All buffers and chemicals were cell culture grade and purchased from Sigma Chemical Company (St. Louis, MO). The NOS inhibitors, N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) and N-omega-nitroL-arginine (L-NNA) were purchased from EMD Millipore (Billerica, MA).

In this study, pulmonary fibroblasts were isolated from 28 - 33 day old female Lewis rats as previously described [25] . Cells were cultured in DMEM (Gibco BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 100 U penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 20 mM HEPES, pH 7.55 (Gibco BRL). For routine passaging and maintenance, cells were grown in serum-supplemented (10% v/v) media in T-75 cell culture flasks from Corning (Tewksbury, MA). It is well established that isolated fibroblasts cultured on a plastic substrate display a myofibroblast phenotype as determined by a-smooth muscle actin expression [3] [26] . Cultured myofibroblasts are known to differentiate under conditions of serum starvation (when growth factors are lacking in the cell culture media) as verified by skeletal muscle protein expression [27] .

Myofibroblasts were seeded at a density of 1 × 10⁵ cells/well, in 6-well plates, and grown for approximately 48 h or until ~75% confluent. Subsequently, the growth media was changed to either fresh 10% FBS (v/v) or 0% (serum-free) DMEM plus either of the NOS inhibitors L-NAME or L-NNA at various concentrations; cells were incubated overnight prior to assay.

Approximately 16 h later, the growth media was aspirated from each well and replaced with phosphate buffered saline (PBS), pH 7.4, containing 100 mM CaCl₂, 1 mM l-arginine, and 1 mM A23187, similar to the method of Leikert et al. [22] . The growth media was removed to avoid interference from serum nitrate as previously reported [23] . Cells were incubated for 3 h at room temperature. This time point was selected after several pilot experiments that determined 1 - 2 h was not long enough for sufficient NO_X accumulation, yet by 4 h of incubation the myofibroblasts had begun to lift off the plate (data not shown). Subsequently, 900 mL from each sample was removed and combined with 100 mL of 1 M Tris buffer, pH 9.2, in a 24-well plate. Samples were incubated with a nitrate reductor (NR) wire for 15 min with stirring to reduce any accumulated nitrate back to nitrite. The NR wire is 3 cm long × 3 mm wide and is made from a metal alloy that catalyzes the reduction of nitrate to nitrite, which is subsequently detected by the electrode (Harvard apparatus). The NR wires were stored in 0.01 M H₂SO₄ when not in use, and were rinsed with deionized water prior to placing into the well with the sample solution.

The NO selective electrode (Innovative Instruments, Tampa, FL) was equilibrated in a 20 mL acid-iodide bath (20 mM potassium iodide, 100 mM sulfuric acid) with constant stirring at room temperature for 30 min prior to calibration. A standard curve in picoamps (pA) was subsequently generated by measuring various known concentrations of sodium nitrite added to the bath. The peak currents were monitored and used to record an amperogram using the inNO-T software v3.2 provided by Innovative Instruments. To measure extracellular NO_X, 100 uL from each NR treated sample was added to the acid iodide bath in which the electrode was immersed. Individual peaks were resolved on the amperogram within 3 - 5 minutes. The signal was allowed to return to baseline before subsequent sample additions. Data points were collected by subtracting the initial baseline from the maximum peak height value for each sample. The NO_X concentration for each sample was extrapolated from a standard calibration curve prepared prior to each experiment.

Pulmonary myofibroblasts were grown in culture as described above in 2.1 in either media containing 10% FBS or 0% FBS. After 48 hours, cells were harvested in PBS and suspended in TRIzol reagent (Invitrogen). Total RNA was isolated according to the manufacturer’s protocol (TRIzol). One µg total RNA was converted to cDNA by the High Capacity cDNA archive kit (Applied Biosystems). Exon-exon boundaries in mRNA sequences for NOS1 [Rn00583793mi], NOS2 [Rn00561646mi], or NOS3 [Rn 02132634si] were identified and submitted to Applied Biosystems, Inc. for Taqman gene expression assay design. Each primer set was chosen so that amplicons would cross exon boundaries to prevent contamination with genomic DNA. Derived experimental Ct values were converted to copy number using a standard curve and subsequently normalized to an endogenous control, β-glucuronidase (GUS) [Rn00566655mi].

It is known that pulmonary myofibroblasts in culture alter their biological function and genetic signature significantly in response serum concentration changes [27] . Therefore, for any assay to be physiologically applicable to myofibroblasts, NO must be quantifiable in media with different serum amounts. When known concentrations of nitrite, first converted to NO in an acid-iodide bath, were measured in culture media that contained either 10% or 0% FBS, using an NO-selective electrode coated with a gas permeable membrane (the amiNO-700 sensor), non-overlapping calibration curves resulted (Figure 1). Although the coefficient of determination (R²) for both samples was >0.90, the resulting linear regressions produced vastly different calibration curves with equations

of y = 502.7x and y = 335.7x for the serum free media and media containing 10% FBS, respectively. To overcome this dilemma, we tested whether NO could be accurately measured in PBS alone. As shown in Figure 2(a) and Figure 2(b), NO is indeed readily detectable by the amiNO-700 sensor in PBS. Therefore, the ability to electrochemically quantify NO production in PBS versus media greatly increases the efficiency of the assay by eliminating the need for multiple media-dependent calibration steps, while also preventing any potential media-induced matrix effects.

The inNO-T Nitric Oxide Measuring System, in addition to the amiNO-700 sensor, contains proprietary nitrate reductor (NR) wires comprised of a reusable metallic catalyst that reduces any nitrate in the samples back to nitrite prior to analysis. Although NO released from the cells is readily detectable using the system, when the nitrate reduction step was added to the protocol, a two-fold amplification in NO was measured (Figure 2(c)). Thus, the simple wire-based reduction step also serves to improve the amperometric assay by increasing its sensitivity and ease; this is in contrast to intricate enzymatic reduction methods previously used with electrochemical probes [23] [24] .

In order to properly use the bioassay in experimental design, it is essential to know which of the three NOS isoforms are expressed in pulmonary myofibroblasts, in particular, since the inducible NOS isozyme has significantly different pharmacokinetic properties and can produce ~1000 times the molar concentration of NO when compared to either constitutive form [19] . When the amount of each NOS mRNA was quantified from cultured pulmonary myofibroblasts, from both 10% FBS and serum free media, the most abundantly expressed isoform was eNOS, regardless of serum, followed by significantly lower levels (~10-fold) of iNOS mRNA; nNOS levels were undetectable (Figure 3). Transcriptional changes of these two NOS variants was reciprocal in that eNOS increased under normal growth conditions, whereas iNOS expression increased when cells were differentiated by the removal of serum. The serum-mediated differential expression implies there may be distinct roles for each isoform requiring different molar concentrations of NO.

When the enhanced assay was used to quantify constitutively produced NO in pulmonary myofibroblasts under both normal growth conditions (10% serum) and after differentiation (0% serum), the assay proved to be reproducible and sensitive; differentiated cells produced ~30-fold more NO under basal conditions than those growing in serum (~20 nM vs ~0.60 nM) (Figure 4(b) and Figure 4(c)). Moreover, treatment of the cells with the constitutive NOS selective inhibitors L-NNA and its methyl ester, L-NAME, dose-dependently reduced NO release from differentiated cells, while proliferating cells were not responsive to either inhibitor (Figure 4(b) and Figure 4(c)) [28] . The variance in the inhibitor response to serum may reflect changes in eNOS subcellular

location since in other cell types, cellular locale strongly affects eNOS’ ability to produce NO and respond to Ca²⁺ [29] . The fact that NO production was not completely abolished in the presence of either inhibitor however, does suggest that iNOS, while expressed 10-fold less than eNOS, contributes somewhat to the overall formation of NO in pulmonary myofibroblasts.