKY PMVSMCs in the absence and presence of Ang II, isoproterenol increased cAMP levels by 831 ± 28 and 3605 ± 370 pmol/mg protein, respectively, i.e., a net change of the isoproterenol-induced cAMP response of 2774 ± 342 pmol/mg protein. In SHR PMVSMCs in the absence of Ang II, isoproterenol increased cAMP levels by 2931 ± 74 pmol/mg protein, whereas in the presence of Ang II, isoproterenol increased cAMP levels by 12171 ± 512 pmol/mg protein, a net change of the isoproterenol-induced cAMP response of 9240 ± 484 pmol/mg protein (P = .004 compared with WKY PMVSMCs; unpaired Student's t test).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of Ang II (0.5 µmol/l) on isoproterenol (Iso; 0.1 µmol/l)-induced cAMP in WKY (open bars) and SHR (solid bars) PMVSMCs. Cells received one of the following four treatments: Ang II alone, Ang II followed by isoproterenol, isoproterenol alone, or vehicles only. In all cases, Ang II or vehicle was added 40 min and isoproterenol or vehicle was added 70 min into the 100 min incubation. Experiments were conducted in the presence of 100 µmol/l IBMX. Isoproterenol-induced cAMP (top) was calculated as cAMP in the isoproterenol group minus cAMP in the untreated group. Ang II enhancement of isoproterenol-induced cAMP was calculated as: (cAMP in the Ang II + isoproterenol group minus cAMP in the Ang II group) minus (cAMP in the isoproterenol group minus cAMP in the untreated group). `a' and `b' represent P < .05 by Student's t tests, n = 9.

To determine whether the differential enhancement by Ang II of isoproterenol-induced cAMP in WKY vs. SHR PMVSMCs was due to strain-related differences in the total adenylyl cyclase activity, cAMP levels were measured in the presence of low (1 µmol/l) and high (5 µmol/l) concentrations of forskolin with and without isoproterenol (0.1 µmol/l). As shown in figure 2, cAMP levels in the presence of both low and high concentrations of forskolin, either without or with isoproterenol, were similar in WKY vs. SHR PMVSMCs. Moreover, in the presence of isoproterenol, the low and high concentrations of forskolin had similar effects on cAMP levels, indicating near maximal stimulation of adenylyl cyclase under these conditions. Although isoproterenol greatly enhanced the effects of forskolin on cAMP levels in both SHR and WKY PMVSMCs, the ability of isoproterenol to enhance the effects of forskolin was independent of strain (P = .162, two-factor ANOVA). Thus, unlike the Ang II/isoproterenol interaction on adenylyl cyclase, the forskolin/isoproterenol interaction was similar in WKY vs. SHR PMVSMCs.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effect of forskolin (1 or 5 µmol/l) in the absence and presence of isoproterenol (Iso; 0.1 µmol/l) on cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Six experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance.

Previous studies (Baukal et al., 1994) demonstrated that Ang II enhances ACTH-induced cAMP in cultured bovine glomerulosa cells and that this effect of Ang II is abolished by inhibition of calcineurin with cyclosporin A or FK506. To determine whether Ang II enhancement of isoproterenol-induced cAMP was mediated by calcineurin, the Ang II/isoproterenol interaction was examined in cells pretreated with FK506 (100 ng/ml for 30 min; four experimental blocks for each strain). Ang II enhancement of isoproterenol-induced cAMP was not attenuated, and in fact was increased, by FK506 in both WKY and SHR PMVSMCs. Ang II enhancement of isoproterenol-induced cAMP in WKY cells without and with FK506 was 260 ± 140 and 443 ± 86 pmol/mg protein, respectively, and Ang II enhancement in SHR cells without and with FK506 was 1369 ± 96 and 1724 ± 35 pmol/mg protein, respectively (strain effect: P < .0001; FK506 effect: P = .017; interaction: P = .393; two-factor ANOVA).

Because Ang II is well known to stimulate vasodilator prostaglandins, it is possible that Ang II enhancement of isoproterenol-induced cAMP is mediated in part by prostaglandins. To address this possibility, Ang II enhancement of isoproterenol-induced cAMP was compared in the absence and presence of the cyclooxygenase inhibitor indomethacin (25 µmol/l added 90 min before the experiment). As illustrated in figure 3, although indomethacin significantly attenuated (P = .035, two-factor ANOVA) Ang II enhancement of isoproterenol-induced cAMP in both WKY and SHR PMVSMCs, even in the presence of indomethacin Ang II markedly enhanced isoproterenol-induced cAMP. Also, the effects of indomethacin were independent of strain (P = .569), and Ang II enhancement of isoproterenol-induced cAMP remained greater in SHR vs. WKY PMVSMCs even in the presence of indomethacin.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of indomethacin (25 µmol/l) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Nine experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance.

Angiotensin type 1 (i.e., AT₁) receptors are coupled to G_i proteins and Ang II releases both alpha-i and beta gamma subunits of G_i proteins. Since both alpha-i and beta gamma subunits of G_i proteins may modify alpha-s-mediated stimulation of adenylyl cyclase activity (Sunahara et al., 1996), it is possible that Ang II/isoproterenol interactions in PMVSMCs involve alpha-i and/or beta gamma subunits of G_i proteins. To test this hypothesis, PMVSMCs were preincubated for 20 hr with pertussis toxin (100 ng/ml) to ADP ribosylate G_i proteins and thus prevent the release of both alpha-i and beta gamma subunits of G_i proteins. Pretreatment with pertussis toxin significantly (P < .001; two-factor ANOVA) increased Ang II enhancement of isoproterenol-induced cAMP in both WKY and SHR PMVSMCs (fig. 4). This increase was statistically more pronounced in SHR, compared with WKY, PMVSMCs (P = .004; two-factor ANOVA) so that the difference between WKY and SHR PMVSMCs vis-á-vis Ang II enhancement of isoproterenol-induced cAMP was enlarged by pertussis toxin. In the same set of experiments, the alpha-2 receptor agonist UK 14,304, which like Ang II is also coupled to G_i, not only failed to potentiate isoproterenol-induced cAMP (fig. 5), but actually caused a significant decrease in isoproterenol-induced cAMP in both WKY and SHR PMVSMCs.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of pertussis toxin (100 ng/ml for 20 hr) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Nine experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance; aP < .05 for differences within the same strain and bP < .05 for between-strain differences by post-hoc testing.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effects of Ang II (0.5 µmol/l) and UK 14,304 (3 µmol/l) on isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Nine experimental blocks were used for both WKY and SHR PMVSMCs; aP < .05 by student's t tests.

The calcium dependency of the interaction between Ang II and isoproterenol was assessed by pretreatment of cells with the intracellular calcium chelator BAPTA-AM (10 µmol/l for 30 min). As illustrated in figure 6, BAPTA-AM significantly (P < .001, two-factor ANOVA) decreased Ang II enhancement of isoproterenol-induced cAMP in PMVSMCs derived from both strains. BAPTA-AM tended to attenuate Ang II enhancement of isoproterenol-induced cAMP more in SHR, compared with WKY, PMVSMCs (P = .052; two-factor ANOVA).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effect of BAPTA-AM (10 µmol/l) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Four experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance.

[Ca²⁺]_i was measured in 68 SHR PMVSMCs and 57 WKY PMVSMCs under basal conditions and during application of Ang II (0.5 µmol/l). The results showed that: 1) PMVSMCs from both strains had similar basal levels of [Ca²⁺]_i; 2) in PMVSMCs from both strains Ang II caused an immediate rise in [Ca²⁺]_i that peaked and then declined to a plateau within two to three min; and 3) neither the peak nor the plateau increase in [Ca²⁺]_i induced by Ang II was strain dependent (data not shown). The effects of BAPTA-AM (10 µmol/l) on Ang II-induced changes in [Ca²⁺]_i were examined in 23 SHR PMVSMCs and 22 WKY PMVSMCs, and in both strains BAPTA-AM abolished the Ang II-induced peak and plateau in [Ca²⁺]_i (data not shown).

The calcium dependency of the interaction between Ang II and isoproterenol was further assessed by pretreatment of cells with the calcium ionophore A23187 (10 µM for 30 min). As illustrated in figure 7, in both WKY and SHR PMVSMCs, A23187 significantly (P < .001; two-factor ANOVA) promoted the potentiating effects of Ang II on isoproterenol-induced cAMP. The ability of A23187 to potentiate the effects of Ang II on isoproterenol-induced cAMP was similar in both strains (P = .851; two-factor ANOVA).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of A23187 (10 µmol/l) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Six experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance.

Chelation of intracellular calcium attenuated and increasing intracellular calcium augmented Ang II enhancement of isoproterenol-induced cAMP. These findings are consistent with an important role for calmodulin in the mechanism of Ang II enhancement of cAMP. In this regard, calmodulin is a well-known modulator of some isoforms of adenylyl cyclase activity (Sunahara et al., 1996), and Kubalak and Webb (1993) reported that in rat aortic smooth muscle cells calmodulin mediates Ang II enhancement of agonist-induced cAMP. Therefore, the possibility that Ang II enhancement of isoproterenol-induced cAMP is mediated by calmodulin in PMVSMCs was investigated. As indicated in figure 8, calmidazolium (10 µmol/l for 30 min), a potent inhibitor of calmodulin, significantly increased, rather than decreased, the Ang II enhancement of the isoproterenol-induced cAMP response in SHR PMVSMCs. In contrast, in WKY PMVSMCs, calmidazolium tended to decrease the Ang II enhancement of the isoproterenol-induced cAMP. Analysis by two-factor ANOVA indicated a highly significant interaction between strain and the effects of calmidazolium on Ang II enhancement of isoproterenol-induced cAMP (P = .0006) with the post-analysis Bonferroni test indicating that the increase in SHR, but not the decrease in WKY, was significant. To further test the role of calmodulin in Ang II enhancement of isoproterenol-induced cAMP, the effect of W-7 (20 µmol/l for 30 min), an alternative calmodulin antagonist, was investigated. Like calmidazolium, W-7 potentiated Ang II enhancement of isoproterenol-induced cAMP in SHR, but not in WKY, PMVSMCs (fig. 9).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effect of calmidazolium (10 µmol/l) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Nine experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance; aP < .05 for differences within the same strain and bP < .05 for between-strain differences by post-hoc testing.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Effect of W-7 (20 µmol/l) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Four experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance; aP < .05 for differences within the same strain and bP < .05 for between-strain differences by post-hoc testing.

Because the experiments with BAPTA-AM and A23187 demonstrated an important role for intracellular calcium yet the experiments with calmidazolium and W-7 did not support a role for calmodulin, we examined the possibility that classic (i.e., calcium/DAG sensitive) protein kinase C mediates the Ang II enhancement of isoproterenol-induced cAMP as recently reported by Klingler et al. (1998) in CHO cells transfected with cDNA for both the AT₁ and V₂ receptors. As shown in figures 10 and 11, treatment of PMVSMCs with the protein kinase C inhibitors calphostin C and chelerythrine chloride abolished the ability of Ang II to enhance isoproterenol-induced cAMP.

View larger version (24K): [in this window] [in a new window]   Fig. 10.   Effect of calphostin C (0.5 µmol/l) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Four experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance; aP < .05 for differences within the same strain and bP < .05 for between-strain differences by post-hoc testing. (Note that in calphostin-treated WKY cells Ang II enhancement was zero.)

View larger version (24K): [in this window] [in a new window]   Fig. 11.   Effect of chelerythrine chloride (5 µmol/l) on Ang II (0.5 µmol/l) enhancement of isoproterenol (Iso; 0.1 µmol/l)-induced cAMP production in WKY (open bars) and SHR (solid bars) PMVSMCs. Four experimental blocks were used for both WKY and SHR PMVSMCs. P values are from two-factor analysis of variance; aP < .05 for differences within the same strain and bP < .05 for between-strain differences by post-hoc testing.

In the interaction studies described above, FK506, pertussis toxin, BAPTA-AM, A23187, W-7, calphostin C and chelerythrine had little or no effect on basal cAMP responses to isoproterenol; however, indomethacin and calmidazolium significantly attenuated basal isoproterenol-induced cAMP responses in PMVSMCs from both strains by ~70% and ~50%, respectively (data not shown).

All of the experiments in the studies described above used 0.5 µmol/l Ang II. It is possible that the dose-response curve to Ang II with regard to isoproterenol-induced cAMP is biphasic, i.e., Ang II might inhibit isoproterenol-induced cAMP at low concentrations while stimulating isoproterenol-induced cAMP at high concentrations. To examine this issue, we determined in SHR isoproterenol-induced cAMP over a 7-log concentration range of Ang II. As shown in figure 12, Ang II at concentrations <10⁹ mol/l had no effect, and at concentrations >10⁹ mol/l Ang II potentiated isoproterenol-induced cAMP without affecting basal levels of cAMP.

View larger version (20K): [in this window] [in a new window]   Fig. 12.   Total cAMP in SHR PMVSMCs in the absence and presence of isoproterenol (Iso; 1 µmol/l) at different concentrations of Ang II. Eight culture wells were used for each data point; aindicates significant difference (P < .01) from all groups not marked with `a'.

	Discussion

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Cultured PMVSMCs were highly and reproducibly responsive to adenylyl cyclase stimulating agents such as isoproterenol and forskolin. As in rat aortic smooth muscle cells (Kubalak and Webb, 1993; McCumbee et al., 1996; Zhang et al., 1997), transfected COS-7 cells (Baukal et al., 1994), transfected CHO cells (Klingler et al., 1998) and bovine adrenal glomerulosa cells (Baukal et al., 1994), Ang II caused a marked potentiation, rather than inhibition, of the cAMP response to isoproterenol in cultured PMVSMCs from both strains.

Ang II enhancement of isoproterenol-induced cAMP was significantly greater in SHR, compared with WKY, PMVSMCs. As illustrated in figure 1, the basal response to isoproterenol, i.e., the response in the absence of Ang II, was greater in SHR, compared with WKY, PMVSMCs. Therefore, it is possible that the greater Ang II enhancement in SHR PMVSMCs was due to the greater basal responsiveness of these cells to isoproterenol. To test this hypothesis, we calculated the mean basal isoproterenol-induced change in cAMP and the mean Ang II enhancement of isoproterenol-induced cAMP for each experimental set of SHR and WKY PMVSMCs, and these pairs of values (n = 20) were subjected to a least squares linear regression analysis which showed no relationship (r² = .11, not significant) between the basal cAMP response to isoproterenol and the magnitude of Ang II enhancement of isoproterenol-induced cAMP. Thus, the differential enhancement by Ang II in SHR vs. WKY PMVSMCs cannot be explained on the basis of different base-line responses to isoproterenol. As illustrated in figure 2, the maximal adenylyl cyclase activity was not different in SHR vs. WKY PMVSMCs, a finding which argues against the hypothesis that the greater Ang II enhancement of isoproterenol-induced cAMP in SHR PMVSMCs was due to a greater capacity of the adenylyl cyclase system in SHR PMVSMCs. It is also unlikely that differences in Ang II receptors account for the differential enhancement by Ang II since radiolabeled ligand binding studies (Chatziantoniou and Arendshorst, 1993; Chatziantoniou et al., 1994) indicate no strain-specific differences in Ang II receptor characteristics in isolated renal resistance vessels or glomeruli from SHR and WKY.

Because the Ang II/isoproterenol interaction was greater in SHR, compared with WKY, PMVSMCs, we explored the mechanism of the Ang II/isoproterenol interaction in PMVSMCs. A known mechanism by which vasoconstrictor peptides increase cAMP levels is through the synthesis of vasodilator prostaglandins (Schramek et al., 1995), which activate Galpha-s. We examined the possible role of prostaglandins by measuring Ang II/isoproterenol interactions in PMVSMCs pretreated with indomethacin, an inhibitor of cyclooxygenase. Because indomethacin decreased the potentiating effect of Ang II on isoproterenol-induced cAMP in PMVSMCs from both strains, Ang II-induced prostaglandin biosynthesis may account for a portion of Ang II enhancement of isoproterenol-induced cAMP. However, an important caveat is that indomethacin at high concentrations may have nonspecific actions (Northover, 1977) so that the inhibitory effect of indomethacin in the present study was not necessarily due to reduced prostaglandin biosynthesis. The possibility that indomethacin caused nonspecific effects is reinforced by the observation that indomethacin reduced basal cAMP responses to isoproterenol by ~70%. Nonetheless, even with high concentrations of indomethacin, Ang II enhancement of isoproterenol-induced cAMP was still strongly evident and remained much greater in SHR. Thus, nonprostanoid mechanisms must have participated and accounted for the greater Ang II enhancement of isoproterenol-induced cAMP in SHR PMVSMCs.

Another potential mechanism to explain Ang II enhancement of isoproterenol-induced cAMP is activation of calcineurin. In cultured bovine glomerulosa cells, Ang II enhances ACTH-induced cAMP via a mechanism that is abolished by the calcineurin blockers cyclosporin A and FK506 (Baukal et al., 1994). However, in cultured PMVSMCs, a high concentration of FK506 failed to attenuate Ang II enhancement of isoproterenol-induced cAMP, a finding which excludes any role for calcineurin with regard to mediating the Ang II/isoproterenol interaction in PMVSMCs.

The pattern of interaction between Ang II and isoproterenol strongly suggests coincident activation of adenylyl cyclase by convergent stimuli (Anholt, 1994; Sunahara et al., 1996). Known mechanisms of synergistic activation of adenylyl cyclase include coincident activation of adenylyl cyclase by alpha-s and beta gamma subunits, alpha-s and calcium-calmodulin and alpha-s and protein kinase C (Sunahara et al., 1996; Taussig and Gilman, 1995). Any of these mechanisms could explain the observed potentiation because all three possible coincident stimuli are activated by Ang II, while isoproterenol provides the activated alpha-s. These possibilities were explored by using pertussis toxin to inhibit release of beta gamma subunits from G_i, UK 14,304 to activate alpha-2-adrenoceptors and release beta gamma subunits from alpha-2-adrenoceptor-coupled G_i, calmidazolium and W-7 to inhibit calmodulin, and calphostin C and chelerythrine to inhibit protein kinase C.

Pertussis toxin increased Ang II enhancement of isoproterenol-induced cAMP in both strains, with a greater enhancement in SHR, suggesting that Ang II enhancement of isoproterenol-induced cAMP is not due to coincident signaling through free beta gamma subunits. Moreover, these results suggest that activation of alpha-i by Ang II limits Ang II enhancement of isoproterenol-induced cAMP. This conclusion is further supported by the effects of the alpha-2 adrenoceptor agonist UK 14,304 which significantly decreased the cAMP response to isoproterenol, indicating that activation of alpha-i has an inhibitory effect on isoproterenol-induced cAMP.

Because calcium-calmodulin is well known to synergize with active alpha-s, the role of calmodulin was investigated by employing calmodulin inhibitors. Neither calmidazolium nor W-7 significantly inhibited the Ang II/isoproterenol interaction in either strain indicating that calmodulin did not mediate Ang II enhancement of isoproterenol-induced cAMP. An unanticipated finding was that both calmidazolium and W-7 actually potentiated Ang II enhancement of isoproterenol-induced cAMP in SHR PMVSMCs. A plausible explanation for the effects of calmidazolium and W-7 may be found by combining known information about the effects of calmodulin antagonists on calcium-ATPase with evidence that there are strain-specific differences in intracellular calcium pools between SHR and WKY rats. Calmidazolium inhibits calcium-ATPase in plasma membranes as well as endoplasmic and sarcoplasmic reticulum, and both calmidazolium and W-7 have been shown to provoke release of stored calcium from thyroid FRTL cells (Tornquist, 1993; Tornquist and Ekokoski, 1996) and in the amoeba Dictyostelium discoideum (Schlatterer and Schaloske, 1996). This effect is reminiscent of the calcium-ATPase pump inhibitor thapsigargin. Neusser et al. (1994), studying calcium storage pools in SHR and WKY vascular smooth muscle cells using thapsigargin and Ang II, have shown that SHR differ from WKY rats in having thapsigargin-sensitive pools that are distinct from Ang II-sensitive pools. Cells that were pretreated with thapsigargin to deplete calcium pools were challenged with Ang II. In SHR, the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the control and thapsigargin-treated cells, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in SHR vascular smooth muscle cells. In contrast, in WKY-derived cells, the calcium response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive calcium pool. This information may be applied to our study. If calmodulin antagonists and Ang II release calcium from distinct storage sites in SHR, preincubation with calmodulin antagonists would result in increased cell calcium levels after the addition of Ang II, whereas, in WKY cells, calmodulin antagonists would not have this effect.

Indeed, our experiments with BAPTA-AM and A23187 support an important role for calcium in Ang II enhancement of isoproterenol-induced cAMP. Chelation of intracellular calcium with concentrations of BAPTA-AM that ablated the intracellular calcium response to Ang II in PMVSMCs markedly reduced Ang II enhancement of isoproterenol-induced cAMP in PGVSMCs from both strains. Conversely, increasing intracellular calcium with A23187 strongly potentiated Ang II enhancement of isoproterenol-induced cAMP in PMVSMCs from both strains. However, our finding that intracellular calcium levels, both basal and Ang II stimulated, were similar in WKY vs. SHR PMVSMCs indicates that the greater Ang II enhancement of isoproterenol-induced cAMP in SHR PMVSMCs was not due to greater basal calcium levels or greater calcium responses to Ang II.

Our results support the conclusion that another factor besides calcium participates in Ang II enhancement of isoproterenol-induced cAMP. This inference is based on the observation that increasing intracellular calcium per se with A23187 did not mimic the effects of Ang II with regards to enhancing isoproterenol-induced cAMP. Also, if Ang II enhancement was mediated exclusively by increases in intracellular calcium, then A23187 would have blocked the enhancing effects of Ang II since intracellular calcium levels would already have been high in the presence of A23187 so that any calcium mediated effects of Ang II would have been obscured. The most likely explanation for Ang II enhancement of isoproterenol-induced cAMP is that calcium-modulates the mechanism that is responsible for Ang II enhancement of isoproterenol-induced cAMP. A likely candidate mechanism would be protein kinase C. Since classical protein kinase C requires both calcium and diacylglycerol for activation, this hypothesis would explain why intracellular calcium modulates Ang II enhancement of isoproterenol-induced cAMP and yet calcium per se does not enhance isoproterenol-induced cAMP. This hypothesis is strengthened by the well-known fact that Ang II receptors are coupled to protein kinase C via G_q. This hypothesis was corroborated in the current study by the observations that two structurally distinct protein kinase C inhibitors, calphostin C and chelerythrine chloride, abolished Ang II enhancement of isoproterenol-induced cAMP in PGVSMCs from both SHR and WKY. Thus, our studies establish that in cultured PGVSMCs protein kinase C mediates coincident signaling between the beta adrenoceptor and the Ang II receptor on adenylyl cyclase activity.

At least two non-mutually exclusive mechanisms could account for the greater Ang II enhancement of isoproterenol-induced cAMP in SHR PGVSMCs. First, it is possible that Ang II increases protein kinase C activity more in SHR PGVSMCs compared with WKY PGVSMCs. Second, because only certain isoforms of adenylyl cyclase are activated by protein kinase C (Sunahara et al., 1996), it is possible that SHR PGVSMCs are enriched in protein kinase C-sensitive isoforms of adenylyl cyclase.

What is the physiological and pathophysiological significance of these findings? With regard to the physiological significance, our findings suggest that protein kinase C-mediated activation of adenylyl cyclase may tend to attenuate Ang II-induced vasoconstriction in the renal microcirculation. Thus, this mechanism may play an important role to limit renal ischemia whenever the renin-angiotensin system is overly activated. This protective mechanism appears to be augmented in genetic hypertension and could thereby function to buffer the prohypertensive effects of Ang II in genetic hypertension. Interestingly, in the intact kidney, Ang II reduces, rather than increases, cAMP (Vyas and Jackson, 1995; Vyas et al., 1996), and this response is augmented in the SHR kidney. The opposite findings in isolated PMVSMCs suggest that countervailing mechanisms regulating adenylyl cyclase are operative in the intact kidney and override the stimulatory effects mediated by protein kinase C. Alternatively, because the site of release of cAMP from intact kidneys cannot be precisely located, it is possible that protein kinase C-mediated activation of adenylyl cyclase is predominant in PMVSMCs even in the intact kidney but that whole kidney cAMP is reduced by Ang II due to inhibition of adenylyl cyclase activity in other renal cells. Additional studies are required to determine whether coincident regulation of adenylyl cyclase in the renal microcirculation importantly contributes to renovascular tone in vivo in normotensive and hypertensive animals.

In summary, in PMVSMCs Ang II potentiates, rather than inhibits, agonist-induced cAMP production, and this potentiation by Ang II is greater in SHR PMVSMCs. The mechanism of Ang II enhancement of agonist-induced cAMP in PMVSMCs is coincident signaling via protein kinase C, and this mechanism is greater in SHR PGVSMCs possibly due to greater activation of protein kinase C activity and/or altered expression of adenylyl cyclase isoforms.