SUMMARY
This study involved 87 male albino rats weighing (180-300g). The animals were housed under standard laboratory conditions (12 h light: 12 h dark controlled photoperiod) at 22 ± 4 Cº, and the animals were supplied standard rat chaw and tap water ad libitum.
The current study focused on the in vitro physiological roles of potassium channels in rat thoracic aorta responses to human urotensin-II (hU-II) in intact and in endothelial impairment induced by mercury chloride (HgCl2). The study included two parallel sets of experiment; intact endothelia groups and endothelial dysfunction (ED) groups.
Each group included incubating aortic segments with one of the following blockers or inhibitors; tetraethylammonium (TEA); a non-selective calcium-activated potassium channel blocker, charybdotoxin (chtx); big and intermediate conductance calcium-activated potassium channels (BKca and IKca) blocker, barium chloride (BaCl2); delay inward rectifier potassium channels blocker, clotrimazole; an intermediate conductance calcium-activated potassium channels (IKca) blocker, glibenclamide; adenosine triphosphates-sensitive potassium channels (KATP) blocker, 4-aminopyridines (4-AP); voltage sensitive potassium channels (KV) blocker.
Other signaling pathways have also been investigated in this experiment that included; indomethacin; a non-selective cyclooxygenase (COX) inhibitor, nifedipine; L-type calcium channels blocker, DL-propargylglycine (PAG); a selective cystathionine-γ-lyase inhibitor, methylene blue (MB); a non-selective soluble guanylate cyclase (GC) inhibitor, L-nitro-arginine methyl ester (L-NAME); an irreversible endothelial nitric oxide synthase (eNOS) inhibitor, melatonin; a potent antioxidant, SB-710411; a selective U-II receptor antagonist, BQ-123; a selective endothelin-1 (ET-1) receptor type-A (ETA) blocker or captopril; angiotensin-converting enzyme (ACE) inhibitor, then cumulative doses of hU-II were applied to each group.
In another set of the experiment, a single dose of half effective concentration (EC50) of HgCl2 was applied to aortic rings pre-incubated with SB-710411, BQ-123, captopril or indomethacin. The present study also included an in vivo investigating the effect of continuous hU-II (a bolus dose of hU-II 200 nM/kg then followed by a continuous infusion of hU-II 50 nM/kg B.W) intravenous (i.v) infusion in anesthetized rats (17 rats) on glomerular filtration rate (GFR) and renal potassium and sodium handling and on mean arterial pressure (MAP). These parameters have also been measured in rats treated with HgCl2.
In the in vitro study, ED was induced by incubation of aortic rings with 1µmol HgCl2 for 40 min prior to run any experimental group. While in in vivo study, ED was induced in conscious rat by daily i.m. injection of 70 ng/kg/day HgCl2 for 50 days.
The statistical analysis of the current study, for the first time, revealed that endothelial impairment induced by HgCl2, which was at least 66 times less than the recommended levels, in rat thoracic aorta remarkably accentuated the vascular responses to hU-II. Another important finding in this study was that potassium channels blockers noticeably changes hU-II efficacy with relatively no changes in the peptide potency with an exception in glibenclamide that high significantly abolished both potency and maximum response to hU-II. However, in the presence of HgCl2, both 4-AP and BaCl2 exhibited revers vascular responses to hU-II in compare with the endothelial intact groups. It is worth noting that HgCl2 did not alter the peptide potency in these experimental groups but significantly deviated the maximum responses indicating that hU-II potency still effective even in such abnormal circumstances.
The maximum responses to hU-II were not only affected by potassium channel blockers, but also by nitric oxide (NO) pathway inhibitors including L-NAME and MB. It is worthy of note to indicate that many other signaling pathways have also seen to modulate the vascular responses to hU-II in the presence of HgCl2. Melatonin, indomethacin and PAG significantly abolished maximum responses to hU-II with no significant changes in the potency in ED group.
Furthermore, SB-710411, captopril and BQ-123 potentially deviated the vascular responses to hU-II, while in the presence of HgCl2 , only captopril significantly abolished the peptide efficacy.
In the current study, we have experimentally proved that a single dose of 3 µmol of HgCl2 significantly contracted the isolated vessels and the possible roles of some vasoactive agents were investigated as well. Each of captopril, BQ-123, SB-710411 or indomethacin significantly reduced the maximum responses to HgCl2 in time depended manner.
In the in vivo experiment of this study, hU-II infusion for one hour significantly reduced GFR and noticeably modulated the renal handling for potassium and sodium. On the other hand, whether long term HgCl2 administration could alter the renal handling for electrolytes and MAP, the results showed that HgCl2 resulted in increased MAP and altered renal potassium and sodium clearance and fractional excretion.
The present study, for the first time, highlighted that endothelial dysfunction potentiated the vascular responses to hU-II and modulated potassium channels contributions to vascular actions of hU-II. The present study also uncovered that angiotensin-II (Ang-II), ET-1, NO, melatonin, H2S and COX pathways might synergistically share the peptide vasomotor actions. These findings provide some pharmacological tools for modulating the peptide behavior, in particular, under pathological conditions.