Male Male Wistar rats (180 - 200 g) were anesthetized and killed by decapitation and the thoracic aortas were isolated. Aortic rings, 4 mm in length, were placed in bath chambers (10 mL) for isolated organs containing physiological salt solution (PSS) at 37˚C, continuously bubbled with 95% O₂ and 5% CO₂, pH 7.4. The system was connected to an F-60 force-displacement transducer, and the contractile responses were recorded on a polygraph (Narco Biosystems Inc., Houston-TX). The aortic rings were submitted to a tension of 1.5 g, which was readjusted every 15 min throughout a 60-min equilibration period before the addition of the given drug. An optimal basal tension of 1.5 g was previously standardized by exposing the vessels to 90 mM KCl under various resting tensions (0.25 - 2.5 g). All the procedures were in accordance with the Ethical Animal Committee of the University of São Paulo, Brazil.

At the beginning of the experiments, the aortas were stimulated with 1 mM norepinephrine to test their functional integrity. In some preparations, the endothelium was mechanically removed by gently rubbing the intimal surface with stainless steel holders. The integrity of the endothelium was tested with acetylcholine (1 mM). Endothelium removal was confirmed by lack of acetylcholine-induced relaxation.

After the endothelial integrity was assessed, aortic rings were pre-contracted with 100 nM phenylephrine. When the plateau was reached, concentration-effect curves to forskolin (0.1 nM to 1 mM) were constructed in the absence or in presence of eNOS inhibitor L-NNA (10 μM), cyclooxigenase inhibitor indomethacin (10 μM) or the combination of both inhibitors in the same concentrations used alone.

Briefly, the aortas were dissected and longitudinally opened and endothelium cells were isolated by gently rake friction in Hanks solution. The resulting cells solution was centrifuged at 200 xg and suspended in Hanks solution. The cells were placed on glass coverslips and kept in a humidified 37˚C incubator gassed with 5% CO₂ for 4 h. The cells were then loaded with 5 mM Fura-2AM in 1 mg/mL BSA for 40 min at 37˚C. Excess dye was removed by washing out the dye with bath solution for 20 min for intracellular desterification of Fura-2AM. Dishes containing Fura-2AM loaded cells were placed in a temperature-regulated (37˚C) chamber mounted on the Nikon inverted microscope [15]. The fluorescence measurement was performed by the window of a dual wavelength spectrofluorimeter (Deltaram, Photon Technology Intl), at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm.

Fura-2 fluorescent signals originated from the cells stimulated with forskolin (1 mM) were collected and stored using a software package from Photon Technology International (Felix). The cells that showed fluorescence above 4 × 10⁵ counts were used. Fura-2 ratio corrected for background fluorescence was converted to [Ca²⁺]_c by the ratio between the two excitation wavelengths (340 and 380 nm) in which Fura-2 fluorescence and measured at 510 nm. Fluorescence emission was collected from the single cell. However, at least three independent experiments were performed using separate cells. Because of the recognized uncertainties inherent to the measurement of absolute [Ca²⁺]_c, in this study the results are expressed as the R340/380 nm ratio of fluorescence.

In order to measure cGMP tissue content, intact rat aortas were incubated with sildenafil (1 µmol/L) for 30 min. The vessels were the exposed to 0.1 μM phenylephrine for 10 min or to 0.1 μM phenylephrine for 10min followed by a 10-min exposure to forskolin (1 mM). In another set of experiments, L-NG-Nitroarginine (L-NNA 10 μM) was incubated for 10 min and the vessel was exposed to phenylephrine (0.1 μM) for 10min, followed by a 10-min exposure to forskolin (1 mM). This was the concentration of forskolin that produced maximum relaxation (Emax) of the aortic rings. The vessel segments were rapidly frozen in liquid nitrogen, homogenized in 6% trichloroacetic acid, and centrifuged. The supernatant was extracted with water-saturated diethyl ether for six times. The samples were then evaporated under nitrogen and the cGMP content was assayed in duplicate by using a commercially available enzyme-immunoassay kit (Amersham Biosciences). Basal cGMP was defined as the cGMP content obtained without stimulation in the homogenized vessel containing intact endothelium. Results are expressed as fentomoles of cGMP per milligram of protein. Protein was determined by using the Folin Ciocaulteau method.

Acetylcholine, Albumin bovine fraction V, fatty acidfree, Albumin bovine fraction V, Dimethyl sulfoxide (Grade I) DMSO, Forskolin (HPLC grade), Indomethacin, Norepinephrine, Phenylephrine hydrochloride, N^G- nitro-L-arginine (L-NNA), and Poly-L-lysine solution were obtained from Sigma. DAF-2DA was purchased from Sigma Chem. Co. (St Louis, MO). Fura-2AM was obtained from Molecular Probes (Eugene, OR). The enzy me-immunoassay Biotrak (EIA) System for measurement of cGMP content was supplied by Amersham Biosciences (GE Healthcare).

The PSS composition was the following (in mM): 130.0 NaCl, 1.6 CaCl₂, 4.7 KCl, 1.17 MgSO₄, 1.18 KH₂PO₄, 14.9 NaHCO₃, 0.026 EDTA, and 5.5 dextrose. The Hanks solution composition was as follows (in mM): 145.0 NaCl, 1.6 CaCl₂ (or without CaCl₂), 5.0 KCl, 1.0 MgCl₂, 0.5 NaH₂PO₄, 10.0 dextrose, and 10.0 HEPES.

The forskolin relaxant effect was measured from the plateau of the contraction induced by phenylephrine and expressed as percent relaxation. The maximum effect (Emax) was considered as the maximal amplitude response reached in the concentration-effect curves for forskolin. The forskolin concentration that produced halfmaximal relaxation amplitude (EC₅₀) was determined and is expressed as pEC₅₀. The pEC₅₀ value is the negative logarithm and expressed as log molar EC₅₀. Data analysis and figure plots were carried out with the aid of software GraphPad PRISM^TM, Version 3.02 (2000) (San Diego, CA). Data are expressed as mean ± SEM. In each set of experiments, n indicates the number of tissues studied. Differences between mean values have been assessed either by Student’s t-test or by one-way analysis of variance (ANOVA), followed by a Newman-Keuls post-hoc test. Values of P < 0.05 were considered to be significant. There are some results in which P is lower than 0.001.

Forskolin induced relaxation in a concentration-dependent way in both endothelium intact and denuded rat aortic rings pre-contracted with phenylephrine. As shown in Figure 1(a), the maximum relaxant effect induced by forskolin (103.8% ± 2.5%, n = 6) was not changed by endothelium removal (100%, n = 6). However, the potency of forskolin in inducing the relaxation in the intact endothelium aortic rings (pEC₅₀: 7.18 ± 0.05) decreased with endothelium removal to 6.99 ± 0.06 (P < 0.05) (Figure 1(b)).

In order to evaluate whether the production of the relaxant endothelial factors NO and PGI₂ are involved in the forskolin-induced relaxation, the intact endothelium aortic rings were incubated with the inhibitors L-NNA, indomethacin or the combination of both inhibitors. LNNA (10 mM) reduced both the maximum effect induced by forskolin to 92.2% ± 3.8% (n = 6, P < 0.05) (Figure 1(a)) and its potency (pEC₅₀: 6.54 ± 0.06, P < 0.001) (Figure 1(b)). On the other hand, indomethacin (10 mM) did not alter the maximum effect (98.3% ± 0.9%, n = 6) (Figure 1(a)) nor its potency (pEC₅₀: 6.93 ± 0.11) (Figure 1(b)). The combination of L-NNA (10 mM) and indomethacin (10 mM) reduced the maximum relaxant effect of forskolin (93.5% ± 4.0%, n = 6, P < 0.05) (Figure 1(a)) and its potency (pEC₅₀: 6.41 ± 0.22, P < 0.01) (Figure 1(b)).

In the isolated rat aortic endothelial cells, forskolin (1 mM) elicited significant increase in the fluorescence intensity of Fura-2AM, from 0.924 ± 0.104 (basal fluorescence) to 1.164 ± 0.015 units (P < 0.01).

As shown in Figures 2(a)-(f), forskolin (1 mM) increase significantly the fluorescence intensity induced by the dye DAF-2T from 0 to 153.3% ± 10.5% (n = 4, P < 0.001), which indicates an increase in [NO]c over the basal levels. In endothelial cells pre-incubated with L-NNA (5 μM), forskolin decreased fluorescence of the dye DAF-2T, related with forskolin-induced NO production from 0 to –5.9% ± 1.2% (n = 3, P < 0.001), below the basal level shown in the graph of Figure 2(g).

In order to evaluate the effect of the inhibition of PKA in a larger number of endothelial cells, it was used flow cytometry in a time-dependent effect of forskolin. Forskolin (1 mM) elicited significant increase in the fluorescence of the dye DAF-2T (blank) in relation to the time immediately after forskolin addition to the cells (time 0: 89.7% ± 12.6% (n = 3, P < 0.01); after 5 min it was increased to 153.8% ± 11.0% (n = 3, P < 0.01) and after 10 min it was increased to 224.6% ± 27.3% (n = 3, P < 0.01). The treatment of the cells with KT5720 (10 μM) inhibited the effect of forskolin in increasing [NO]c with the IF of 60.3% ± 1.0% at T0 (n = 3, P < 0.05), after 5 min (107.9% ± 5.4%, n = 3, P < 0.004), and after 10 min (168.4% ± 15.5%, n = 3, P < 0.05) (Figure 3) in relation to forskolin.

As shown in Figure 4, forskolin elicited a significant increase in cGMP production (28.0 ± 1.8 fmol of protein) as compared to the basal levels in homogenized vessel containing intact endothelium (E⁺) (16.2 ± 1.8 fmol of protein) (P < 0.05). The production of cGMP stimulated with forskolin was significantly inhibited by L-NNA (18.8 ± 0.4 fmol of protein) (P < 0.05), which values were comparable to the basal levels.