Chemicals Buffer reagents AP collagenase


Chemicals Buffer reagents AP collagenase

Postby admin » Mon Jul 17, 2017 7:00 am

Buffer reagents, 4-AP, collagenase (Type F and H), dithioerythritol, KT 5720, KT 5823 and papain were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All drugs and reagents were dissolved in distilled water unless otherwise noted. KMUP-1 was dissolved in 10% absolute alcohol, 10% propylene glycol, and 2% 1N HCl at 10mM. Serial dilutions were made in phosphate buffer solution, with the final solvent concentration ≤ 0.01%.
Data analysis and statistics
Data are expressed as the mean ± standard error and n indicates the number of cells. Repeated measure analysis of variances compared values at a given voltage. When appropriate, a Tukey–Kramer pairwise comparison was used for post hoc analysis. All p values ≤ 0.05 were considered statistically significant.
KMUP-1 prevents UTP-mediated depolarization and cytosolic Ca2+ elevation
Figure 1
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UTP downregulation of KDR currents
Using conventional whole cell patch-clamp and pipette solutions that minimize large-conductance Ca2+ activated K+ (BKCa) channel activity, the KDR current was isolated in rat PASMCs. Positive voltage steps to –30 mV activated KDR without an induction of inactivation (Figure 2A). KDR currents in pulmonary artery myocytes are comprised of two slowly inactivating components that vary in their sensitivity to 4-AP (5mM). Perfusion with 4-AP significantly inhibited this current (39.5 ± 4.3%, n = 6) at +40 mV. 4-AP partially suppressed the KDR current, thus exhibiting both 4-AP-sensitive and 4-AP-insensitive components as previously described in cerebral smooth muscle AUY922 [9]. Like 4-AP, superfused UTP (30μM) also had a significant effect on the KDR current (41.3 ± 5.2%, n = 6) at +40 mV, with peak inhibition occurring at 20 minutes. Interestingly, in the presence of 4-AP (5mM), UTP had no effect on the remaining KDR current (Figure 2B), suggesting that UTP modulates the 4-AP sensitive component of the KDR current.
Figure 2
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Figure 2. Uridine 5′-triphosphate (UTP) inhibits the 4-aminopyridine (4-AP)-sensitive component of the KDR current. (A) Representative KDR current recordings before and after the addition of UTP (30μM). (B) Net I-V relationship under control conditions and in the presence of UTP or UTP + 4-AP (5mM) (n = 6). KDR = delayed rectifying K+ (KDR). * Significant difference from control.
Y27632 and KMUP-1 prevent UTP inhibition of KDR currents
Previous observations showed that the UTP-sensitive P2Y receptor can activate the RhoA/ROCK pathway [9]. Subsequent experiments examined whether this signaling protein enables UTP to suppress the 4-AP-sensitive component of the KDR current. As shown in Figure 3, application of the ROCK inhibitor Y27632 (30μM) [9] showed no significant effects on the KDR current in comparison with the control. Y27632 had a concentration-dependent manner to prevent UTP-suppressed KDR current (data not shown), but it abolished the activity of UTP at ≥ 30μM. The restoration of UTP inhibited KDR current by 30μM Y27632 is shown in Figure 3. Similar to the ROCK inhibitor experiments, KMUP-1 (30μM) alone showed no significant effects on the KDR current, but it abolished the response of UTP-suppressed KDR current under this concentration. The concentration-effect relationship of KMUP-1 on UTP is not presented in Figure 4. To study the additive effect of KMUP-1 and Y27632 on UTP-inhibited KDR currents, applied KMUP-1 + Y27632 to UTP perfusate showed effect similar to that of KMUP-1 abolished UTP only, i.e. no additive effect by combining both agents. Therefore, we suggest that KMUP-1, like Y27632, modulates the ROCK signaling resulting in prevention of UTP effects as well (Figure 4).
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