A xanthine-based KMUP-1 with cyclic GMP enhancing and K(+) channels opening activities in rat aortic smooth muscle

Abstract

1. KMUP-1 (1, 3, 5 mg kg(-1), i.v.), a xanthine derivative, produced dose-dependent sustained hypotensive and short-acting bradycardiac effects in anaesthetized rats. This hypotensive effect was inhibited by pretreatment with glibenclamide (5 mg kg(-1), i.v.). 2. In endothelium-intact or denuded aortic rings preconstricted with phenylephrine, KMUP-1 caused a concentration-dependent relaxation. This relaxation was reduced by endothelium removal, the presence of NOS inhibitor L-NAME (100 microM) and sGC inhibitors methylene blue (10 microM) and ODQ (1 microM). 3. The vasorelaxant effects of KMUP-1 was attenuated by pretreatment with various K(+) channel blockers TEA (10 mM), glibenclamide (1 microM), 4-AP (100 microM), apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). 4. Increased extracellular potassium levels (30 - 80 mM) caused a concentration-related reduction of KMUP-1-induced vasorelaxations. Preincubation with KMUP-1 (1, 10, 100 nM) increased the ACh-induced maximal vasorelaxations mediated by endogenous NO release, and enhanced the potency of exogenous NO-donor SNP. 5. The vasorelaxant responses of KMUP-1 (0.01, 0.05, 0.1 microM) together with a PDE inhibitor IBMX (0.5 microM) had an additive action. Additionally, KMUP-1 (100 microM) affected cyclic GMP metabolism since it inhibited the activity of PDE in human platelets. 6. KMUP-1 induced a dose-related increase in intracellular cyclic GMP levels in rat A10 vascular smooth muscle (VSM) cells, but not cyclic AMP. The increase in cyclic GMP content of KMUP-1 (0.1 - 100 microM) was almost completely abolished in the presence of methylene blue (10 microM), ODQ (10 microM), and L-NAME (100 microM). 7. In conclusion, these results indicate that KMUP-1 possesses the following merits: (1) stimulation of NO/sGC/cyclic GMP pathway and subsequent elevation of cyclic GMP, (2) K(+) channels opening, and (3) inhibition of PDE or cyclic GMP breakdown. Increased cyclic GMP display a prominent role in KMUP-1-induced VSM relaxations.

Figure 1

Chemical structure of KMUP-1.

Figure 2

Effects of KMUP-1 (1, 3, 5 mg kg⁻¹) on mean arterial blood pressure (MAP) and heart rate (HR) in Wistar rats, anaesthetized with pentobarbital. 10% Propylene glycol plus 10% ethanol mixed with 2% HCl was used for KMUP-1 as control. Vertical bars represent s.e.mean change from the baseline value, which was 115±10 mmHg and 370±21 b.p.m. for MAP and HR, respectively. Each point represents the mean±s.e.mean of eight experiments. ^*Indicates P<0.05, Two way repeated measures ANOVA followed by Student-Newman-Keuls test.

Figure 3

Effects of intravenous injection of KMUP-1 and levcromakalim induced hypotensive responses before and after i.v. injection of glibenclamide (5 mg kg⁻¹) in anaesthetized rats. Each value represents the mean±s.e.mean of 6 – 8 experiments. ^*Indicates P<0.05, Paired Student's t-test.

Figure 4

Vasorelaxations induced by cumulative concentrations of KMUP-1 in endothelium-intact and endothelium-denuded rat aortic rings preconstricted with phenylephrine (1 μM). KMUP-1 induced relaxant responses were significantly different in endothelium-intact and endothelium-denuded aortic rings at the indicated KMUP-1 concentrations. Each point represents the mean±s.e.mean of 8 – 10 experiments. ^*Indicates P<0.05, Two way repeated measures ANOVA followed by Student-Newman-Keuls test.

Figure 5

Effects of KMUP-1 on vasorelaxations induced by cumulative concentrations of ACh in endothelium-intact (E⁺) or SNP in endothelium-denuded (E⁻) rat aortic rings preconstricted with phenylephrine (10 μM, left panel) and KCl (30 mM, right panel). Each point represents the mean±s.e.mean of 6 – 8 experiments. ^*Indicates P<0.05, ANOVA followed by Student-Newman-Keuls test.

Figure 6

The vasorelaxant effects of KMUP-1 and levcromakalim on rat thoracic aortic rings precontracted with different concentrations of KCl (30 – 80 mM). Each point represents the mean±s.e.mean of eight experiments.

Figure 7

Effects of KMUP-1 and levcromakalim on rat aortic rings precontracted by phenylephrine in the absence and presence of various K⁺ channel blockers. Each point represents the mean±s.e.mean of 6 – 8 experiments. ^*Indicates P<0.05, two way repeated measures ANOVA followed by Student-Newman-Keuls test.

Figure 8

The vasorelaxant effects of KMUP-1 on rat aortic rings precontracted by phenylephrine in the absence and presence of L-NAME (100 μM), methylene blue (10 μM) and ODQ (1 μM). Each value represents the mean±s.e.mean of eight experiments. ^*Indicates P<0.05, ^**indicates P<0.01, paired Student's t-test.

Figure 9

Additive effects of KMUP-1 and IBMX on rat aortic rings precontracted by phenylephrine. Each value represents the mean±s.e.mean of eight experiments. ^*Indicates P<0.05 as compared with the control value, ANOVA followed by Dunnett's test.

Figure 10

Effects of sodium nitroprusside, sildenafil and KMUP-1 on cyclic GMP levels in rat A10 vascular smooth muscle cells. Each value represents the mean±s.e.mean of three independent triplicate experiments. ^*Indicates P<0.05, ^**indicates P<0.01 as compared with the control value, ANOVA followed by Dunnett's test. CTL: solvent control (10% alcohol+10% propylene alcohol+2% HCl).

Figure 11

Effects of KMUP-1 on cyclic GMP levels in rat A10 vascular smooth muscle cells in the absence and presence of L-NAME (100 μM), ODQ (10 μM) and methylene blue (10 μM). The amount of basal release of cyclic GMP was 1750±154 fmol 10⁵ cells⁻¹. Each value represents the mean±s.e.mean of three independent triplicate experiments. ^*Indicates P<0.05, ^**indicates P<0.01, ANOVA followed by Bonferroni t-test.

Figure 12

Effects of KMUP-1 and isoprenaline on cyclic AMP levels in rat A10 vascular smooth muscle cells. Each value represents the mean±s.e.mean of three independent triplicate experiments. ^*Indicates P<0.05, ^**indicates P<0.01 as compared with the control value, ANOVA followed by Dunnett's test. CTL: solvent control (10% alcohol+10% propylene alcohol+2% HCl).

Figure 13

Proposed mechanism of action of KMUP-1 on the intracellular cyclic GMP synthesis and metabolism, K⁺ channels opening and phosphodiesterase inhibition.