Vasorelaxing Activity of Ulmus davidiana Ethanol Extracts in Rats: Activation of Endothelial Nitric Oxide Synthase

Ulmus davidiana var. japonica Rehder (Urticales: Ulmaceae) (UD) is a tree widespread in northeast Asia. It is traditionally used for anticancer and anti-inflammatory therapy. The present study investigated the effect of an ethanol extract of UD on vascular tension and its underlying mechanism in rats. The dried root bark of UD was ground and extracted with 80% ethanol. The prepared UD extract was used in further analysis. The effect of UD on the cell viability, vasoreactivity and hemodynamics were investigated using propidium iodide staining in cultured cells, isometric tension recording and blood pressure analysis, respectively. Low dose of UD (10~100microg/ml) did not affect endothelial cell viability, but high dose of UD reduced cell viability. UD induced vasorelaxation in the range of 0.1~10microg/ml with an ED50 value of 2microg/ml. UD-induced vasorelaxation was completely abolished by removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. UD inhibited calcium influx induced by phenylephrine and high K+ and also completely abolished the effect of L-NAME. Intravenous injection of UD extracts (10~100 mg/kg) decreased arterial and ventricular pressure in a dose-dependent manner. Moreover, UD extracts reduced the ventricular contractility (+dP/dt) in anesthetized rats. However, UD-induced hypotensive actions were minimized in L-NAME-treated rats. Taken together, out results showed that UD induced vasorelaxation and has antihypertensive properties, which may be due the activation of nitric oxide synthase in endothelium.

Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase

The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

Impaired endothelium-dependent relaxation is mediated by reduced production of nitric oxide in the streptozotocin-induced diabetic rats

To evaluate the involvement of nitric oxide production on the endothelium-dependent relaxation in diabetes, we have measured vascular and endothelial function and nitric oxide concentration, and the expression level of endothelial nitric oxide synthase in the streptozotocin-induced diabetic rats. Diabetic rats were induced by the injection of streptozotocin (50 mg/kg i.v.) in the Sprague-Dawley rats. Vasoconstrictor responses to nonrepinephrine (NE) showed that maximal contraction to norepinephrine (10(-5) M) was significantly enhanced in the aorta of diabetic rats. Endothelium-dependent relaxation induced by acetylcholine was markedly impaired in the aorta of diabetic rats, these responses were little improved by the pretreatment with indomethacin. However, endothelium-independent relaxation induced by nitroprusside was not altered in the diabetic rats. Plasma nitrite and nitrate (NO2/3) levels in diabetic rats were significantly lower than innon-diabetic rats. Western blot analysis using a monoclonal antibody against endothelial cell nitric oxide synthase (eNOS) revealed that the protein level was lower in the aorta of diabetic rats than in non-diabetic rats. These data indicate that nitric oxide formation and eNOS expression is reduced in diabetes, and this would, in part, account for the impaired endothelium-dependent relaxation in the aorta of streptozotocin-induced diabetic rats.

Alteration of 4-aminopyridine-sensitive, voltage-dependent K+-channel in arterial smooth muscle cells of one-kidney, one-clip Goldblatt hypertensive rats

Using the patch-clamp technique, we investigated the alteration of 4-aminopyridine(4-AP)-sensitive, voltage-dependent K+ channel (KV) in the mesenteric arterial smooth muscle cell (MASMC) of renovascular hypertensive model, one-kidney one-clip Goldblatt hypertensive rat (GBH). To isolate KV current, internal pipette solution contained 5 mM ATP and 10 mM EGTA. Under these condition, MASMC was depolarized by 4-AP, but charybdotoxin did not affect membrane potential. Membrane potential of hypertensive cell (- 40.3 +/- 3.2 mV) was reduced when compared to that of normotensive cell (-59.5 +/- 2.8 mV). Outward K+ current of hypertensive cell was significantly reduced when compared to normotensive cell. At 60 mV, the outward currents were 19.10 +/- 1.91 and 14.06 +/- 1.05 pA/pF in normotensive cell and hypertensive cell respectively. 4-AP-sensitive K+ current was also smaller in hypertensive cell (4.28 +/- 0.38 pA/pF) than in normotensive cell (7.65 +/- 0.52 pA/pF). The values of half activation voltage (V1/2) and slope factor (k1) as well as the values of half inactivation voltage (V1/2) and slope factor (k1) were virtually similar between GBH and NTR. These results suggest that the decrease of 4-AP-sensitive K+ current contributes to a depolarization of membrane potential, which leads to development of vascular tone in GBH.

Decreased voltage dependent K+ currents in cerebral arterial smooth muscle cells of one-kidney, one-clip Goldblatt hypertensive rat

The Kv channel activity in vascular smooth muscle cell plays an important role in the regulation of membrane potential and blood vessel tone. It was postulated that increased blood vessel tone in hypertension was associated with alteration of Kv channel and membrane potential. Therefore, using whole cell mode of patch-clamp technique, the membrane potential and the 4-AP-sensitive Kv current in cerebral arterial smooth muscle cells were compared between normotensive rat and one-kidney, one-clip Goldblatt hypertensive rat (1K,1C-GBH rat). Cell capacitance of hypertensive rat was similar to that of normotensive rat. Cell capacitance of normotensive rat and 1K,1C-GBH rat were 20.8+/-2.3 and 19.5+/-1.4 pF, respectively. The resting membrane potentials measured in current clamp mode from normotensive rat and 1K,1C-GBH rat were -45.9+/-1.7 and -38.5+/-1.6 mV, respectively. 4-AP (5 mM) caused the resting membrane potential hypopolarize but charybdotoxin (0.1 muM) did not cause any change of membrane potential. Component of 4-AP-sensitive Kv current was smaller in 1K,1C-GBH rat than in normotensive rat. The voltage dependence of steady-state activation and inactivation of Kv channel determined by using double-pulse protocol showed no significant difference. These results suggest that 4-AP-sensitive Kv channels play a major role in the regulation of membrane potential in cerebral arterial smooth muscle cells and alterations of 4-AP-sensitive Kv channels would contribute to hypopolarization of membrane potential in 1K,1C-GBH rat.

Mechanism of membrane hyperpolarization by extracellular K+ in resistance-sized cerebral arterial muscle cell of rabbit

We sought to find out the mechanism of vascular relaxation by extracellular K+ concentration ((K+)o) in the cerebral resistant arteriole from rabbit. Single cells were isolated from the cerebral resistant arteriole, and using voltage-clamp technique barium-sensitive K+ currents were recorded, and their characteristics were observed. Afterwards, the changes in membrane potential and currents through the membrane caused by the change in (K+)o was observed. In the smooth muscle cells of cerebral resistant arteriole, ion currents that are blocked by barium, 4-aminopyridine (4-AP), and tetraethylammonium (TEA) exist. Currents that were blocked by barium showed inward rectification. When the (K+)o were 6, 20, 60, and 140 mM, the reversal potentials were -82.7+/-1.0, -49.5+/-1.86, -26+/-1.14, -5.18+/-1.17 mV, respectively, and these values were almost identical to the calculated K+ equilibrium potential. The inhibition of barium-sensitive inward currents by barium depended on the membrane potential. At the membrane potentials of -140, -100, and -60 mV, Kd values were 0.44, 1.19, and 4.82 muM, respectively. When (K+)o was elevated from 6 mM to 15 mM, membrane potential hyperpolarized to -50 mV from -40 mV. Hyperpolarization by K+ was inhibited by barium but not by ouabain. When the membrane potential was held at resting membrane potential and the (K+)o was elevated from 6 mM to 15 mM, outward currents increased; when elevated to 25 mM, inward currents increased. Fixing the membrane potential at resting membrane potential and comparing the barium-sensitive outward currents at (K+)o of 6 and 15 mM showed that the barium-sensitive outward current increased at 15 mM K+. From the above results the following were concluded. Barium-sensitive K+ channel activity increased when (K+)o is elevated and this leads to an increase in K+ -outward current. Consequently, the membrane potential hyperpolarizes, leading to the relaxation of resistant arteries, and this is thought to contribute to an increase in the local blood flow of brain.

Effect of pH on the ATP-sensitive K+ channel in aortic smooth muscle cells from rats

The effects of pH on K+ currents were investigated in single smooth muscle cells isolated from the thoracic aorta of Wistar-Kyoto rats. Whole-cell K+ currents were recorded in the conventional configuration of the voltage-clamp technique. Pinacidil (10muM) activated the whole-cell current and the pinacidil-activated current was completely inhibited by glibenclamide (10muM), an inhibitor of ATP-sensitive K+ channel (KATP channel). Pinacidil-activated current was reversed at near the K+ equilibrium potential. This current was time- and voltage-independent and reduced by elevating intracellular ATP. Pinacidil-activated current was reduced by lowering the external pH. However, alteration of internal pH has controversial effects on pinacidil-activated current. When the single cell was dialyzed with 0.1 mM ATP, alteration of internal pH had no effect on pinacidil-activated K+ current. In the contrast, when the single cell was dialyzed with 3 mM ATP, pinacidil-activated current was increased by lowering internal pH. Our results suggest that K+ channel activated by pinacidil may be KATP channel and internal H+ may reduce the inhibitory effect of ATP on KATP channel.