Elevated contractile responses to acetylcholine in organ cultured rabbit carotid artery

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum- free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.     

Clusters of proliferating endothelial cells and smooth muscle cells in rabbit carotid arteries

Schwarz and Benditt found clustering of replicating cells in aortic endothelium in 1976 and discussed how homeostasis of the arterial wall is maintained through this nonrandom distribution of replicating cells. However, it is still unclear how cells of vascular walls turnover. In order to address this issue, we evaluated distribution of the cells in mitotic cycle, labeled by Ki67‐immunostaining, in serial histological sections of twelve carotid arteries of six adult male Japanese rabbits. As a result, a total of 1713 Ki67‐positive endothelial cells (ECs) and 1247 Ki67‐positive smooth muscle cells (SMCs) were identified. The Ki67‐positivity rate in ECs and SMCs were about 0.048% and 0.0027%, respectively. Many of the Ki67‐positive cells clustered in two (EC, 37%; SMC, 33%), three to four (EC, 8%; SMC, 28%), and five to eight cells (EC, 5%; SMC, 10%). Clusters having more than eight cells were not found. Thus, it can be speculated that the cell division of proliferating ECs and SMCs occur four times at most. These novel findings offer great insights for better understanding of the mechanism that underlies cell number regulation of the blood vessel.     

Anomalous artery directly connecting the external and internal carotid arteries

An anomalous artery directly connecting the external with the internal carotid artery was encountered on the right side of a 68-year-old Japanese female cadaver. This anomalous artery (5 mm in diameter, 12 mm in length) branched out from the posterior aspect of the external carotid at the level of the origin of the lingual artery, ran obliquely upward posteriorly along the course of the hypoglossal nerve, and was confluent with the anterior aspect of the internal carotid artery. No other variations were found in the morphological aspects of, or in the anatomical relationships between, the carotid arteries and their surrounding structures on either side. The carotid body-like structure was observed at the carotid bifurcation and was innervated by small branches of the glossopharyngeal, the vagus and the sympathetic trunk. Embryologically, it is conceivable that this anomalous artery may have derived from the right second branchial arch artery, although there is no abnormality in other derivative structures of the second pharyngeal arch. There may have been no effect from this anomaly on the functions of the arterial blood flow and blood supply under normal circumstances in the present case, but this report may be of embryological significance and contribute some insight into the mechanisms of the formation of the carotid circulation systems.     

褪黑素减轻吸烟大鼠颈动脉的氧化损伤

目的探讨褪黑素能否减轻吸烟大鼠颈动脉的氧化损伤。方法将20只大鼠随机分为对照组、吸烟组(建立大鼠吸烟模型)和褪黑素(5 mg/kg)干预组。7 d后取颈动脉,荧光探针2',7'-DCFH-DA检测颈动脉内活性氧(ROS)含量;Western blot检测颈动脉内NF-κB、eNOS、MCP-1、VCAM-1和ICAM-1的表达。结果吸烟能明显诱导大鼠颈动脉内ROS的增加(P<0.01);褪黑素干预组ROS含量明显回降(P<0.01);吸烟组颈动脉内eNOS的表达明显低于对照组(P<0.01);褪黑素干预组颈动脉内eNOS的表达明显回升(P<0.01);吸烟组颈动脉内的NF-κB、MCP-1、VCAM-1和ICAM-1的表达明显高于对照组(P<0.01);褪黑素干预组的NF-κB、MCP-1、VCAM-1和ICAM-1的表达明显回降(P<0.05)。结论吸烟能明显诱导大鼠颈动脉内ROS的增多及氧化损伤,褪黑素能通过NF-κB通路减轻吸烟大鼠颈动脉的氧化损伤。     

Fluctuations in carotid arterial distensibility during the menstrual cycle do not influence cardiovagal baroreflex sensitivity

Aim: Fluctuations in autonomic nervous functions throughout the menstrual cycle and the underlying mechanism concerning them are not well known. This study was designed to test the hypothesis that fluctuations in cardiovagal baroreflex sensitivity (BRS) throughout the menstrual cycles of young women are due to fluctuations in carotid arterial distensibility. Methods: In eight eumenorrhoeic healthy young women (18–24 years), we determined the variations in the carotid arterial distensibility coefficient (DC; via simultaneous ultrasonography and applanation tonometry), cardiovagal BRS (phase IV of the Valsalva manoeuvre and the sequence method; up‐ or down‐sequence spontaneous BRS), and serum oestradiol and progesterone concentrations at five points in the menstrual cycle (menstrual = M, follicular = F, ovulatory = O, early luteal = EL, and late luteal = LL). Results: Serum oestradiol and progesterone levels were consistent with the predicted cycle phases. Carotid arterial DC fluctuated cyclically, increasing significantly from the M (52.4 ± 4.9 × 10^?3 kPa^?1, mean ± SE) and F (52.7 ± 4.4) phases to the O (57.6 ± 4.4) phase and declining sharply in the EL (46.0 ± 4.0) and LL (45.1 ± 3.0) phases (F = 6.37, P < 0.05). Contrary to our prediction, however, cardiovagal BRS by the Valsalva manoeuvre (P = 0.73) or sequence method (up‐sequence spontaneous BRS; P = 0.84: down‐sequence spontaneous BRS; P = 0.67) did not change significantly during the menstrual cycle. Conclusion: The results suggest that, although carotid arterial distensibility fluctuates with the changes in ovarian hormone levels that occur during the menstrual cycle, the fluctuations in carotid arterial distensibility do not influence cardiovagal BRS.     

The effect of experimental frost-bite on the responses of the rabbit ear-artery to adrenergic and electrical stimulation

The aim of the present investigation was to study the effect of sub-zero temperatures on the adrenergic activated, smooth muscle-contraction of a peripheral blood-vessel. The central ear-artery of the rabbit was used for this purpose. The artery was stimulated to contract in vitro by activation of phentolamine sensitive, post-junctional, a-adrenoceptors by use of noradrenaline, or by noradrenaline released from noradrenergic nerves in the blood-vessel following electrical field stimulation. The effect of freezing the tissue in vivo for 15 min at sub-zero temperatures (–4, - 6 and – 9 ^oC) was studied in vitro. Exposure to – 4 and – 6 ^oC did not alter the apparent affinity (ED₆₀) of noradrenaline significantly, when measured immediately, or 2 or 6 days after exposure. The maximal response to noradrenaline was reduced by approximately 54, 74 and 100% following exposure to – 4 , – 6 and – 9 ^oC, respectively. The response was completely restored after 2 and 6 days of regeneration in vivo following exposure to – 6 ^oC, whereas the response after exposure to – 9 ^oC was restored by only about 8 and 30% after 2 and 6 days regeneration, respectively. The maximal response to electrical field stimulation, which was completely inhibited by tetrodotoxin and phentolamine, was reduced by approximately 92% after exposure for 15 min to –4 and – 6 ^oC, while it was completely inhibited after exposure to – 9 ^oC. The response was restored by only 15–20% following 2 and 6 days in vivo after exposure to – 6 ^oC. No regeneration of the contraction induced by the electrical stimulation was observed after 2 or 6 days following exposure to –9 ^oC. The results of the present study suggest that noradrenergic nerves in the central ear-artery of the rabbit are more sensitive to subzero temperatures than are the post-junctional, noradrenergic α-receptors and the smooth muscle of the blood-vessel. The damage induced by freezing appeared to be both time and temperature dependent and there was only limited restoration of the nervous function 6 days after severe freezing.     

Clinical trial of argatroban, a direct thrombin inhibitor, as an anticoagulant in cardiopulmonary support and apheresis in emergency patients: a preliminary report

 To evaluate the safe and effective use of argatroban, a competitive direct thrombin inhibitor, as an alternative anticoagulant for percutaneous cardiopulmonary support (PCPS) and continuous hemofiltration or hemodiafiltration (CHF/CHDF), a preliminary multicenter clinical trial was conducted between October 1999 and September 2000. Nine patients who underwent PCPS and/or CHF/CHDF were enrolled in the study during this period. The dosage of argatroban was controlled so that the activated clotting time (ACT) was maintained at around 180 to 200 s. The mean duration of argatroban administration was 82 ± 92 h, and the mean dose was 0.67 ± 0.40 μg kg⁺¹ min⁻¹. Severe hemorrhagic complications requiring the discontinuation of argatroban administration were not observed in any of the patients. Platelet loss was prevented to some degree, and plasma levels of fibrinogen were well preserved during PCPS/CHDF. Except for two patients undergoing CHDF, clot formation within the extracorporeal circulation circuit was not identified macroscopically after the discontinuation of the procedures. We conclude that argatroban might be useful as an alternative anticoagulant in cases where heparin cannot be safely used because of the increased risk of bleeding complications, thrombocytopenia, and/or hypofibrinogenemia. Although the optimal dose of argatroban has not been established, we propose an initial starting dose of 0.7 to 1.0 μg kg⁻¹ min⁻¹, followed by adjustments to maintain an ACT of between 180 and 250 s. Received: December 17, 2001 / Accepted: June 1, 2002 Acknowledgments We thank Dr. S. Miyamoto (St. Marianna University School of Medicine), Dr. Y. Okada (Showa University School of Medicine), Dr. M. Shimizu, and Dr. J. Ishikawa (Yokohama City University School of Medicine) for providing data used in this study. We also thank Dr. S. Kanesaka (Showa University School of Medicine Fujigaoka Hospital) and Dr. S. Imaki (St. Marianna University School of Medicine, Yokohama City Seibu Hospital) for their helpful advice. Correspondence to:K. Akashi     

Hypercapnic vasodilatation in isolated rat basilar arteries is exerted via low pH and does not involve nitric oxide synthase stimulation or cyclic GMP production

The relaxant effect of hypercapnia (15% CO₂) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-l -arginine (l -NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this relaxation by 54% and 70%, respectively. The effect of l -NOARG was completely reversed by l -arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO₂ elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response to hypercapnia. l -NOARG and MB, but not TTX, significantly reduced the basal cGMP content in cerebral vessels. Adding 1.5% halothane to the incubation medium did not result in a significant increase in cGMP content. Lowering the pH by cumulative application of 0.12 m HCl resulted in relaxation identical to that obtained by lowering the pH with 15% CO₂. In vessel segments in which the endothelium had been removed beforehand 15% CO₂ induced relaxation that was not different from that seen in vessels with intact endothelium. l -NOARG had no affect in endothelium denuded vessels. The results suggest that high CO₂ elicits vasodilatation of isolated rat basilar arteries by a mechanism independent of nitric oxide synthase (NOS) activity. The markedly reduced basal cGMP levels in cerebral vessels by l -NOARG and MB suggest that there exists a basal NO formation in the cerebral vessel wall.     

Activation of the non-receptor tyrosine kinase cSrc in macrophage-rich atherosclerotic plaques of human carotid arteries

To determine the involvement of the non-receptor tyrosine kinase cSrc in plaque destabilization in carotid atherosclerosis (CAS), which is responsible for cerebral infarction, we performed quantitative and morphological detection of phosphorylated active cSrc (p-cSrc) and histopathological examination in CAS lesions. We examined carotid endarterectomy specimens obtained from 32 CAS patients. Each specimen was used for immunoblot and immunohistochemical analyses of p-cSrc, histopathological analysis, and image analysis of macrophage content. There was a strong positive correlation between cSrc activation on blots and macrophage content on sections. When we defined the macrophage-rich plaque (MRP) and the macrophage-poor plaque (MPP) as having macrophage content more and less than 5%, respectively, the p-cSrc density and the occurrence of plaque hemorrhage and thrombus formation were significantly increased in the MRP group (n=18) compared to the MPP group (n=14). p-cSrc immunoreactivity was localized in lesional endothelial cells, macrophages, and smooth muscle cells, which contained proinflammatory substances: the upstream oxidized low density lipoprotein, tissue factor and osteopontin, and the downstream active forms of extracellular signal-activated kinase and p38 and nuclear factor-kappaB. Our results suggest that cSrc activation in lesional cells contributes to plaque destabilization in CAS via persistent inflammation.     

Hyperglycaemia alters the endothelium‐dependent relaxation of canine coronary arteries

The aim of the present study was to investigate the effects of experimental diabetes and hyperglycaemia per se on the endothelium‐dependent relaxation of isolated canine coronary arteries and to analyse the possible involvement of the cyclooxygenase pathway in the alterations induced by hyperglycaemia. Rings from the left anterior descending coronary arteries of 18 metabolically healthy, six alloxan‐diabetic and six insulin‐treated alloxan diabetic dogs were set up for isometric tension recording. Diabetic coronaries as well as healthy vessels subjected to in vitro hyperglycaemia (25.5 mmol L^–1 glucose) showed impaired (P < 0.05) relaxation to acetylcholine (3 nmol L^–1–10 μmol L^–1) compared with normoglycaemic, i.e. metabolically healthy and insulin‐treated diabetic controls, either before or after indomethacin (3 μmol L^–1) administration. The maximal dilation elicited by acetylcholine was further decreased (P < 0.05) by the cyclooxygenase inhibitor in the diabetic coronaries only. Relaxation to sodium nitroprusside did not differ among groups. These results suggest that hyperglycaemia may result in impaired endothelium‐dependent dilation of coronary arteries. Diminished relaxation of diabetic coronaries is worsened by the inhibition of the synthesis of vasodilator cyclooxygenase products.     

Vasodilator responses to alpha-human-atrial natriuretic peptide in isolated omental and pulmonary arteries from rabbit and man

In isolated vessels from the pulmonary and mesenteric/omental arterial circulations of rabbit and man, the effects of synthetic α-human atrial natriuretic peptide (α-hANP) were investigated. The vessels had an outer diameter of 0.6–1.2 mm and were contracted by noradrenaline (NA) and prostaglandin (PG) F_2α, (mesenteric/omental arteries) or by 5–hydroxytryptamine (5–HT) and PGF_2α (pulmonary arteries). It was found that in rabbits, mesenteric vessels contracted by NA and PGF_2α were not significantly relaxed by α-hANP. Rabbit pulmonary vessels contracted by PGF_2α or 5–HT were concentration-dependently relaxed by α-hANP (maximum 70%). Human omental vessels contracted by NA and PGF_2α showed a moderate (30%) relaxation after addition of the peptide. Human pulmonary arteries contracted by 5–HT were relaxed by α-hANP almost to baseline, while PGF_2α-contracted vessels showed a maximum relaxation of 65%. It is concluced that α-hANP has a relatively selective effect on pulmonary arterial vessels from rabbit as well as man. The results suggest that the peptide may be involved in the regulation of pulmonary vascular tone.     

The influence of the sympathetic impulse pattern on contractile responses of rat mesenteric arteries and veins

Contractile responses to electrical field stimulation of excised small mesenteric arteries and veins of the rat were compared when stimuli were delivered in irregular bursts or at regular intervals. Spontaneously occurring skin vasoconstrictor impulses in a few-unit median nerve recording in man were stored on tape and used to trigger a stimulator. Two irregular stimulation sequences at average frequencies of 1.6 and 1.8 Hz, respectively, were used. In the arteries, average contractile responses were significantly greater at an irregular than at an even stimulation frequency, but in the veins, similar degrees of contraction were obtained with the two modes of stimulation. The frequency-response relationships to continuous regular stimulation showed the artery to respond less than the vein at low frequencies. This apparently explains the differences in behaviour between the vessels to irregular stimulation. The results show that not only the number of impulses, but also their pattern of occurrence, may influence the degree of vasoconstriction. Thus, the normal irregular sympathetic discharge pattern in itself has a bearing on the physiology of neuro-effector control mechanisms.     

喉罩对颈总动脉和颈内动脉血流动力学的影响

目的 研究喉罩对颈总动脉(CCA)和颈内动脉(ICA)内径、血流速率和血流量等血流动力学参数的影响.方法 随机选择择期全麻下行腹腔镜胆囊切除术患者60例,为美国麻醉医师协会(ASA)Ⅰ～Ⅲ级.根据不同年龄分为中青年组(A组,20～59岁)和老年组(B组,60～85岁).按不同的喉罩套囊内压力将A组和B组分为4个亚组,即A1、B1(套囊内压为20～30 cm H2O)(1 cm H2O=0.098 kPa)和A2、B2(套囊内压为40～50 cm H2O),每组15例.所有患者术毕后送入ICU复苏,尚未清醒时拔除气管导管,而后置入喉罩.记录置入喉罩前(T0),置入喉罩后3 min(T1)、10 min(T2)和拔除喉罩后(T3)各时点CCA和ICA的内径、血流速率和血流量等血流动力学参数以及生命体征参数.结果 CCA、ICA的内径和CCA血流量的基础值B组比A组要大(P<0.05).与T0比较,在T1和T2时点,A组和B组CCA内径分别减少9.5%～12.9%和14.5%～24.3%(P<0.05或P<0.01),其中以B2组减少最为显著,A2组和B2组ICA内径分别减少10.9%和16.3%(P<0.05).CCA和ICA的血流速率无明显变化(P>0.05).与T0比较,A组和B组,T1和T2时CCA的血流量分别减少9.3%～10.7%和12.2%～19.1%(P<0.05),其中以B2组减少最为显著,A组和B组ICA血流量分别减少10.0%～13.5%和13.9%～16.6%(P<0.05).在T3时点各观察指标均恢复至T0水平.结论 喉罩通气时,CCA和ICA的内径有所缩小,其血流量相应减少,老年患者减少更为明显,而其血流速率则无明显改变.     

Chronic increases in transmural pressure reduce NO‐mediated dilations in isolated resistance arteries of the hamster

It is unclear whether the impairment of NO‐mediated dilation in hypertension is the cause or the consequence of high blood pressure. We therefore studied in isolated resistance arteries whether elevated transmural pressure affects NO‐mediated dilation. Arteries (n=5–7) were perfused at hydrostatic pressures of either 45, 120 or 160 mmHg for 48 h. Subsequently, diameter and calcium responses (fura 2) were studied at a transmural pressure of 45 mmHg. Pre‐exposure to 120 and 160 mmHg reduced resting diameters and minimal diameters after stimulation with noradrenaline and significantly increased corresponding intracellular free calcium levels in vascular smooth muscle. Moreover, the NO‐mediated dilation in response to acetylcholine was significantly reduced although the increase in endothelial calcium was not altered. Dilations induced by the NO donor SNP were not affected. It is concluded that chronically elevated pressure per se impairs endothelial NO production by a mechanism distal to receptor‐dependent calcium increases.     

Rhythmic contractions of isolated small arteries from rat: influence of the endothelium

Small arteries of the mesenteric arcade from Wistar rats display rhythmic oscillations superimposed on the tonic contractile response when exposed to submaximal doses of noradrenaline. We have previously shown that mechanical removal of the endothelium abolishes these oscillations. In the present study different methods to eliminate or modify the influence of the endothelium were used in order to further characterize the mechanisms behind rhythmic contractions in these vessels. Endothelium was removed either mechanically or chemically by perfusing the vessels with 0.3% CHAPS. The absence of functional endothelium enhanced noradrenaline sensitivity and simultaneously abolished oscillations in tension and membrane potential, but did not affect resting membrane potential. The rhythmic activity was also reduced or abolished by exposure to haemoglobin, methylene blue, LY83583 or l -NNA. Indomethacin and propranolol were without effect. Sodium nitroprusside or the permeant analogue of cyclic GMP, 8-bromo cyclic GMP, restored rhythmic activity in precontracted endothelium-denuded vessels. The data suggest that release of nitric oxide from the endothelium, and subsequent generation of cyclic GMP in the smooth muscle, activates oscillations in membrane potential and tension; the oscillator itself appears to be located within the smooth muscle cells.