Fig. 1. Western blot showing the expression of proteins involved in cGMP turn-over and signaling in dissociated VSM cells from the tunica media of rat aorta. (A) GC 1β, (B) PKG I, (C) PDE5, and (D) MHC II were detected only in early passage rat aortic VSM cells. TM (Tunica Media) denotes smooth muscle cell layer before dissociation. P0 and P1 refer to passage number, subscript numbers denote number of days in culture. 40 μg of total protein was loaded per lane. GAPDH = internal loading standard.

Fig. 2. Immunohistochemical comparison of untransfected, and Cygnet-2.1 transfected smooth muscle cells. (A) Immunohistochemical staining of two cells (1 and 2) using a Cy5-linked PKG antibody, which recognizes both endogenous PKG and transfected Cygnet-2.1. (B) Citrine fluorescence of the Cygnet-2.1 transfected cell 1. (C) Total immunoreactivity of cells 1 and 2 as determined by their average pixel intensities. (D) Model of cGMP binding induced conformational change of the FRET-based indicator, Cygnet-2.1 (em.= emission wavelength, ex.= excitation wavelength).

Fig. 3. Kinetics of NO-evoked cGMP transients in early passage, Cygnet-2.1 transfected VSM cells. 485/525 nm emission ratios indicating intracellular levels of cGMP in response to (A) 500 nM DEA/NO and (B) 100 μM DETA/NO, which correspond to 370 nM and 330 nM of maximal NO as measured by a NO-electrode (red traces), respectively. Green, blue, and violet traces represent regions within Cygnet-2.1 transfected cells (see insets) monitored for ECFP and Citrine emission intensities. (C) Estimation of intracellular cGMP by an [¹²⁵I]-radioimmunoassay at indicated time points following addition of 500 nM DEA/NO.

Fig. 4. NO concentration dependency of cGMP transients. 480/535 nm emission ratios from Cygnet-2.1 in VSM cells responding to (A) 200 nM and (B) 10 μM DEA/NO. (C) Linear correlation between DEA/NO concentration (200 nM–10 μM) and half-maximal cGMP peak width (P_1/2). The inset: correlation of 485/535 nm emission ratios upon 50 nM–400 nM exposures to DEA/NO. (D) 485/535 nm emission ratios of 2 and 10 μM DEA/NO applications (black traces) aligned with predicted NO concentrations (NO_calc) emanating from these concentrations (red traces). Inset: plot of P_1/2 against NO_calc for 1, 2, and 10 μM DEA/NO. Colored traces represent different regions within the imaged cells.

Fig. 5. Maximal and steady-state cGMP levels. (A) P0 VSM cells were treated with 1 μM or 10 μM DEA/NO corresponding to plateau cGMP responses at indicated times and total cGMP levels were determined using a radioimmunoassay system (see Materials and methods). B = basal cGMP in untreated cells. (B) Cyan/yellow emission ratio of a typical VSM cell responding to 100 μM of DEA/NO, followed by 100 μM Sildenafil and (C) 1 μM DEA/NO followed by 50 μM YC-1. Colored traces represent different regions within the imaged cells.

Fig. 6. Sildenafil eliminates the decay phase of cGMP peaks. (A) 100 μM Sildenafil slightly increased resting cGMP levels indicating a basal PDE5 activity in VSM cells. Addition of peak-inducing 400 nM DEA/NO (see Fig. 2A) resulted in sustained cGMP elevations. Inset: radioimmunoassay measurements of untreated VSM cells (Basal) and treated with 100 μM Sildenafil for 5 min, and 1 μM DEA/NO (2 min), followed by Sildenafil (5 min). Measurements are expressed as fmoles of cGMP normalized to μgs of total protein. (B) The Sildenafil/NO plateau of cGMP was also demonstrated in cells which initially showed a transient response to 400 nM DEA/NO.

Fig. 7. Guanylyl cyclase inhibition terminates cGMP responses. (A) Failure of VSM cells to respond to maximal, 100 μM DEA/NO dose in the presence of 10 μM ODQ. (B) Broadened cGMP transient executed with 1 μM DEA/NO (compare to Fig. 2 and Fig. 3A, B). (C) cGMP peak conditions as shown in (B), however, immediately after maximum NO activation (30 s) application of 10 μM ODQ resulted in instantaneous decay of cGMP.

Fig. 8. NO-induced cGMP transients in the presence of elevated intracellular cGMP. (A) Western blot analysis of Phospho-PDE5 at time points corresponding to peak, decay phase, and new baseline following application of 500 nM DEA/NO (see Fig. 2A). 35 μg of total protein was loaded per lane. P-PDE5 levels were also normalized to GAPDH and total PDE (not shown). (B) 10–15% emission ratios elicited with either 10 μM 8-Br-cGMP or, (C) 5 μM HMR 1766, followed by DEA/NO induced cGMP peak responses.

Fig. 9. Multifold cGMP transients in response to various NO concentrations. (A) Sequential applications of 500 nM DEA/NO. Following three consecutive stimuli, DEA/NO was washed out before a final NO dose was applied. (B) Restimulation of cGMP peak through addition of a second 200 nM DEA/NO application during the rapid decay of a cGMP transient. (C) Sustained peaks of cGMP using 1 μM DEA/NO were also elicited in rapid succession.

Analysis of NO/cGMP kinetics in vascular smooth muscle cells

Numbers in parentheses indicate number of experiments. ¹⁾L_T = mean lag time; time from stimulus to a 5% response. ²⁾T_p = time-to-peak; time interval from 5–95% of the response. ³⁾FRET = mean amplitude of the % emission ratio change. ⁴⁾P_1/2 = half-maximal peak width. ⁵⁾t_1/2 = half-maximal decay rate.⁶⁾VSM cells were preincubated with 100 μM Sildenafil. ⁷⁾ODQ (10 μM) was added at the maximal FRET ratio response (30 s postapplication). NA = not applicable.