Actions of 4-chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth muscle and endothelial cells

1. Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca²⁺, apparently through ryanodine-sensitive Ca²⁺ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca²⁺, by use of Ca²⁺ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells.
2. After phenylephrine-(PE, 10 μM) sensitive Ca²⁺ stores were depleted by maximal PE stimulation in Ca²⁺-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca²⁺ was restored for a 30 min refilling period. At the end of this period, Ca²⁺ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca²⁺ store.
3. In a concentration-dependent manner (30, 100 and 300 μM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca²⁺-free media. This suggests that Ca²⁺ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca²⁺-containing or Ca²⁺-free Krebs solution.
4. Restoring Ca²⁺ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca²⁺. The contraction of tissues pretreated with 300 μM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 μM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca²⁺-free and Ca²⁺-containing medium suggesting a rapid reversal of CEP effects.
5. Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K⁺ in the absence of and after 30 min pre-incubation with 30, 100 and 300 μM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K⁺ (77.6, 41.1 and 10.8% of control).
6. Some arterial rings were pre-incubated with ryanodine (30 μM) for 30 min before the construction of PE concentration-response curves. In Ca²⁺-free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9±1.2% of 100 mM K⁺ contraction, n=11) in the presence of external Ca²⁺. EC₅₀ values for PE in ryanodine-treated tissues (1.7±0.25 μM, n=16) were not significantly different from controls (2.5±0.41 μM, n=22). Maximum contractions to PE (118.5±4.4% of 100 mM K⁺ contraction, n=16) were also unaffected by ryanodine when compared to controls (129±4.2%, n=23).
7. When fura-2 loaded smooth muscle cells (n=13) and endothelial cells (n=27) were imaged for Ca²⁺ distribution, it was observed that 100 and 300 μM CEP in Ca²⁺-free medium caused Ca²⁺ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 mM) reversed the effect of CEP on intracellular Ca²⁺ to control values.
8. These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca²⁺ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5-HT and K⁺. The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca²⁺ elevation.

     

Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery

1. The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves.
2. Vasopressin (10⁻¹⁰–10⁻⁷ M) caused concentration-dependent contractions (pD₂=8.36±0.09). The V₁-receptor antagonist d(CH₂)₅Tyr(Me)AVP (10⁻⁹–10⁻⁸ M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA₂ value of 9.83 with a slope of 1.10±0.14.
3. Vasopressin (3×10 ⁻¹⁰ and 10⁻⁹ M) caused concentration-dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration-response curve for noradrenaline. The V₁-receptor antagonist induced concentration-dependent inhibitions of potentiation induced by vasopressin. The selective V₁-receptor agonist [Phe^*, Orn⁸]-vasotocin (3×10 ⁻¹⁰ and 10⁻⁹ M) induced potentiation of electrical stimulation-evoked responses which was also inhibited in the presence of the V₁ antagonist (10⁻⁸ M). In contrast, the V₂-receptor agonist deamino-8-D-arginine vasopressin (desmopressin 10⁻⁸–10⁻⁷ M) did not modify the electrical stimulation-induced responses and the V₂-receptor antagonist [d(CH₂)₅, D-Ile^*, Ile⁴, Arg⁸]-vasopressin (10⁻⁸–10⁻⁷ M) did not affect the potentiation evoked by vasopressin.
4. In artery rings contracted by 10⁻⁶ M noradrenaline in the presence of 10⁻⁶ M guanethidine and 10⁻⁶ M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency-dependent relaxations which were unaffected by 10⁻⁹ M vasopressin but abolished by 10⁻⁶ M tetrodotoxin.
5. Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl₂ to KCl depolarized vessels. The augmenting effects were inhibited by the V₁ antagonist.
6. In the presence of the calcium antagonist nifedipine (10⁻⁶ M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl.
7. The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V₁ receptor stimulation which brings about an increase in calcium entry through dihydropyridine-sensitive calcium channels.

     

Characterization of α1-adrenoceptor subtypes mediating contractions to phenylephrine in rat thoracic aorta,mesenteric artery and pulmonary artery

1. The subtype of α₁-adrenoceptor mediating contractions to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery were investigated by use of antagonists which show selectivity between the cloned α₁-adrenoceptor subtypes in binding studies.
2. Cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat thoracic aorta by prazosin (pA₂ 9.9), WB4101 (pA₂ 9.6), 5-methylurapidil (pA₂ 8.1), benoxathian (pA₂ 9.2) and indoramin (pA₂ 7.4). These compounds were also competitive antagonists in the mesenteric and pulmonary arteries (except for 5-methylurapidil in the pulmonary artery), (prazosin pA₂ 9.9 and 9.7; WB4101 pA₂ 9.8 and 9.6; 5-methylurapidil pA₂ 7.9 and pK_B estimate 8.0; benoxathian pA₂ 8.8 and 9.3; indoramin pA₂ 7.2 and 7.5, respectively).
3. RS 17053 was not a competitive antagonist in any blood vessel as Schild plot slopes were greater than unity. The pK_B estimates for RS 17053 were 7.1 in aorta, 7.0 in the mesenteric artery and 7.7 in the pulmonary artery.
4. The α_1D-subtype selective antagonist BMY 7378 appeared to be non-competitive with shallow Schild plot slopes. The data were better fitted with two lines in all tissues, with Schild plot slopes that were no longer different from unity, except in the pulmonary artery. The higher affinity site for BMY 7378 in the aorta had a pA₂ of 9.0, while it was 8.8 and 8.9 in the mesenteric and pulmonary arteries, respectively.
5. MDL73005EF acted in a non-competitive manner in all three blood vessels, with shallow Schild plot slopes. The pK_B estimates for MDL73005EF were 8.4 in aorta, 7.5 in the mesenteric artery and 8.0 in the pulmonary artery.
6. In all three blood vessels the functionally determined antagonist affinity estimates correlated best with published pK_i values for their displacement of [³H]-prazosin binding on membranes expressing cloned α_1d-adrenoceptors compared with α_1a- or α_1b-adrenoceptors. The antagonist affinity estimates in the aorta, mesenteric and pulmonary arteries correlated highly with their previously published pA₂ values in rat aorta (α_1D) and less well with those for α_1A- and α_1B-adrenoceptors mediating contraction of the rat epididymal vas deferens and rat spleen, respectively.
7. The results of this study suggest that the contraction to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery are mediated in part via the α_1D-subtype of adrenoceptor. The data for both BMY 7378 and MDL73005EF in all three blood vessels are consistent with receptor heterogeneity. However, the identity of the second site is unclear.

     

Laparoscopic end-to-end aortobifemoral bypass with reimplantation of the inferior mesenteric artery

Background: Colic ischemia is a serious complication that can occur after abdominal aortic surgery. It has been described in two patients after laparoscopic aortic surgery. The goal of the current experiment was to determine the feasibility of inferior mesenteric artery (IMA) reimplantation during laparoscopic aortobifemoral bypass (LAFB). Methods: Six piglets were submitted to the laparoscopic approach according to the ``apron' technique previously described. The infrarenal aorta was clamped and an LAFB was performed using a dacron graft. The IMA was reimplanted in the body of the graft with a running 5-0 polypropylene suture. Results: Mean operation and dissection times were 282.5 min (range, 270–310 min) and 123 min (range, 110–140 min), respectively, with a mean blood loss of 108 ml (range, 80–150 ml). Aortic clamping and anastomotic times were 123 min (range, 110–135 min) and 33 min (range, 24–45 min), respectively. The IMA reimplantation took 55 min (range, 45–70 min). At autopsy, all anastomoses were patent with no stenosis nor leak. Conclusion: Laparoscopic IMA reimplantation during laparoscopic aortobifemoral bypass is feasible. Received: 10 July 1998/Accepted: 15 November 1998     

Mesenteric cystic neoformations: Report of two cases

(Received for publication on Apr. 28, 1997; accepted on May 15, 1998)     

Analysis of the action of idazoxan calls into question the reliability of the rat isolated small mesenteric artery assay as a predictor for α1-adrenoceptor-mediated pressor activity

We have studied the effects of idazoxan in rat aorta and small mesenteric artery. In the aorta, idazoxan behaved as a partial agonist (pK_A=6.30). Prazosin produced rightward shift (pA₂=9.88) and steepening of the idazoxan curve. In contrast, idazoxan had no effect of basal tension in the mesenteric artery, but shifted the noradrenaline curve to the right in a parallel manner (pA₂=6.12). The selective al-adrenoceptor agonist, indanidine, also behaved as a partial agonist in the aorta and produced no significant contractions of the small mesenteric artery. Since idazoxan and indanidine have been reported to raise blood pressure in the pithed rat via an action at vascular ₁-adrenoceptors, these results call into question the reliability of the small mesenteric artery assay as a predictor for ₁-adrenoceptor-mediated pressor activity in vivo.     

依那普利对2型糖尿病大鼠肠系膜血管内皮细胞的保护作用

目的　糖尿病血管病变的初始原因是血管内皮细胞损害。本研究观察依那普利对糖尿病大鼠肠系膜血管内皮细胞是否有保护作用。方法　大鼠用高脂饮食饲养 4周后 ,ip链佐霉素 30mg·kg- 1诱导 2型糖尿病 ,继续饲以高脂饮食 4周后 ,依那普利组给予依那普利 10mg·kg- 1·d- 1,ig ,连续 4周。采用大鼠离体肠系膜血管灌流技术 ,用去甲肾上腺素 1μmol·L- 1预收缩血管 ,再给予乙酰胆碱 (ACh) 1μmol·L- 1使血管舒张。观察ACh的舒张率来反映内皮细胞功能。结果　糖尿病大鼠肠系膜血管ACh舒张率为 (33±8) % ,较对照组 (79± 8) %明显降低 ,依那普利治疗组血管ACh舒张率为 (5 2± 6 ) % ,较糖尿病组明显改善。用皂素去内皮后 ,三个组肠系膜血管对ACh舒血管的反应性均明显降低 ,三组间无显著性差异。去内皮前后 ,三个组肠系膜血管对硝普钠舒张血管的反应性无显著变化。结论　依那普利对 2型糖尿病大鼠肠系膜血管内皮细胞具有保护作用。     

老年冠心病患者肠系膜上动脉造影影像分析

目的提高对老年冠心病患者并发肠缺血的认识,完善冠心病防治规则。方法选择经临床和影像检查确诊的36例老年(65~74岁)冠心病患者(观察组)与31例中青年(35~50岁)冠心病患者(对照组),观察比较2组肠系膜上动脉造影结果,分析年龄、冠状动脉病变程度与肠系膜上动脉病变之间的关系。结果观察组发现肠系膜上动脉病变19例(53%),对照组发现3例(10%)(P<0.05)。观察组冠状动脉多支血管病变者24例(67%),对照组多支血管病变者7例(23%)(P<0.05)。2组中冠状动脉多支血管病变31例(46%),合并肠系膜上动脉病变19例(19/22,86%);单支血管病变36例(54%),合并肠系膜上动脉病变3例(3/22,14%)(P<0.05)。结论冠心病患者的年龄越大,冠状动脉损害越严重,肠系膜上动脉损害多在冠状动脉损害较严重时发生,应重视老年冠心病患者并发肠缺血的存在。     

肠系膜上静脉血栓形成的诊断和治疗

目的探讨肠系膜上静脉血栓形成(MVT)的诊断及治疗。方法总结15例MVT的诊治经验,从病因、诊断和治疗等方面进行分析。结果初诊多数表现为急腹症,症状和体征不相符。9例经手术病理证实,6例术前确诊,3例误诊,7例治愈,2例死亡。6例急性MVT诊断确立后立即采用抗凝溶栓等保守治疗,5例治愈,1例中转手术治愈。结论该病症状和体征无特异性。诊断更主要依靠影像学检查(彩色超声多普勒、CT、磁共振成像、血管造影)发现肠系膜上静脉的栓子征象来确立。急性MVT在肠缺血尚未导致透壁性肠坏死、肠穿孔时,抗凝溶栓治疗可行,如有急性腹膜炎时中转手术。在肠坏死时,早期切除坏死肠段,包括静脉阻塞的肠系膜部分,术中、术后抗凝治疗,是提高治愈率的重要措施。     

Assessment of duodenal circular drainage operations to treat superior mesenteric artery syndrome

To assess the clinical value of duodenal circular drainage operation to treat superior mesenteric artery syndrome(SMAS). Methods Forty two cases of SMAS were treated with duodenal circular drainage operation from 1959 to 2001. Clinical data were analyzed retrospectively. Results In this group,37 cases were treated with duodenal circular drainage operation,and had good effect after follow- up of 1～15 years,the other 5 cases were first treated with anterior repositioning of the duodenum (1 case),gastrojejunostomy (1 case) ,duodeojejunostomy (2 cases) .subtotal gastrectomy and Billroth Ⅱ gastrqjejunostomy( 1 case), but vomiting was not relieved until duodenal circular drainage operation was performed again. Follow-up of 9～10 years revealed good effect. Conclusion In SMAS, if the reversed peristalsis is stronger and continuous,and vomiting appears frequently, the symptom can not be relieved even if the obstruction of duodenum is removed by operation. The key treatment is the relief of reversed peristalsis. Only the duodenal circular drainage operation could resolve the drainage direction of duodenal content and relieve the symptom of vomiting.