Nerve growth factor decreases soluble guanylate cyclase in rat pheochromocytoma PC12 cells.

Nitric oxide (NO) modulates neurotransmission in the central and peripheral nervous systems. NO acts, in part, by stimulating cGMP production by soluble guanylate cyclase (sGC), an obligate heterodimer composed of α and β subunits. To investigate mechanisms that regulate responsiveness to NO in the nervous system, sGC regulation was examined in a rat pheochromocytoma cell line (PC12) exposed to nerve growth factor (NGF). NGF decreased sGC α1 and β1 subunit mRNA and protein levels as well as NO-stimulated sGC enzyme activity. The NGF-mediated decrease in sGC subunit mRNA levels was blocked by 5′-deoxy-5′-methylthioadenosine (an inhibitor of NGF-induced tyrosine phosphorylation). NGF did not decrease sGC subunit mRNA levels in PC12 cells containing a mutant Ras protein that blocks Ras-dependent intracellular signaling. Incubation of PC12 cells with a transcription inhibitor (actinomycin D) or protein synthesis inhibitors (anisomycin or cycloheximide) attenuated the ability of NGF to decrease sGC subunit mRNA levels. Moreover, sGC subunit mRNA levels decreased more rapidly in NGF-treated cells than in actinomycin D-treated cells, suggesting that NGF decreases sGC subunit mRNA stability. Thus, NGF decreases sGC subunit mRNA levels via mechanisms that are dependent on protein tyrosine phosphorylation and Ras activation. The effect of NGF on sGC subunit mRNA stability appears to be transcription- and translation-dependent. Modulation of sGC subunit levels and enzyme activity in PC12 cells suggests that NO responsiveness may be regulated in the nervous system by NGF.

the posterior pituitary (3). Many of the effects of NO on neuronal functions are mediated by the intracellular second messenger, cGMP. cGMP regulates neurotransmitter release (4) and appears to have an important role in long term potentiation in hippocampal pyramidal neurons (5). In addition, cGMP has been reported to repress gonadotropin-releasing hormone gene expression in a hypothalamic cell line (6). Moreover, NO has been observed to increase viability of trophic factor-deprived PC12 cells and sympathetic neurons via a cGMP-dependent mechanism (7).
NO stimulates soluble guanylate cyclase (sGC) to synthesize cGMP. sGC is an obligate heterodimer composed of ␣ and ␤ subunits with two isoforms of each subunit identified in the rat genome: ␣1, ␣2, ␤1, and ␤2 (8). cGMP interacts with several intracellular targets including protein kinases, ion channels, and phosphodiesterases. cGMP is metabolized to relatively inactive GMP by phosphodiesterases.
Although the regulation of NO production in the nervous system has been extensively investigated, the mechanisms regulating responsiveness to NO are less completely understood. We (9) and others (10) observed that agents which increase intracellular cAMP decrease sGC subunit mRNA levels and decrease the ability of cells to synthesize cGMP in response to NO-donor compounds. Ujiie et al. (11) reported that agents which increase intracellular cGMP concentrations also decrease sGC enzyme activity and subunit mRNA levels in rat medullary interstitial cells. Whether or not NO responsiveness is regulated in the nervous system has not been reported.
The rat pheochromocytoma cell line, PC12, is an extensively characterized model used for the study of cell differentiation and proliferation in response to receptor-mediated tyrosine kinase activation (12). Recently, Peunova and Enikolopov (13) reported that differentiation of PC12 cells in response to nerve growth factor (NGF) was associated with increased expression of NO synthases. In the present study, we investigated the effect of NGF on sGC function in PC12 cells. sGC subunit mRNA and protein levels as well as sGC enzyme activity decreased in PC12 cells exposed to NGF. Evidence is presented that NGF decreases sGC subunit mRNA levels via mechanisms that are tyrosine kinase-and Ras-dependent.
Cell Culture-PC12 rat pheochromocytoma cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 culture medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 112 units/ml penicillin, and 112 * This work was supported by National Institutes of Health Grants T32 HL07208 (to H. L.) and HL55377 (to K. D. B.) and by a grant to the Cardiovascular Research Center from Bristol Myers Squibb Pharmaceuticals. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  ¶ Established Investigators of the American Heart Association. 1 The abbreviations used are: NO, nitric oxide; sGC, soluble guanylate cyclase; NGF, nerve growth factor; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; MeSAdo, 5Ј-deoxy-5Ј-methylthioadenosine; IBMX, isobutylmethylxanthine; L-NAME, N G -nitro-L-arginine methyl ester; PMA, phorbol 12-myristate 13-acetate; MEK, mitogen-activated protein kinase kinase; ERK, Ras/extracellular signalregulated kinase. units/ml streptomycin. Adherent cells were passaged every 3-4 days into 100-mm tissue culture dishes at a density of 1 ϫ 10 7 cells/plate, and cells were used 2 days following passage.
M-M17-26, a PC12 cell line expressing a dominant inhibitory mutant Ras (a point mutation in codon 17 resulting in the substitution of serine by asparagine) (14), was generously provided by Dr. G. M. Cooper (Dana Farber Cancer Institute, Boston, MA). M-M17-26 cells were maintained in the same medium as PC12 cells supplemented with geneticin (G418, 0.4 mg/ml).
RNA Blot Hybridization-RNA was isolated from PC12 cells by the guanidine isothiocyanate-cesium chloride method (15). Fifteen g of RNA were fractionated in 1.5% agarose-formaldehyde gel, transferred to MAGNA CHARGE membranes (Micron Separations, Westborough, MA), and cross-linked by exposure to UV light. Membranes were hybridized overnight at 42°C with either a 32 P-radiolabeled 0.9-kilobase EcoRI/SacI restriction fragment of the rat sGC ␣1 subunit cDNA or a 32 P-radiolabeled 1.4-kilobase KpnI/BglII restriction fragment of the rat sGC ␤1 subunit (both cDNAs generously provided by Dr. M. Nakane, Abbott) (16). Membranes were washed at high stringency in a solution containing 3 mM sodium citrate, 30 mM sodium chloride, and 0.1% sodium dodecyl sulfate at 65°C and were exposed to x-ray film. To quantitate the amount of RNA loaded on the agarose-formaldehyde gels, the membranes were subsequently hybridized with a 10-fold molar excess of a 32 P-radiolabeled oligonucleotide complementary to rat 18 S ribosomal RNA (17). In some experiments, RNA blots were also hybridized with radiolabeled probes derived from cDNAs that encoded rat c-jun or c-fos (both kindly provided by Dr. T. Curran, Roche Institute of Molecular Biology, Nutley, NJ) (18). Autoradiograms were scanned using a Color Image Scanner (Seiko Epson Corp., Japan). All RNA blots shown are representative of at least three similar experiments.
Measuring sGC Subunit Protein Levels-PC12 cells were washed twice with 10 ml of ice-cold phosphate-buffered saline and harvested by scraping with a rubber policeman into buffer that contained 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride (TED buffer). Cell membranes were disrupted by passing through a 22-gauge needle 10 times. Cell extracts were centrifuged at 100,000 ϫ g for 30 min at 4°C. Cell supernatants containing 50 g of protein were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose filters (Micron Separations). Filters were blocked in phosphate-buffered saline containing 5% nonfat milk at room temperature for 1 h and then incubated with an antiserum directed against the rat ␣1 sGC subunit (provided by Dr. M. Nakane) or with an immunoaffinity-purified polyclonal antiserum directed against the sGC ␤1 subunit 2 for 1 h at room temperature. Bound mouse and rabbit antibodies were detected by incubation of filters with goat anti-mouse immunoglobulin-horseradish peroxidase (Amersham Life Sciences, Inc.) and horseradish peroxidase-protein A (Boehringer Mannheim), respectively, for 1 h at room temperature and were visualized using chemiluminescence (Enhanced Chemiluminescence Kit, Amersham Life Sciences, Inc.).
Protein concentrations in cell extracts were measured using the Bio-Rad dye concentration reagent (Bio-Rad) and bovine serum albumin as a standard.
Soluble Guanylate Cyclase Enzyme Activity-sGC activity was measured as described previously (20). Briefly, PC12 cells were extracted in TED buffer, and cell supernatants were prepared as described above. Cell extracts (10 g) were incubated in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 4 mM MgCl 2 , 0.5 mM IBMX, 7.5 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 1 mM GTP with or without 1 mM sodium nitroprusside for 10 min at 37°C. The reaction was terminated by addition of 0.9 ml of ice-cold 0.05 M HCl and boiling for 3 min. The concentration of cGMP in the reaction mixture was measured using a commercial radioimmunoassay kit (Biomedical Technologies Inc., Stoughton, MA). sGC enzyme activity is expressed as pmol of cGMP produced/min/mg of protein in the cell extract supernatant.

Characterization of Soluble Guanylate Cyclase Regulation by Nerve Growth Factor in a Rat Pheochromocytoma Cell Line
NGF Decreases sGC Subunit mRNA Levels in PC12 Cells-To investigate the effect of NGF on sGC subunit gene expression, sGC subunit mRNA levels were measured in PC12 cells incubated with and without 100 ng/ml NGF for 2-24 h. NGF decreased sGC ␣1 and ␤1 subunit mRNA levels ( Fig. 1, Panel A). Decreases in sGC subunit mRNA levels were evident within 2 h after exposure to NGF, and minimum levels were detected at 4 h. After 24 h of continuous exposure to NGF, sGC subunit mRNA levels returned toward baseline. The NGF-mediated decrease in sGC subunit mRNA levels was concentration-dependent ( Fig. 1, Panel B). Decreased sGC subunit mRNA levels were evident in PC12 cells exposed for 4 h to as low as 1 ng/ml NGF, and 10 ng/ml produced a near-maximal effect.
NGF Decreases NO-activated sGC Enzyme Activity in PC12 Cells-To ascertain whether the NGF-mediated decrease in sGC subunit mRNA levels was associated with changes in sGC enzyme function, basal and NO-stimulated sGC enzyme activities were measured in extracts of PC12 cells exposed to NGF. Basal sGC enzyme activity in PC12 cells was low and was not altered in PC12 cells exposed to 100 ng/ml NGF for 2-24 h. In extracts from untreated PC12 cells, sodium nitroprusside, a NO-donor compound, increased sGC enzyme activity 25-fold. NO-stimulated sGC enzyme activity decreased in PC12 cells exposed to NGF for 24 h but not in cells exposed for 2, 4, and 8 h (data not shown). The effect of NGF on NO-stimulated sGC enzyme activity was dose-dependent; exposure of PC12 cells to 10 and 100 ng/ml NGF decreased NO-stimulated sGC enzyme activity by 25 and 50%, respectively (*, p Ͻ 0.05 and **, p Ͻ 0.01, respectively) (Fig. 2). RNA extracted from PC12 cells exposed to 100 ng/ml NGF for 0, 2, 4, and 24 h was fractionated on a formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized with sGC ␣1 and ␤1 subunit cDNA probes and with an oligonucleotide complementary to 18 S ribosomal RNA. Panel B, dose response. RNA extracted from PC12 cells exposed to 0 -1000 ng/ml NGF was hybridized with sGC ␣1 and ␤1 subunit cDNA probes and with an oligonucleotide complementary to 18 S ribosomal RNA. sGC subunit protein levels were measured in PC12 cells incubated in the presence and absence of NGF. Consistent with the observation that prolonged exposure to NGF was necessary to decrease NO-stimulated sGC enzyme activity, decreased sGC subunit protein levels were evident in PC12 cells incubated with NGF for 24 h but not in cells exposed for 2, 4, and 8 h (data not shown). The effect of NGF on levels of both sGC subunits was dose-dependent; decreased subunit protein levels were detected in cells exposed to NGF in concentrations of 10 ng/ml or greater (Fig. 3).

Signal Transduction Pathways Regulating sGC Subunit
Gene Expression in PC12 Cells Exposed to NGF Modulation of sGC Subunit Gene Expression Is Tyrosine Phosphorylation-dependent-The biological response to NGF is initiated by autophosphorylation of its high affinity receptor (p140) on tyrosine residues (21). To determine whether the mechanisms involved in the regulation of sGC subunit gene expression are tyrosine phosphorylation-dependent, sGC subunit mRNA levels were measured in PC12 cells pretreated with 3 mM MeSAdo (a methyltransferase inhibitor that inhibits tyrosine phosphorylation of the NGF receptor as well as other proteins in PC12 cells exposed to NGF) (21). At this concentra-tion, MeSAdo effectively inhibited NGF-induced tyrosine phosphorylation in PC12 cells (data not shown). MeSAdo also blocked the NGF-induced decrease in sGC subunit mRNA levels (Fig. 4). Incubation of PC12 cells with two other growth factors that signal through receptor tyrosine phosphorylation, EGF and bFGF, in concentrations sufficient to induce protein tyrosine phosphorylation (22) and c-fos gene expression in PC12 cells (14), failed to alter sGC subunit gene expression (Fig. 5).
Regulation of sGC Subunit Gene Expression by NGF Is cGMP-, cAMP-, Calcium-, NO-, and Protein Kinase C-independent-To further characterize the intracellular signaling mechanisms participating in the regulation of sGC subunit gene expression by NGF, sGC subunit mRNA levels were measured in PC12 cells exposed to agents that modulate several regulatory pathways. Incubation of PC12 cells with membranepermeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, did not decrease sGC subunit mRNA levels (Fig. 5). Moreover, pretreatment of PC12 cells with 1 mM L-NAME, a NO synthase inhibitor) did not attenuate the ability of NGF to decrease sGC subunit mRNA levels, suggesting that NO synthase activity did not account for the effect of NGF (Fig. 6). Soluble guanylate cyclase ␣1 and ␤1 subunit levels were measured in PC12 cells exposed to 0 -100 ng/ml NGF for 24 h. Proteins in the soluble fraction of cell extracts were fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Immunoreactive sGC ␣1 subunit (82 kDa) was detected using a monoclonal antibody directed against ␣1 subunit purified from rat lung. Immunoreactive sGC ␤1 subunit (70 kDa) was detected using an affinity-purified polyclonal antibody directed against a pMAL-␤1 subunit fusion protein produced in bacteria. PC12 cells were also exposed to PMA (100 nM), an activator of protein kinase C, and A23187 (5 M), a calcium ionophore. sGC subunit mRNA levels were not altered in PC12 cells exposed to either PMA or A23187 (Figs. 5 and 6). In addition, pretreatment of PC12 cells with 10 M bisindolylmaleimide I, a protein kinase C inhibitor, did not block the effect of NGF on sGC subunit gene expression (data not shown).
To determine the effect of cAMP on sGC subunit mRNA levels in PC12 cells, cells were incubated for 4 h with 1 mM dibutyryl cAMP (a membrane-permeable cAMP analogue), 10 M forskolin (an adenylate cyclase agonist), or 1 mM IBMX (a phosphodiesterase inhibitor). All three agents decreased sGC subunit mRNA levels (Fig. 5). However, incubation of PC12 cells with 100 ng/ml NGF for 1-30 min did not increase intracellular cAMP levels, whereas 10 M forskolin increased intracellular cAMP levels 15-fold after 30 min (data not shown). These results suggest that although increased intracellular cAMP concentrations can decrease sGC subunit mRNA levels, they do not account for the effect of NGF on sGC subunit gene expression.
NGF Regulates sGC Subunit Gene Expression via Ras Activation-Ras activation has a critical role in signaling many of the effects of NGF binding to its receptor. To study the role of Ras in the regulation of sGC subunit gene expression by NGF, M-M17-26, a PC12 cell line expressing a dominant inhibitory Ras mutant, was used. Whereas NGF decreased sGC subunit mRNA levels in wild-type PC12 cells (Fig. 7, Panel A, lanes 3  and 4), sGC subunit mRNA levels did not differ in M-M17-26 cells incubated in the presence or absence of NGF (lanes 5-8).
In contrast, increased intracellular cAMP decreased sGC subunit mRNA levels in M-M17-26 cells (Fig. 7, Panel B), suggesting that regulation of sGC subunit gene expression by cAMP is not mediated via Ras activation. The inability of NGF to decrease sGC subunit mRNA levels in M-M17-26 cells was not due to absence of the NGF receptor since tyrosine phosphorylation of cellular proteins, including a 140-kDa protein likely to be the NGF receptor, was observed in M-M17-26 cells exposed to NGF (data not shown). Furthermore, exposure of M-M17-26 cells to NGF stimulated c-fos gene expression in a time-dependent manner (Fig. 7, Panel C), as described previously (14).

Molecular Mechanisms Involved in the Regulation of sGC Subunit Gene Expression in NGF-treated PC12 Cells
Destabilization of sGC Subunit mRNAs by NGF Is Dependent on Gene Transcription-To investigate the role of mRNA sta-bility on the effect of NGF on sGC subunit mRNA levels, we examined the effect of actinomycin D, an RNA polymerase inhibitor, on sGC subunit gene expression in PC12 cells. The levels of sGC ␣1 and ␤1 subunit mRNAs did not change in PC12 cells exposed to 10 M actinomycin D for up to 6 h. In contrast, c-jun mRNA levels decreased more than 50% within 1 h (Fig. 8, left panel). sGC subunit levels decreased more rapidly in PC12 cells exposed to NGF than in cells exposed to actinomycin D (see Fig. 1, Panel A), suggesting that NGF decreases sGC subunit mRNA stability. Moreover, incubation of PC12 cells with actinomycin D blocked the ability of NGF to decrease sGC subunit mRNA levels (Fig. 8, right panel). These results suggest that NGF decreases the stability of sGC subunit mRNAs via a transcription-dependent mechanism.
Destabilization of sGC Subunit mRNAs by NGF Is Dependent on Protein Synthesis-To further examine the mechanisms by which NGF decreases sGC subunit mRNA stability, PC12 cells were pretreated with cycloheximide or anisomycin, protein synthesis inhibitors, before exposure to NGF. Inhibition of protein synthesis completely blocked the ability of NGF to decrease sGC ␤1 subunit mRNA levels and partially blocked the decrease in ␣1 subunit mRNA levels (Fig. 9). These data suggest that NGF decreases sGC subunit mRNA levels through mechanisms that involve both RNA transcription and de novo protein synthesis.

DISCUSSION
Soluble guanylate cyclase is a critical component in NOmediated signal transduction. In this study, regulation of sGC subunit gene expression was investigated in a rat pheochromocytoma cell line, PC12 cells, exposed to the neurotrophic factor NGF. NGF decreased levels of both sGC ␣1 and ␤1 subunit mRNAs in a dose-and time-dependent manner. The half-maximal effect of NGF on sGC subunit gene expression was observed between 1 and 10 ng/ml, which is consistent with the reported dissociation constants (K d ) of the two classes of NGF receptors (approximately 10 Ϫ9 M, 25 ng/ml) (23,24) and the EC 50 for the activation of mitogen-activated protein kinase by NGF (3 ϫ 10 Ϫ10 M, 10 ng/ml) (25) in PC12 cells. Decreased sGC subunit mRNA levels were observed within 2 h after addition of NGF and reached lowest levels within 4 h. Decreased sGC subunit mRNA levels were associated with decreased sGC subunit protein levels and NO-activated enzyme activity. However, the decrease in subunit protein levels and enzyme activity was detectable only after 24 h of continuous exposure to NGF. These results suggest that both sGC subunits are relatively stable cellular proteins in PC12 cells.
Although two forms of NGF receptor p75 and p140, are expressed in PC12 cells, one form, p140, appears to be required for NGF-induced receptor tyrosine kinase activity, Ras activation, and immediate early gene expression (including c-fos) (26). Me-SAdo, a methyltransferase inhibitor, inhibits NGF-induced tyrosine kinase activation of the NGF receptor as well as other cellular proteins (21). MeSAdo blocked the ability of NGF to decrease sGC subunit mRNA levels. These observations suggest that receptor tyrosine kinase activation, the initiating event in NGF signal transduction, is involved in the NGF regulation of sGC subunit gene expression in PC12 cells. Moreover, the tyrosine kinase-mediated regulation of sGC subunit gene expression appeared to be NGF-selective, because EGF and bFGF, agonists for two other receptor tyrosine kinases in PC12 cells, failed to modulate sGC subunit mRNA levels.
NGF is known to generate cellular responses via multiple signal transduction pathways. It has been reported that NGF increases the half-life of GAP43 mRNA in PC12 cells through a protein kinase C-dependent mechanism (27). Protein kinase C activation alone was insufficient to account for the NGF-induced decrease in sGC subunit mRNA levels because incubation of PC12 cells with PMA, a protein kinase C agonist, did not alter sGC subunit gene expression. Moreover, the inability of bisindolylmaleimide I, a protein kinase C inhibitor, to block the effect of NGF on sGC subunit mRNA levels suggested that protein kinase C activation was not required for the regulation of sGC subunit gene expression by NGF.
Similar to observations in rat fetal lung fibroblasts (9) and rat aortic smooth muscle cells (10), agents that increase intracellular cAMP were found to decrease sGC subunit gene expression in PC12 cells. However, consistent with the observations of Hatanaka et al. (28) and Buskirk et al. (29), cAMP levels were not increased in PC12 cells exposed to NGF. These results suggested that regulation of sGC gene expression by NGF is not dependent on cAMP.
Several observations led us to consider the possibility that NO and cGMP may mediate the effect of NGF on sGC subunit gene expression. First, Peunova and Enikolopov (13) observed that NGF stimulated NO synthase expression in PC12 cells. Second, Ujiie et al. (11) reported that NO donor compounds and agents that increase intracellular cGMP levels decreased sGC subunit mRNA and enzyme levels in rat medullary interstitial cells. Finally, we recently observed that sGC subunit mRNA and protein levels and sGC enzyme activity were decreased in rat pulmonary artery smooth muscle cells exposed to NO-donor compounds. 2 In the present study, incubation of PC12 cells with membrane-permeable cGMP analogues did not decrease sGC subunit mRNA levels. Moreover, pretreatment of PC12 cells with L-NAME did not block the effect of NGF on sGC subunit mRNA levels. These results suggested that the effect of NGF on sGC subunit gene expression was not mediated by NO or cGMP.
Ras appears to have an important role in the regulation of sGC subunit mRNA levels by NGF. Ras is located at the inner surface of the plasma membrane and transduces signals from tyrosine kinase receptors to intracellular target molecules (30). Intrinsic GTPase activity regulates the levels of active (GTPbound) and inactive (GDP-bound) Ras. To investigate the role of Ras in the regulation of sGC subunit gene expression, the M-M17-26 cell line (14), a PC12 cell line stably transfected with the mutant p21(Asn-17)Ha-ras gene, was used. The encoded mutant Ras is inactive due to a high affinity for GDP and likely competes with normal Ras for guanine nucleotide exchange factors, sequestering them into nonfunctional complexes (19). Incubation of M-M17-26 cells with NGF did not decrease sGC subunit mRNA levels, suggesting that the NGF effect was Ras-dependent. The presence of a functional NGF receptor in M-M17-26 cells was confirmed by the observations that NGF induced tyrosine phosphorylation of cellular proteins and stimulated c-fos gene expression. Ras activates several signaling pathways, at least one of which, the Raf/MEK/ERK protein kinase cascade, participates in PC12 cell differentiation (12). It remains to be determined whether or not NGF regulation of sGC subunit gene expression is Raf/MEK/ERK-dependent. Of note, agents that increase cAMP concentrations were able to decrease sGC subunit mRNA levels in M-M17-26 cells, suggesting that the effect of cAMP on sGC subunit gene expression is Ras-independent.
To further investigate the mechanisms regulating sGC subunit gene expression in PC12 cells, the effect of actinomycin D on NGF-mediated regulation of sGC subunit mRNA levels was measured. sGC subunit mRNA levels decreased more rapidly in PC12 cells exposed to NGF than in PC12 cells exposed to actinomycin D. These results suggest that NGF decreased sGC subunit mRNA levels, at least in part, by decreasing sGC subunit mRNA stability. sGC subunit mRNA levels did not differ in PC12 cells exposed to actinomycin D alone or in cells exposed to actinomycin D with NGF. These results suggest that NGF decreases sGC subunit mRNA stability via a mechanism that is dependent on RNA transcription. Exposure of PC12 cells to agents that inhibit protein synthesis blocked, at least partially, the ability of NGF to decrease sGC subunit mRNA levels, suggesting that regulation of mRNA stability was protein synthesis-dependent. Of note, we have observed that exposure of rat pulmonary artery smooth muscle cells to NOdonor compounds destabilizes sGC subunit mRNAs via a similar transcription/translation-dependent mechanism. 2 These results suggest that regulation of sGC subunit mRNA stability is an important mechanism involved in modulating sGC function in multiple cell types. Similar factors may be involved in the coordinate destabilization of sGC ␣1 and ␤1 subunit mRNAs in PC12 cells exposed to NGF and rat pulmonary artery smooth muscle cells exposed to NO.
Although other components of the NO/cGMP signal transduction system contribute to NO responsiveness, the NGFinduced decrease in sGC function may be expected to decrease responsiveness to NO by decreasing cGMP synthesis. Since cGMP modulates neurotransmission (1,6), changes in sGC function are likely to permit regulation of the ability of NO to alter neuronal activity. Exposure of PC12 cells to NGF activates expression of a program of genes leading to neuronal differentiation with neurite outgrowth and cessation of cell proliferation. Recent evidence suggests that NGF induces expression of NO synthases (beginning after 24 h of exposure), which leads to cytostasis, thereby permitting differentiation (13). It is unknown whether or not the NO-mediated inhibition of PC12 cell proliferation is cGMP-dependent. Incubation of PC12 cells with NGF appears to decrease sGC function before induction of NO synthases. It is possible that the NGF-induced decrease in sGC activity prevents excess signaling via cGMP under conditions associated with high levels of NO production.
In summary, a neurotrophic factor, NGF, decreased sGC subunit mRNA and protein levels and decreased NO-activated sGC subunit enzyme activity. NGF-mediated regulation of sGC subunit gene expression appeared to require NGF receptorstimulated tyrosine kinase activity and Ras activation. The effect of NGF on sGC subunit mRNA levels did not depend on NO, cGMP, or protein kinase C. Although agents that increase cAMP levels decreased sGC subunit mRNA levels, NGF did not increase cAMP levels, suggesting that NGF-mediated regulation of sGC subunit gene expression is cAMP-independent. NGF decreased sGC ␣1 and ␤1 subunit mRNA levels coordinately, at least in part by decreasing mRNA stability. Inhibitors of RNA transcription and protein synthesis attenuated the ability of NGF to decrease sGC subunit levels, suggesting that NGF induces synthesis of a factor that selectively decreases subunit mRNA stability. The decrease in sGC function in PC12 cells exposed to NGF appeared to precede the induction of NO synthases. These results suggest that NGF-induced changes in NO responsiveness as well as NO synthesis may contribute to the neuronal differentiation of PC12 pheochromocytoma cells.