Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Although angiotensin (Ang) II is a potent vasoconstrictor in vivo, recent studies indicate that in vascular smooth muscle cells cultured from conduit arteries, Ang II enhances the accumulation of cAMP in response to agonists that stimulate adenylyl cyclase. Kubalak and Webb (1993) reported that Ang II enhances cAMP production in response to isoproterenol, adenosine, and prostacyclin in cultured rat aortic smooth muscle cells, a finding later confirmed by McCumbee et al. (1996), who showed that Ang II enhances isoproterenol-induced cAMP production 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 levels (Brizzolara-Gourdie and Webb, 1997).

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 was, until very recently, unknown. Studies from our laboratory have demonstrated marked Ang II enhancement of isoproterenol-induced cAMP production in cultured preglomerular microvascular smooth muscle cells (PMVSMCs) obtained from both normotensive Wistar Kyoto rats and spontaneously hypertensive rats (Mokkapatti et al., 1998).

Which Ang receptor mediates Ang II enhancement of agonist-induced cAMP production in vascular smooth muscle cells has not been carefully evaluated. Ang II per se interacts with two receptor subtypes designated AT₁ and AT₂ receptors (Timmermans et al., 1993). Because AT₂ receptors, similar to cAMP, may cause vasodilation (Scheuer and Perrone, 1993; Arima et al., 1997), it is possible that Ang II enhancement of agonist-induced cAMP production is mediated by AT₂ receptors. It is also possible that Ang II is metabolized by vascular smooth muscle cells to Ang (3-8) and/or Ang (1-7), two peptides that cause vasodilation via their own receptors distinct from AT₁ and AT₂ receptors (Kramar et al., 1997; Iyer et al., 1998).

Because Ang II enhancement of agonist-induced cAMP production may represent an important mechanism to protect vascular smooth muscle cells from too much Ang II-induced vasoconstriction and/or proliferation, it is important to fully characterize the Ang receptor subtypes that mediate this interesting effect. Accordingly, the purpose of this investigation was to elucidate the Ang receptor subtypes that mediate Ang II enhancement of agonist-induced cAMP production in culture vascular smooth muscle cells obtained from preglomerular microvessels.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Cell culture supplies were obtained from Gibco Laboratories (Grand Island, NY), with the exception of donor-defined fetal calf serum, which was purchased from Hyclone Laboratories (Miami, FL). Reagents and drugs were obtained as follows: iron oxide and chloroacetaldehyde were obtained from Aldrich Chemical Co. (Milwaukee, WI); Ang II, Ang III, Ang (3-8), Ang (1-7), (±)-isoproterenol and 3-isobutyl-1-methyxanthine (IBMX) were obtained from Sigma Chemical Co. (St. Louis, MO); PD 123319 was obtained from Research Biochemicals International (Natick, MA); L 158809 was obtained from Merck and Co. (West Point, PA); and 1-propanol was obtained from J.T. Baker (Phillipsburg, NJ). Immunochemicals for cell characterization were obtained from sources described by Dubey and coworkers (Dubey et al., 1992). All other chemicals were of the highest grade available.

Animals. In this study, PMVSMCs were obtained from male spontaneously hypertensive rats (12-14 weeks of age) purchased from Taconic Farms (Germantown, NY). PMVSMCs from spontaneously hypertensive rats were used because in a previous study we determined that Ang II enhancement of isoproterenol-induced cAMP production was much greater in PMVSMCs from spontaneously hypertensive rats compared with normotensive Wistar Kyoto rats (Mokkapatti et al., 1998). Animals were housed in the University of Pittsburgh Animal Facility with controlled temperature, relative humidity, and light cycle (22°C, 55%, and lights on 7 AM to 7 PM, respectively). Rats were fed Prolab RMH 3000 obtained from PMI Feeds (St. Louis, MO) and were given tap water ad libitum. Animal care conformed with institutional guidelines, and studies had prior approval of the Institutional Animal Care and Use Committee.

Cell Culture. PMVSMCs were cultured, purified, and characterized as previously described in detail (Dubey et al., 1992; Mokkapatti et al., 1998). Briefly, renal microvessels were isolated by injecting iron oxide particles into the kidneys, mincing and dispersing the renal cortical tissue, and using a magnet to retain the iron-laden vessels. Microvessels were digested with collagenase and passed through a hypodermic needle to shear off glomeruli. The arteriolar fraction retained after sieving (80 µm mesh) was suspended in supplemented DMEM with 20% fetal calf serum, plated, and incubated (37°C in 5% CO₂, 95% air, and 98% humidity). The medium was changed daily until cells attained confluence. The PMVSMCs were repeatedly submitted to selective plating (Aviv et al., 1983) to reduce fibroblast contamination. Experiments were conducted at confluence in the third to fifth passage. At this passage level, cells retain their phenotypic characteristics and grow exponentially. PMVSMCs were characterized by morphology (hill-and-valley pattern of growth), functional responses (contraction to Ang II and norepinephrine), and immunofluorescence staining (positive for smooth muscle-specific - and -isoactin, heavy chain myosin and desmin, and negative for von Willebrand factor).

Concentration-Response Curve to Ang Peptides. Medium was aspirated from cell monolayers, and PMVSMCs were washed twice with 1 ml of Dulbecco's PBS. The medium was replaced with 500 µl of PBS containing IBMX (1 mM), isoproterenol (1 µM), and a given concentration (0, 0.1, 1, 10, 100 or 1000 nM) of a specific Ang peptide [Ang II, Ang III, Ang (3-8), or Ang (1-7)]. In these experiments IBMX was included in the medium to inhibit phosphodiesterase and augment any cAMP responses to the Ang peptides. IBMX was included in the concentration-response study because we wished to detect responses to very low concentrations of Ang peptides. Experiments were terminated 30 min after the addition of isoproterenol and/or Ang peptides by aspiration of supernatants and immediate freezing of cells and supernatants. Cellular cAMP was extracted with 1 ml of 1-propanol at 4°C for 30 min with continuous shaking. The 1-propanol was then evaporated, and samples were resuspended in phosphate buffer for assay of cAMP. Protein in PMVSMCs was assayed with the bicinchoninic acid assay (Pierce, Rockford, IL).

Effects of Ang Receptor Antagonists on Ang II and Ang III Enhancement of Isoproterenol-Induced cAMP Production in PMVSMCs. Medium was aspirated from cell monolayers, and PMVSMCs were washed twice with 1 ml of Dulbecco's PBS. The medium was replaced with 480 µl of PBS containing either L 158809 (10 nM) or PD 123319 (300 nM) or no drug. In these experiments IBMX was not included in the medium to avoid any potential interactions with the nonpeptide antagonists. Because high concentrations of Ang peptides were used, cAMP responses could be easily detected in the absence of phosphodiesterase inhibition. After 15 min of pretreatment with the receptor antagonists, PMVSMCs were treated with either isoproterenol (1 µM final concentration) or its vehicle (PBS) and with Ang II, Ang III, or their vehicles (PBS). These treatments were administered in a total volume of 20 µl to bring the final volume in the culture wells to 500 µl. Experiments were terminated 30 min after the addition of isoproterenol and/or Ang peptides by aspiration of supernatants and immediate freezing of cells and supernatants. Cellular cAMP was extracted with 1 ml of 1-propanol at 4°C for 30 min with continuous shaking. The 1-propanol was then evaporated, and samples were resuspended in phosphate buffer for assay of cAMP. Protein in PMVSMCs was assayed with the bicinchoninic acid assay.

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 fluorometric detection. Samples were prepared by adding 10 µL of acetate buffer (0.5 mol/liter sodium acetate, pH 4.35), 10 µL of a 1 µM solution of internal standard (adenine-9--D-arabinofuranoside), and 10 µL of 50% chloroacetaldehyde followed by incubation for 1 h 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 mM 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 approximately 0.12 pmol/injection.

Statistical Analyses. The levels of cAMP in the medium (extracellular) and PMVSMCs (intracellular) were normalized to total protein. Concentration-response curves to the Ang peptides were fitted to the following sigmoidal function: cAMP = Basal + (Maximal-Basal)/(1 + 10^{LogEC50-Log[peptide]}). Curve fitting was performed with Prism Version 2.0 (GraphPad Software, San Diego, CA). Log EC₅₀s for Ang II and Ang III were compared with an unpaired Student's t test using Prism.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Figure 1 summarizes the effects of Ang II, Ang III, Ang (3-8), and Ang (1-7) on intracellular (Fig. 1A) and extracellular (Fig. 1B) cAMP levels in PMVSMCs treated with isoproterenol (1 µM) and IBMX (1 mM). Neither Ang (3-8) nor Ang (1-7) increased intracellular or extracellular cAMP levels. In contrast, both Ang II and Ang III markedly increased cAMP levels. When these data were fitted to a sigmoidal concentration-response curve, the basal and maximal intracellular and extracellular cAMP levels were similar for Ang II compared with Ang III (basal intracellular cAMP levels, 1683 ± 90 and 1643 ± 42 pmol/mg protein for Ang II and Ang III, respectively; maximal intracellular cAMP levels, 3088 ± 68 and 2896 ± 55 pmol/mg protein for Ang II and Ang III, respectively; basal extracellular cAMP levels, 980 ± 38 and 1019 pmol/mg protein for Ang II and Ang III, respectively; maximal extracellular cAMP levels, 1956 ± 34 and 2039 ± 26 pmol/mg protein for Ang II and Ang III, respectively). Ang II was approximately 10-fold more potent compared with Ang III in increasing both intracellular and extracellular cAMP levels, and the log EC_50s (molar concentrations) for Ang II (extracellular, 8.68 ± 0.11; intracellular, 9.04 ± 0.16) were significantly less (p < .001) than the log EC_50s (molar concentrations) for Ang III (extracellular, 7.86 ± 0.06; intracellular, 7.96 ± 0.11).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of Ang II (), Ang III (), Ang (3-8) (), and Ang (1-7) () on extracellular (A) and intracellular (B) cAMP levels in PMVSMCs treated with isoproterenol (1 µM). Values indicate means ± S.E.M. (n = 4).

Figure 2 illustrates the effects of L 158809 on Ang II (100 nM) and Ang III (100 nM) enhancement of isoproterenol (1 µM)-induced intracellular and extracellular cAMP levels. L 158809 blocked the enhancement of isoproterenol-induced cAMP levels by both Ang II and Ang III (p-values < .0001 for all comparisons). In contrast, PD 123319 had no detectable effect on either Ang II or Ang III enhancement of isoproterenol-induced cAMP levels (Fig. 3).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Effects of L 158809 on 100 nM Ang II (A) and 100 nM Ang III (B) enhancement of isoproterenol (1 µM)-induced cAMP levels in PMVSMCs in extracellular (top) and intracellular (bottom) compartments. Values indicate means ± S.E.M. (n = 4).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Effects of PD 123319 on 100 nM Ang II enhancement of isoproterenol (1 µM)-induced cAMP levels in PMVSMCs in extracellular (A) and intracellular (B) compartments. Values indicate means ± S.E.M. (n = 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies have documented that Ang II enhances agonist-induced cAMP production in macrovascular (rat aorta) smooth muscle cells (Kubalak and Webb, 1993; McCumbee et al., 1996), and we recently extended this concept to microvascular (rat renal microvessels) smooth muscle cells (Mokkapatti et al., 1998). The present study demonstrates that Ang III, like Ang II, enhances agonist-induced cAMP production in cultured microvascular smooth muscle cells and unequivocally establishes that the AT₁ receptor is solely responsible for Ang II and Ang III enhancement of agonist-induced cAMP production in cultured microvascular smooth muscle cells. Finally, the present study shows that neither Ang (3-8) nor Ang (1-7) enhances agonist-induced cAMP production in cultured microvascular smooth muscle cells.

Ang (3-8) and Ang (1-7) are biologically active Ang peptides. Therefore, it is conceivable that Ang II enhancement of agonist-induced cAMP production is mediated by metabolism of Ang II to Ang (3-8) and/or Ang (1-7). However, the present study demonstrates that Ang (3-8) and Ang (1-7) have no effect on agonist-induced cAMP production even at very high concentrations. This finding virtually eliminates the possibility that the effects of Ang II on agonist-induced cAMP production are mediated by conversion of Ang II to Ang (3-8) and Ang (1-7) in cultured PMVSMCs. Therefore, in cultured PMVSMCs, neither the Ang (3-8) receptor nor the Ang (1-7) receptor mediates the effects of Ang II on agonist-induced cAMP production. However, because we do not know whether cultured PMVSMCs possess Ang (3-8) or Ang (1-7) receptors, we cannot logically exclude the possibility that Ang (3-8) and/or Ang (1-7) receptors enhance agonist-induced cAMP production in other cell types/tissues.

Our concentration-response study demonstrates that Ang II is 10-fold more potent than Ang III with in enhancing agonist-induced cAMP production. If all the effects of Ang II are mediated by conversion to Ang III, then the potency of Ang II would be less than or equal to the potency of Ang III. Because this is not the case, Ang II must enhance agonist-induced cAMP at least in part independent of conversion to Ang III. Thus, both Ang II and Ang III directly activate Ang receptors that cross-talk in some manner with adenylyl cyclase.

The observation that Ang II is 10-fold more potent than Ang III in the enhancement of agonist-induced cAMP production is consistent with the AT₁ receptor mediating the effects of Ang II and Ang III on cAMP levels in cultured PMVSMCs. In general, in cases in which the AT₁ receptor mediates a given biological effect, Ang II is usually approximately 10-fold more potent than Ang III (Pendleton et al., 1991; Fujimoto et al., 1992; Robertson et al., 1992; Li et al., 1995, 1996, 1997). However, the reduced potency of Ang III relative to Ang II is at least in part due to increased susceptibility of Ang III to degradation by aminopeptidases (Fujimoto et al., 1992; Robertson et al., 1992), and Ang II and Ang III are equipotent in stimulating aldosterone secretion by the adrenal gland (Campbell et al., 1974), inducing pressor responses when injected into the cerebral ventricles (Wright et al., 1996) and causing vasoconstriction of the cat pulmonary circulation (Cheng et al., 1994). Also, the relative potencies of Ang II and Ang III for AT₂ receptor-mediated effects are not well characterized, because few biological responses have been discovered for the AT₂ receptor. Therefore, the relative potencies of Ang II and Ang III do not definitively implicate the AT₁ receptor.

To test the hypothesis that AT₁ receptors mediate Ang II and Ang III enhancement of agonist-induced cAMP production in PMVSMCs, we examined the effects of Ang II and Ang III on agonist-induced cAMP production in the presence and absence of L 158809, a potent and highly selective AT₁ receptor antagonist with an IC₅₀ of 0.3 nM for the AT₁ receptor versus an IC₅₀ of >10,000 nM for the AT₂ receptor (Chang et al., 1992). In our study, 10 nM L 158809 markedly inhibited Ang II and Ang III enhancement of agonist-induced cAMP production. Because the concentration of L 158809 used in the present study was far below the IC₅₀ of L 158809 for the AT₂ receptor but 33-fold greater than the IC₅₀ of L 158809 for the AT₁ receptor, these results strongly implicate the AT₁ receptor as that Ang receptor subtype mediating Ang II and Ang III enhancement of agonist-induced cAMP production in PMVSMCs.

A final experiment was conducted to directly assess the role of the AT₂ receptor in Ang II enhancement of agonist-induced cAMP production. PD 123319 is a potent and highly selective AT₂ receptor antagonist with an IC₅₀, as determined by competition binding studies, of 21 nM for the AT₂ receptor versus an IC₅₀ of >10,000 nM for the AT₁ receptor (Dudley et al., 1990). In our study, 300 nM PD 123319 had no effect on Ang II enhancement of agonist-induced cAMP production. It is highly unlikely that this negative result was due to partial agonist effects of PD 123319, because isoproterenol-induced cAMP production was nearly identical in the presence and absence of PD 123319 (2274 versus 2169 pmol/mg protein in the absence and presence of PD 123319, respectively). Because the concentration of PD123319 used in the present study was 15-fold greater than the IC₅₀ of PD 123319 for the AT₂ receptor, these results rule out any involvement of the AT₂ receptor in Ang II enhancement of agonist-induced cAMP production. Although we did not test the effects of PD 123319 on Ang III enhancement of agonist-induced cAMP production, given the observation that L 158809 completely abolished Ang III enhancement of agonist-induced intracellular cAMP levels, it is unlikely that any receptor subtype other than the AT₁ receptor mediates Ang III enhancement of agonist-induced cAMP production in PMVSMCs.

Our conclusion that the AT₁ receptor mediates Ang II and Ang III enhancement of agonist-induced cAMP production in cultured PMVSMCs is entirely consistent with recent published studies using cloned and expressed AT₁ receptors. Baukal et al. (1994) reported that Ang II enhanced adrenocorticotropin-induced cAMP production in COS-7 cells transfected with the rat AT₁ receptor. Similarly, Klingler et al. (1998) found that Ang II enhanced vasopressin-induced cAMP production in Chinese hamster ovary cells transfected with cDNA for both AT₁ and V₂ receptors. Thus, these important studies indicate that in transfected cells, AT₁ receptors can enhance agonist-induced cAMP production. Our studies extend this concept to native AT₁ receptors residing in functional microvascular smooth muscle cells.

An important issue is the mechanism by which Ang II and Ang III enhance agonist-induced cAMP levels. Because the concentration-response study was conducted in the presence of a very high concentration of a "broad spectrum" phosphodiesterase inhibitor, the enhancement in cAMP levels caused by Ang II and Ang III cannot be attributed to changes in the rate of cAMP breakdown by phosphodiesterases. Moreover, because Ang II and Ang III enhanced both intracellular and extracellular levels of cAMP, changes in cAMP levels cannot be attributed to changes in cAMP egress from the cells. Therefore, the results of the present study implicate an effect of Ang II and Ang III on the synthesis of cAMP. Recently, we discovered that in PMVSMCs Ang II enhancement of isoproterenol-induced cAMP levels is mediated by protein kinase C (Mokkapatti et al., 1998). Most likely Ang II and Ang III, via protein kinase C, augment the effects of agonists on adenylyl cyclase activity.

An important unresolved issue is the physiological significance of AT₁ receptor-mediated enhancement of agonist-induced cAMP production. It is possible that in vivo Ang II enhancement of agonist-induced cAMP production protects vascular smooth muscle cells from the provasoconstrictive, progrowth, and promigratory effects of Ang II. If so, this would represent an extremely important regulatory mechanism that, if malfunctioning, could lead to cardiovascular diseases such as hypertension, heart failure, and atherosclerosis.

In summary, the findings of the current study clearly indicate that Ang II and Ang III enhance agonist-induced cAMP production in PMVSMCs exclusively via the AT₁ receptor. Future studies are needed to elucidate the physiological significance of this interesting interaction between Ang II and agonists that stimulate adenylyl cyclase.