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

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 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