Introduction

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The balance between RNA synthesis and degradation pathways dictates steady state levels of gene expression in cells (1). In eukaryotic cells, mRNA half-lives can range from seconds to many hours (for a review, see Ref. 2). For example, the expression of several short-lived cytokine and proto-oncogene mRNAs can be induced transiently by gene transcription, but rapid mRNA degradation prevents their accumulation inside the cell (3). A general picture has emerged in which these unstable mRNAs are transcribed into an environment in which the destabilizing machinery preexists in a fully active form.

The mechanisms responsible for controlling the activities of iron-response element binding proteins and their interactions with mRNA provide what is now the most well understood class of gene expression control in which mRNA stability is controlled by changing extracellular conditions (4). Here, the intracellular concentration of iron, and perhaps oxidative metabolites, regulates the ability of responsive trans-acting mRNA binding proteins to control mRNA translation and degradation (5, 6). There are relatively few examples in which stabilization and destabilization of specific mRNAs have been documented in response to receptor-mediated activation of intracellular signaling pathways (for a recent review, see Ref. 7). Second messenger activity has been implicated in the negative control of mRNAs encoding some cell surface G protein-coupled receptors, including muscarinic (8), thyrotropin-releasing hormone (9), and ₁-adrenergic receptors (10). Studies by Malbon et al. (11, 12) suggest the ₂-adrenergic receptor mRNA is degraded in trans as a long term response (>24 hr) to cAMP elevation. This is correlated with the induction of cytoplasmic mRNA binding proteins interacting with AU-rich mRNA motifs in the ₂-adrenergic receptor mRNA (13-15). However, in other studies, ₂-adrenergic receptor mRNA destabilization has not been observed under the conditions of short term agonist exposure (16, 17), although the confounding influence of transcriptional control of the gene by the same signals may obfuscate any early effects of agonist on mRNA stability. Similar complications were found in studies of regulation of the mRNAs encoding the plasminogen activator inhibitor (18) and tyrosine aminotransferase (19), which together represent the few known examples in which changes in cAMP levels appear to be capable of destabilizing specific mRNAs.

Recent studies of the regulation of VSMC AT₁-R reveal it to be an interesting model system for the study of regulated mRNA decay processes. VSMC AT₁-R gene expression is down-regulated by a diverse group of extracellular signals, include angiotensin II and other agonists of related G_q-coupled receptors; by growth factors; and by several synthetic agents used as activators or even inhibitors of signal transduction cascades (20-22). The current data indicate that down-regulation of the AT₁-R mRNA in response to growth factors and G_q-coupled receptor agonists is, like the ₂-adrenergic receptor, mediated by superimposed processes involving both transcriptional repression and enhanced mRNA decay. Although there is some evidence in the literature that AT₁-R gene expression can be down-regulated by cAMP-elevating agents, it has been unclear whether this effect is a consequence of transcriptional repression, mRNA destabilization, or both (22, 23). In this report, we show that cAMP-elevating agents do not affect AT₁-R gene transcription in VSMC, yet they rapidly induce a loss of steady state mRNA levels. These observations provide clear evidence of a role for PKA activity in the control of VSMC AT₁-R mRNA stability by extracellular signals.

	Experimental Procedures

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Materials. [-³²P]UTP, [-³²P]dCTP, [-³²P]ATP, [³H]adenine, and Hybond N membranes were from Amersham (Arlington, Heights, IL). TRI-reagent was from Molecular Research Center (Cincinnati, OH). Ro20-1724 was from Research Biochemicals (Natick, MA). Isoproterenol, DRB, forskolin, Bt₂-cAMP, Bt₂-cGMP, salts, and buffers were from Sigma Chemical (St. Louis, MO) or Life Technologies (Gaithersburg, MD). Antibiotics, serum, and cell culture media were from Life Technologies. Isoproterenol, Bt₂-cAMP, and Bt₂-cGMP were dissolved in sterile water. DRB was prepared as 10 µg/µl stock in highly purified DMSO. Forskolin was prepared as a 10 mM stock and also diluted in DMSO. The same concentration of solvent was used as vehicle in all experiments.

Rat VSMC culture. Rat thoracic aorta VSMCs are kept as a continuous cell line and were kindly provided by Dr. R. W. Alexander (Emory University); they were cultured in Dulbecco's modified Eagle's medium with 3.7 g/liter of NaHCO₃, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated calf serum in an incubator in 5% CO₂ at 37°. After confluence, cells were growth-arrested before experiments through culture in serum-free media for 24 hr. Cells were used between passages 10-25 after primary explant.

cAMP production assay. VSMCs in 35-mm-diameter six-well plates were incubated for 2 hr at 37° with 1 ml of [³H]adenine (5 µCi/ml) prepared in Dulbecco's modified Eagle's medium with 10 mM HEPES, pH 7.4, before exposure to agonists for 5 min in the presence of the phosphodiesterase inhibitor Ro20-1724 (0.1 mM). Production of cAMP was terminated by aspiration and the addition of 1 ml of 5% trichloroacetic acid to the wells. [³H]cAMP was isolated by sequential Dowex and alumina chromatography and measured by scintillation counting (24). Production of cAMP was calculated as a percentage of conversion, with the cpm in [³H]cAMP fractions divided by the sum of cpm in the [³H]cAMP and [³H]ATP fractions.

PKA assay. The protocol used in this study is a modification of one provided by Dr. Lee Graves (University of North Carolina, Chapel Hill) (25). VSMCs grown onto 35-mm dishes were rinsed and exposed for 5 min at 37° to the indicated concentrations of isoproterenol in the absence of phosphodiesterase inhibitors. Next, the dishes were aspirated and rinsed twice with 1 ml of ice-cold phosphate-buffered saline, pH 7.4. After an additional 1 ml rinse in homogenization buffer (50 mM -glycerophosphate, pH 7.4, 1.5 mM EGTA, 1 mM dithiothreitol, 0.1 mM Ro20-1724), the cells were scraped, collected, and sonicated briefly in 300 µl of homogenization buffer before centrifugation for 10 min at 10,000 × g and 4° to remove debris. Aliquots of the supernatant (10 µl) were assayed in triplicate for PKA activity over 15 min at 30° in a buffer (30 µl final volume) containing 25 mM -glycerophosphate, pH 7.4, 1.25 mM EGTA, 10 mM MgCl₂, 0.5 mM dithiothreitol, 0.17 mM Kemptide, and 2 µCi of [-³²P]ATP (3000 Ci/mmol). The reactions were terminated by spotting with 25 µl of the reaction mix on squares (2 × 2 cm) of P-81 phosphocellulose paper. The paper was washed four or five times in 300 ml of 150 mM phosphoric acid and once in ethanol, dried to air, and then counted by scintillation. PKA phosphorylation is defined as the difference in radioactive counts between aliquots assayed in the absence or presence of protein kinase I (5-22) inhibitor peptide (15 µM). Total PKA activity was determined in each experiment by measuring Kemptide phosphorylation in the presence of 50 µM cAMP.

RNA isolation and hybridization. Confluent 2- to 3-day cultures on 35- or 60-mm dishes were grown in serum-free medium for an additional 24 hr before challenge with vehicle or agonists for the indicated time. Total RNA was extracted using TRI reagent and quantified by measuring absorbancy at 260 nm. In some experiments, RNA (10 µg) was separated by formaldehyde-agarose gel electrophoresis before transfer to Hybond nylon membranes. In other experiments, using a slot-blot apparatus, duplicate samples of total RNA (5-10 µg) was denatured by mixing with equal volumes of formaldehyde (37%) and 20× SSC (1× contains 150 mM sodium chloride, 15 mM sodium citrate, pH 7.4) and heating at 65° on a heating block for 15 min. Samples were then diluted in 0.5 ml of diethylpyrocarbonate-treated 10× SSC and transferred to a nylon membrane under gentle vacuum. RNA was cross-linked onto the membrane under ultraviolet light (1200 kJ) in a Stratalinker 2400 (Statagene, La Jolla, CA).

Nuclear run-on. The nuclear run-on method was modified from that reported by others (26) as described previously (20). Cells in 150-mm-diameter dishes were grown to confluence in growth media and then for an additional 24 hr in serum-free conditions before treatment for either 45 min or 4 hr with either vehicle or the indicated concentration of forskolin. The cells were harvested and nuclei were isolated as described previously (20). Nuclei (1-3 × 10⁷/reaction) were mixed with 450 µCi of [-³²P]UTP (>3000 µCi/mmol) and 100 µM concentrations of ATP, CTP, and GTP in a final volume of 300 µl. The run-on reaction took place at 30° for 30 min before the radiolabeled transcripts were extracted with 800 µl of TRI-reagent and resuspended in 50 µl of diethylpyrocarbonate-treated water.

	Results

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The time-dependent effect of cAMP-elevating agents on AT₁-R mRNA levels in VSMC were examined by treating the cells for various periods of time with a single concentration (10 µM) of forskolin, as a direct activator of adenylyl cyclase. This effect was compared with that for vehicle (0.1% DMSO) alone and for cells pretreated for 30 min with 50 µg/ml DRB, an inhibitor of transcriptional elongation (28) to block AT₁-R mRNA synthesis, in the absence or presence of forskolin. The results of a representative hybridization slot-blot are shown in Fig. 1, and the cumulative results of several experiments are shown in Fig. 2. Forskolin induces a time-dependent loss of AT₁-R mRNA levels that is evident as early as 2 hr after agonist treatment and continues until ~6 hr after treatment, after which AT₁-R mRNA levels approach a new, lower steady state.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Representative slot-blot AT1-R mRNA hybridization signal time course comparing the effects of vehicle (0.1% DMSO) with those of forskolin (10 µM), DRB (50 µg/ml), and forskolin after a 30-min pretreatment of VSMCs with DRB. Each slot contains 5 µg of total VSMC RNA. Reproduction of a PhosphorImage file that has been sharpened to remove background grayness and improve contrast. The AT1-R transcripts detected in this hybridization protocol have been extensively characterized in other Northern hybridization studies by formaldehyde gel electrophoresis (20, 21).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Destabilization of AT1-R mRNA by forskolin and the effect of a transcriptional inhibitor. VSMCs were administered a single dose of forskolin (10 µM; ) or vehicle (0.1% DMSO; ). At the zero-hour time point, VSMC and RNA samples were subsequently collected at the indicated times. Other cells were pretreated for 30 min with 50 µg/ml DRB before the addition of 10 µM forskolin () or vehicle () at the zero-hour time point and collection of RNA samples at the indicated times. Points, relative hybridization signal normalized to the zero-hour vehicle time point and is expressed as mean ± standard error of five independent experiments. All treatments were performed in parallel.

In the presence of DRB, AT₁-R mRNA steady levels are reduced to ~50% of the level in vehicle-treated cells within 6 hr, which is consistent with previous observations with the use of actinomycin D instead (20, 21). This treatment reveals the intrinsic decay rate of the AT₁-R mRNA under nonstimulated conditions, but the possibility that DRB treatment is also affecting components of the processes involved in AT₁-R mRNA decay cannot be excluded. Treatment with forskolin induces a faster rate of decay of AT₁-R mRNA levels than that observed in the presence of DRB, in which mRNA levels are reduced to ~50% of the control level within 3 hr after the addition of forskolin. Because the rate of forskolin-stimulated AT₁-R mRNA decay is greater than the rate of intrinsic decay, this comparison alone suggests that forskolin treatment enhances the degradation of, or destabilizes, the AT₁-R mRNA. DRB pretreatment abolishes the effect of forskolin. One interpretation of this observation is that forskolin stimulates the transcription of a gene or genes that mediate AT₁-R mRNA destabilization.

Nuclear run-on assays were performed to assess whether forskolin can repress transcription of the AT₁-R gene. Confluent VSMCs were treated with vehicle or 10 µM forskolin for 45 min or with 1 or 30 µM forskolin for 4 hr before nuclear harvest and in vitro run-on transcription. As shown in Fig. 3, forskolin treatment under any of these conditions does not affect significantly transcription of the AT₁ receptor gene. On the basis of these results, the reductions in the levels of the intact cell AT₁-R mRNA after treatment with forskolin are entirely attributable to destabilization of the mRNA. For each of these nuclear run-on experiments, GAPDH was used as an internal control only to ensure the integrity of the in vitro transcription system. However, in each run-on experiment we have conducted after a 4-hr forskolin treatment, GAPDH transcription is enhanced significantly, reaching >200% of control levels.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Forskolin does not affect AT1-R gene transcription. VSMCs were treated with vehicle (0.1% DMSO) (Veh) or 10 µM of forskolin (FSK) for 45 min or for 4 hr with vehicle, 1 µM forskolin, or 30 µM forskolin before isolation of nuclei. Radiolabeled nuclear RNA was isolated after a 30-min in vitro run-on in the presence of [32P]UTP and used as a probe for hybridization to blots cross-linked with 5 µg each of plasmids for the AT1-R cDNA Ca18b in pBluescript (AT1-R), pBluescript alone as negative control (pBS), or GAPDH in pIBIP as positive control for the run-on assay. A, Collage of PhosphorImages from representative 45-min or 4 hr run-on assays. B, Results of quantification of the hybridization signals for all assays conducted for each conditions expressed as a percentage ± range of the response to vehicle. Three run-on assays were performed after a 45-min treatment, and two run-on experiments were performed for each concentration of forskolin in the 4-hr treatments.

Isoproterenol was used as an agonist of ₂-adrenergic receptors; it activates adenylyl cyclase by coupling through heterotrimeric G_s and is used to evaluate the effect of receptor-stimulated adenylyl cyclase on AT₁-R mRNA levels. The expression level of ₂-adrenergic receptors in VSMCs is very low (only at 10 fmol/mg of membrane protein).¹ However, when VSMCs are treated with isoproterenol, a dose-dependent rise of cAMP production is seen, with an EC₅₀ value of 355 ± 59 nM (mean ± standard error; nine experiments) (Fig. 4). Maximal levels of cAMP production are achieved with 10 µM isoproterenol, which represents a 16-fold response over basal levels of production. In contrast, isoproterenol is much more potent than this in reducing AT₁-R mRNA levels, with an EC₅₀ value of 0.4 ± 0.3 nM (six experiments). As shown in Fig. 4, the maximal reduction of isoproterenol treatment over 4 hr in AT₁-R mRNA levels represents 60 ± 6% (six experiments) of the levels in control cells. Attenuated hybridization signals are evident at 10 nM concentrations of isoproterenol. Interestingly, this concentration of isoproterenol does not yield cAMP production levels that are significantly greater than those observed with vehicle-treated controls.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Dose-dependent effects of isoproterenol on cAMP production and AT1-R mRNA level in VSMCs. For the cAMP production assays, the VSMCs were prelabeled with [3H]adenine and exposed to indicated concentrations of isoproterenol for 5 min before fractionating radiolabeled nucleotides. cAMP production () was calculated as a percentage of the conversion rate of [3H]ATP to [3H]cAMP normalized to the highest response to isoproterenol (100%) within each separate experiment. Points, mean ± standard error of nine independent experiments in which maximal percent conversion was 16 ± 4-fold over basal. AT1-R mRNA levels () were analyzed after a 4-hr treatment with either vehicle or the indicated concentration of isoproterenol. Points, relative hybridization signal (mean ± standard error) normalized to the treatment with vehicle (100%) from six independent experiments.

No upper plateau of cAMP production in response to forskolin is observed in dose-effect stimuli, which is typical of its effect, so an EC₅₀ value cannot be calculated for its ability to produce cAMP. Similar to isoproterenol, forskolin reduces VSMC AT₁-R mRNA levels in a dose-dependent manner (Fig. 5). Forskolin treatment for 4 hr down-regulates AT₁-R mRNA levels with an EC₅₀ value of 66 ± 28 nM (four experiments). Similar to isoproterenol-stimulated cells, AT₁-R mRNA levels are clearly attenuated at forskolin concentrations (0.1 µM) that fail to elicit a detectable increase in cAMP production. AT₁-R mRNA levels are maximally reduced at 1.0 µM forskolin, a concentration that yields only a ~2-fold increase over basal levels of cAMP production. The data shown in Fig. 6 indicate that Bt₂-cAMP application down-regulates AT₁-R mRNA in a dose-dependent manner, whereas Bt₂-cGMP does not reduce AT₁ receptor mRNA levels at concentrations as high as 1 mM. These data suggest that cAMP- but not cGMP-dependent protein kinases are involved in down-regulation of AT₁-R mRNA levels in VSMCs. Taken together, these dose-effect results suggest that the levels of AT₁-R mRNA are very sensitive to minor increments in the levels of cAMP in VSMCs and suggest a role for PKA in the modulation of AT₁-R mRNA stability.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Dose-dependent effects of forskolin on cAMP production and AT1-R mRNA level in VSMCs. For the cAMP production assays, the VSMCs were prelabeled with [3H]adenine and exposed to indicated concentrations of forskolin for 5 min before fractionating radiolabeled nucleotides. cAMP production () was calculated as a percentage of the conversion rate of [3H]ATP to [3H]cAMP normalized to the highest response to forskolin (100%) within each separate experiment. Points, the relative hybridization signal normalized to the treatment with vehicle (100%; mean ± standard error of four independent experiments) in which maximal percent conversion was 20 ± 5-fold over basal. AT1-R mRNA levels () were analyzed by Northern hybridization after a 4-hr treatment with vehicle or the indicated concentration of forskolin.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Bt2-cAMP but not Bt2-cGMP decreases VSMC AT1-R mRNA levels in VSMCs. Cells were treated for 4 hr with either vehicle or the indicated concentrations of Bt2-cAMP or Bt2-cGMP. Total RNA was isolated, and AT1-R mRNA levels were quantified by Northern analysis. The data are normalized to the response observed in the vehicle-treated samples. Points, mean ± standard error of the effects observed in three independent experiments.

Assays were performed to determine whether PKA is activated at concentrations of isoproterenol and forskolin that reduce mRNA levels but do not elicit detectable cAMP production. VSMCs were stimulated for 5 min with vehicle or various doses of the drugs before preparation of extracts to assess PKA activity. As shown in Fig. 7, increased PKA activity is observed at 1 nM isoproterenol and maximally at 10 nM isoproterenol. Similarly, 1 µM forskolin is sufficient to stimulate PKA maximally. Thus, low concentrations of isoproterenol and forskolin that do not lead to detectable increases in cAMP production clearly affect both PKA activity and AT₁-R mRNA levels (compare data in Fig. 7 with that in Figs. 4 and 5). It is of interest to note, however, that the maximal level of PKA activation by isoproterenol and forskolin is only 50% greater than the activity seen in unstimulated cells. Furthermore, the maximal levels of PKA activity in extracts of the stimulated cells are equivalent to that which can be stimulated with 50 µM cAMP in vitro. These observations demonstrate that although basal levels of PKA activity are quite high in these cells, relatively modest increases in PKA activity by cAMP-elevating agents are sufficient to induce AT₁-R mRNA decay and disrupt the steady state for AT₁-R gene expression.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   PKA activity is stimulated maximally in VSMCs by low doses of isoproterenol and forskolin. VSMCs were stimulated for 5 min with the indicated concentrations of isoproterenol () or forskolin () before assay for PKA activity in 10 µl of cell extracts as described in the text. PKA-dependent phosphorylation of Kemptide substrate is defined as the difference between that in the absence and presence of 15 µM protein kinase I(5-22) peptide. Points, mean ± standard error of triplicate phosphorylation determinations from three experiments. Basal PKA activity is 36,711 ± 8,736 cpm/min/ml, whereas PKA activity in cells treated with 10 µM isoproterenol is 57,107 ± 9,642 cpm/min/ml.

	Discussion

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Because there is no effect of cAMP-elevating agents on transcription of the AT₁-R gene, the present data provide clear evidence that degradation of an mRNA can be controlled by cAMP-mediated signaling. The known systems in which evidence exists for either positive or negative mRNA stability control by cAMP-elevating agents tend to be complicated by redundant or competing transcriptional control processes. Such examples include studies of mRNAs for the ₂-adrenergic receptor (11, 16, 17), follicle-stimulating hormone receptor (29), hepatic enzymes (30), c-myc protooncogene (31), some T cell-derived cytokines (31), and pulmonary surfactant proteins (32).

Of these, regulation of the ₂-adrenergic receptor mRNA in hamster smooth muscle cells by cAMP signaling is among the most thoroughly studied. As shown in this study, forskolin-induced destabilization of the AT₁-R mRNA in VSMCs occurs within the first several hours of treatment. In contrast, destabilization of the ₂-adrenergic receptor mRNA is evident only after a more prolonged exposure to cAMP-elevating agonists (11, 12, 15, 33) but not within the first several hours after stimulation with cAMP-elevating agents (16, 17). It has been suggested (15) that the trans-acting mRNA binding proteins implicated in regulated ₂-adrenergic receptor mRNA turnover (13, 14) might also target other receptor mRNAs, including that for the AT₁-R. Although cAMP-induced AT₁-R mRNA binding proteins in VSMCs are incompletely characterized at this time,² angiotensin II stimulation induces a group of AT₁-R mRNA binding proteins that differ in several characteristics from the ₂-adrenergic receptor mRNA binding proteins (21). A role for PKA in this response to the hormone is a matter of current investigation, but it is of interest that angiotensin II stimulates cAMP production by 2-3-fold in these VSMCs.² Thus, it is unclear whether a relationship exists between the cAMP-dependent processes regulating ₂-adrenergic receptor mRNA stability and those regulating AT₁-R mRNA levels.

The dose-effect studies (Figs. 4 and 5) reveal an almost 3-order of magnitude discrepancy between the EC₅₀ values of the isoproterenol-mediated mRNA effect and the cAMP production response. The sensitivity of the AT₁-R mRNA to these agonists is consistent with long-standing observations in many systems in which downstream PKA-dependent hormonal responses can occur without detectable changes in cAMP levels relative to basal (see, for example, Refs. 34-36) and are reminiscent of a classic signal-amplification process typical for a PKA-activated catalytic cascade (37). However, the tight correlation between the PKA activity and mRNA dose-response curves implicates PKA as a mediator of the mRNA decay response to isoproterenol and argues against the presence of additional catalytic mediators downstream of PKA. The failure to observe detectable changes in cAMP production at agonist concentrations that clearly have an effect on both AT₁-R mRNA levels and PKA activity is most likely explained by our use of a relatively insensitive assay for cAMP production. The finding of maximal activation of PKA at low concentrations of agonist explains how cAMP-elevating agents do not further decrease AT₁-R mRNA levels beyond those observed with low concentrations of agonists, even as cAMP production is further increased, and argue that PKA mediates the AT₁-R mRNA destabilization response after treatment with cAMP-elevating agents.

A paradox exists in that although this study demonstrates that activation of PKA triggers AT₁-R mRNA decay, the basal levels of PKA activity in the cells are already elevated to ~60% of maximum in unstimulated cells. If control of AT₁-R mRNA stability is a dominant pathway, one would expect AT₁-R mRNA expression levels to be quite low in this environment under unstimulated conditions; yet the mRNA is expressed robustly. AT₁-R gene expression has clearly adapted to these unique conditions in the cells. It is of interest to note that this high level of AT₁-R expression is quite unusual and is rarely observed in primary cultures of VSMCs. One explanation for the paradox is that high basal levels of PKA activity may also serve to facilitate AT₁-R mRNA synthesis indirectly, through effects on other pathways of AT₁-R gene expression control. Stimulation of cell surface receptors coupled to phospholipase C down-regulates transcription of the AT₁-R gene (20, 22), and PKA activation inhibits the mitogen-activated protein kinase cascade in VSMCs (25). Perhaps basal mitogen-activated protein kinase cascade activity serves normally to attenuate AT₁-R gene transcription, and the high levels of PKA activity provide an inhibitory tone on this pathway, thus allowing for stronger transcription than would occur if basal PKA activity were lower. This is consistent with the observation that treatment of these cells with cAMP-elevating agents does not induce an unchecked obliteration of the AT₁-R mRNA levels but rather stimulates a readjustment toward a new steady state in which ongoing mRNA synthesis is matched by the now more active mRNA decay process. In addition to maximal stimulation of PKA activity and enhancement of the rate of AT₁-R mRNA decay, other reasons may account for the approach to this new steady state asymptote ~6 hr after stimulation. It may represent destruction of the agonists or desensitization of the cells to their effects.

A previous study suggested that both PKA and protein kinase C activities are involved in down-regulation of AT₁-R gene expression in smooth muscle cells (23), but this conclusion is drawn largely from an analysis that used isoquinoline-based protein kinase inhibitors. It has been shown recently that the isoquinoline compound H-7 effectively inhibits gene transcription by blocking phosphorylation of the carboxyl-terminal domain of RNA polymerase II (38). This observation renders as inappropriate this and related compounds for use in studies of signaling mechanisms involved in gene expression control. We observed a broad dose-dependent reduction of AT₁-R mRNA levels in VSMCs using the isoquinoline derivative H-89 (data not shown), a compound purported to have cAMP-dependent kinase selectivity (39). The net responses observed with these kinase inhibitors on mRNA levels likely represent a composite of effects in which they act as general transcriptional inhibitors, as inhibitors of kinases such as PKA and protein kinase C, and also perhaps as inhibitors of related kinases (40). The results of the current study resolve at least one aspect of the issue for a role of kinases in regulation of VSMC AT₁-R gene expression by demonstrating mRNA destabilization as a mechanism for down-regulation of AT₁-R gene expression in response to cAMP-elevating agents. Previous observations have shown a significant repression of VSMC AT₁-R gene transcription in response to stimulation with growth factors and angiotensin II and that these effects are superimposed on apparent mRNA destabilization processes also elicited by these agonists (20-22). It will now be important to return to these other classes of agonists to understand how components of their more complex signaling pathways control AT₁-R gene expression.

To the extent that other mRNA molecules encoding for proteins involved in the differentiated contractile function in VSMCs may also serve as substrates for this regulated cAMP-dependent mRNA decay process, PKA-mediated mRNA destabilization may play a central role in orchestrating patterns of gene expression in VSMCs in response to persistent or skewed exposure to many classes of neurotransmitters, hormones, and growth factors. In this regard, mRNA destabilization may play a role in facilitating phenotypic rearrangements in VSMCs and, thus, vascular remodeling. In addition, the cardiovascular effects of angiotensin II are mediated exclusively by the cell surface AT₁-R. Inhibitors of AT₁-R function are widely effective in the treatment of cardiovascular diseases such as hypertension and congestive heart failure, but there is little incontrovertible evidence for genetic defects in any components of the renin-angiotensin system. It is possible that subtle defects in the cellular processes involved in regulation of the expression of a normal AT₁-R gene can exacerbate whatever role angiotensin II may play in disease progression. Establishment of the mechanisms involved in the control of VSMC AT₁-R gene expression can thus specifically contribute to understanding of the role played by VSMCs and the AT₁-R in the development of cardiovascular disease and generally is more important as a model system with which to elucidate mechanisms of mRNA stability control processes regulated by extracellular signals.