Preincubation with papaverine leads to decreases in phosphorylation of the mypt1 in HSV.

<p>HSV rings were suspended in a muscle bath and equilibrated in Kreb’s bicarbonate buffer for 2 h. Rings were untreated (Basal), treated with 1 μM norepinephrine (NE) for 5 minutes, 1 mM papaverine (Pap) for 10 min followed by 1 μM norepinephrine for 5 min or 1 mM papaverine (Pap) for 10 min. Tissues were snap frozen and protein lysates were separated on 4–20% criterion gels and transferred to nitrocellulose membrane. Mypt1 and phospho mypt1 were identified by western blotting using antibodies against mypt1(Santa Cruz, 1:500) and phospho mypt1(thr 696) antibodies (cell signal, 1:500), respectively, and the ratio of p-mypt1/total mypt1 was calculated. Representative western blot for mypt1 are shown in Panel A with the cumulative bar graph shown in Panel B. * Significant, N = 4, p<0.05.</p

Papaverine pretreatment for 2 hrs prior to organ culture attenuated intimal thickening in HSV.

<p>HSV were fixed at day 0 for preculture (basal) conditions and then at 14 days with either control treatment or pretreatment with 1 mM papaverine for 2 hrs. Tissue was fixed after 14 days of organ culture and the intimal thickness was measured. Representative staining shown in 5 x magnification are shown in Panel A, with 10 x magnification shown in Panel B. Black line indicates the intimal thickening. The cumulative quantification of intimal thickness is displayed in Panel C. * Significant compared to control, (N = 4), p<0.05 The area of the thickness was measured using Matlab software and the cumulative data is shown in Panel D. * Significant compared to control, N = 4, p<0.05.</p

Pre incubation with papaverine, blocks NE induced isometric force generation (blue, Panel A and B) and intracellular Ca²⁺ release (red, Panel A and B) in HSV.

<p>HSV was suspended on a Fluroplex apparatus and loaded with Fura-2AM for 4 hrs, then treated with either norepinephrine alone or papaverine (1mM) pretreatment followed by norepinephrine. Tissue treated with papaverine had significantly lower changes in fluorescence ratio (N = 3, * Significant compared to NE, p<0.05 Panel C).</p

Preincubation with papaverine inhibits norepinephrine (NE) induced contractions in human saphenous vein.

<p>Saphenous vein rings were suspended in a muscle bath and equilibrated in Kreb’s bicarbonate buffer for 2 h. Panel A: Representative force tracings of rings treated with 0.5 μM NE, 1mM papaverine (Pap), or 1 mM papaverine for 10 min followed by 0.5 μM NE. Panel B: Cumulative data obtained when the force was converted to stress and decrease in stress was converted to a percentage of the maximal initial KCl contraction which was set as 100%. Means ± SE, n = 6 P < 0.05. * Significant compared to NE. Panel C:</p

Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction

A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (<i>i.e.</i>, vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity <i>in vitro</i> as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein <i>ex vivo</i>. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides

Preincubation with forskolin reduces phosphorylation of cofilin in PCA.

<p>PCA rings were treated with the same conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060986#pone-0060986-g003" target="_blank">Figure 3</a>. Proteins were extracted and separated on SDS polyacrylamide gels and p-cofilin was detected by western blotting. Panel A: Representative western blot of p-cofilin and np-cofilin. Panel B: Cumulative data representing the relative amounts of p-cofilin over the total cofilin and then normalized to the basal. Quantitative values were obtained when the phosphorylated and non-phosphorylated bands were quantitated densitometrically. n = 4, p<0.05. *significant compared to all other conditions.</p

Dose dependent effect of forskolin on force, MLC phosphorylation, F-actin levels and HSP20 phosphorylation during suppression of force.

<p>PCA rings were suspended in a muscle bath and equilibrated in Kreb’s bicarbonate buffer for 2 h and rings were pretreated with either nothing, 1, 5, or 10 µM forskolin followed by 5 µM histamine. Force was measured and the tissue was frozen and analysed for MLC phosphorylation, F-actin levels, and HSP20 phosphorylation, Panel A: Cumulative data obtained when the force was converted to stress. Panel B: Cumulative data representing the relative amounts of the p-MLC over the total MLC obtained when the phosphorylated and non-phosphorylated bands were quantitated densitometrically. Panel C: F-actin concentration was calculated and cumulative data is plotted. Panel D: Cumulative data representing the relative amounts of p-HSP20 over total HSP20, obtained when the phosphorylated and non-phosphorylated bands were quantitated densitometrically.</p

Preincubation with forskolin suppressed histamine-induced force in porcine coronary artery.

<p>PCA rings were suspended in a muscle bath and equilibrated in Kreb’s bicarbonate buffer for 2 h. Panel A–D: Representative force and intracellular calcium tracings of rings pretreated with either nothing, 1, 5, or 10 µM forskolin followed by 5 µM histamine. Panel E: Comparison of concurrent change in flourescent ratio (340/380 nm) to Stress (N/m²) from histamine-induced contraction with various doses of forskolin added as a pretreatment. Panel F: Reversibility of Forskolin-mediated force suppression. Contraction to 5 µM histamine was assesed after washing off the 5 µM forskolin. The sample was rechallanged with histamine every 10 min for the first 50 min to determine recovery of contraction. Samples were also challenged after two hours of washing which resulted in the full recovery of histamine induced contraction.</p

Preincubation with forskolin abolish histamine induced paxillin phosphorylation in PCA.

<p>Conditions are the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060986#pone-0060986-g003" target="_blank">Figure 3</a>. Proteins were extracted and separated on SDS polyacrylamide gels and p-paxillin and np- paxillin were detected by western blotting. Panel A: Representative western blot of p-paxillin and np-paxillin. Panel B: Cumulative data representing the relative amounts of p-paxillin over total paxillin and normalized to the basal. Quantitative values were obtained when the phosphorylated and non-phosphorylated paxillin bands were quantitated densitometrically n = 4. p<0.05. *significant compared to all other conditions.</p

Preincubation with forskolin induces phosphorylation of VASP.

<p>Conditions are the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060986#pone-0060986-g003" target="_blank">Figure 3</a>. Proteins were extracted and separated on SDS polyacrylamide gels. Phosphorylation of VASP causes mobility shift in the SDS gel and p- and np- VASP forms were detected by western blotting using VASP antibody that recognizes both forms. Panel A: Representative western blot of p- and np-VASP. Panel B: Cumulative data representing the relative amounts of p-VASP/total VASP and then normalized to the basal. Quantitative values were obtained when the phosphorylated and non-phosphorylated bands were quantitated densitometrically. n = 4, p<0.05. *significant compared to histamine.</p