Effects Of 5-(N, N-dimethyl) Amiloride On Vascular Tone Of Isolated Thoracic Aortic Rings In Rats

Objective: Although the exciting effects of 5-[N, N-dimethyl] amiloride (DMA) have been studied in rat isolated aorta cardiac, its pharmacological properties in vascular beds and underlying mechanism(s) are still not well clarified. The present study was designed to observe the effects of DMA, an NHE inhibitor, on the contractions in rat isolated aorta and to get an insight into its mechanism.Methods: The rats were sacrificed by luxation and the thoracic aorta was quickly removed from each animal and placed in physiological salt solution (PSS). The aorta was carefully cleaned of adherent fat and connective tissue. The vessel was then cut into rings of about 4mm length. Two stainless-steel wires were passed through the lumen of each ring. One stirrup was connected to an isometric force transducer to measure tension in the vessels. The rings were placed in a 10ml organ chamber containing PSS. The rings were stretched until they exerted an optimal basal tension of 2g, and then were allowed to equilibrate for 1 hour with the bath fluid being changed every 15 minutes. The solution was bubbled with 100% O2. Rings of the isolated rat thoracic aorta were mounted in organ baths and isometric contractile force was measured. Isotonic tension of thoracic aortic rings precontracted by potassium chloride (KCl, 30mmol/L) or norepinephrine (NE, 1×10-6mol/L) was recorded. The response action of DMA and effect of various drugs were observed in the rings.Results: (1) DMA did not significantly affect the resting tone of rat thoracic aortic rings. (2) DMA concentration-dependently contracted the thoracic aortic rings precontracted by 30mmol/L potassium chloride; the maximal contractions by 1×10-6 mol/LDMA was (14.46±5.27)% and by 3×10-5mol/L DMA was (30.49±4.11) % respectively. EC50=(5.38±1.68)μmol/L。(3) DMA (1×10-7mol/L~3×10-5mol/L) produced concentration-dependent relaxations of endothelium-intact (endothelium+) or denude endothelium(endothelium-) rat isolated aortic rings precontracted by 1μM norepinephrine; the maximal relaxation was (88.96±5.05)% and (43.54±8.22)% respectively, EC50=(1.82±0.89)μmol/L. (4) There was significant difference between the rings intact and denude endothelium (P<0.05). (5) During the thoracic aortic rings placed in PSS solution eliminating CaCl2 the effect of CaCl2 contracted the thoracic aortic rings was enhanced and the concentration-response curve shifted to the leftward when pretreatmente with DMA (3×10-5 mol/L) compared without DMA. (6) DMA did not significantly affect the tone of rat thoracic aortic rings pretreatment with 30mmol/L potassium chloride without CaCl2. (7) The contraction was prevented by pretreatment with the NCX inhibitors KB-R7943(1×10-6 mol/L), nicardipine (1×10-9 mol/L L-calcium channel blocker), and digitalis (1×10-6 mol/L a Na+-K+ pump inhibitor). (8) The relaxation was not affected by 1×10-6 mol/L digitalis (a Na+-K+-ATP synthetase inhibitor), 1×10-11 mol/L nicardipine (a L-type calcium channel inhibitor), but was nearly depressed by 1×10-4 mol/L NG-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthetase inhibitor), 1×10-7 mol/L KB-R7943 (a Na+/Ca2+exchanger inhibitor) and 1×10-5 mol/L indomethacin (a cyclo-oxygenase inhibitor).Conclusions: (1) DMA did not significantly affect the resting tone of rat thoracic aortic rings. (2) DMA contracted the thoracic aortic rings precontracted by 30mmol/L potassium chloride in a concentration-dependent manner; the effect was not related to the intracellular Ca2+; DMA augmented the contraction of thoracic aortic rings induced by Ca2+ releasing from Ca2+ stores and influx from extracellular, respectively; Further more, it is suggested that sarcolemmal L-type Ca2+ channels, Na+-K+ pump and Na+/Ca2+ exchanger may be involved in the mechanisms via which DMA concentration-dependently contracted the thoracic aortic rings precontracted by 30mmol/L potassium chloride. (3) DMA produces a concentration-dependent vasorelaxation in endothelium-intact (endothelium+) or denude endothelium (endothelium-) rat isolated aortic rings precontracted by norepinephrine, and the vasorelaxation was relevant to the production of NO and prostacyclin, was relevant to arcolemmal Na+/Ca2+ exchanger, but was not relevant to L-type Ca2+ channels and Na+-K+ pump.