pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

RA 642, a pyrimido-pyrimidine-derivative with vasodilating and hypertensive potency

Summary 2,2-[(4,8-bis(diethylamino)-pyrimido [5,4-d]-pyrimidine-2,6-diyl)di-(2-methoxyethyl)imino]diethanol), RA 642, combines hypertensive and vasodilating effects. In anaesthetized animals arterial blood pressure was increased by i.v. doses of 0.25–4 mg/kg in cats and 0.025–0.25 mg/kg in dogs. In conscious dogs, 25 mm increase of mean blood pressure was achieved with 0.2 mg/kg i.v. and 18.8 mg/kg p.o. Cerebral blood flow was enhanced and calculated cerebral vascular resistance was reduced by RA 642. Total peripheral resistance was diminished by 0.25–1.0 mg/kg i.v. A vasodilatation of femoral and coronary vessels was shown after intraarterial injection. This effect as well as a BaCl₂-antagonism in the isolation ileum is explained by a papaverine-like relaxant effect on smooth muscle. Activity on peripheral adrenergic receptors was excluded. Hypertension was abolished in spinalized cats, indicating a central mechanism of this effect.     

Openers of small conductance calcium-activated potassium channels selectively enhance NO-mediated bradykinin vasodilatation in porcine retinal arterioles

Background and purpose:

Small (SK_Ca or K_Ca2) and intermediate (IK_Ca or K_Ca3.1) conductance calcium-activated potassium channels are involved in regulation of vascular tone and blood pressure. The present study investigated whether NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) and CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine), which are selective openers of SK_Ca and IK_Ca channels and of SK_Ca2 and SK_Ca3 channels, respectively, enhance endothelium-dependent vasodilatation in porcine retinal arterioles.

Experimental approach:

In porcine retinal arterioles, SK_Ca3 and IK_Ca protein localization was examined by immunolabelling. Endothelial cell calcium was measured by fluorescence imaging. For functional studies, arterioles with internal diameters of 116 ± 2 µm (n = 276) were mounted in microvascular myographs for isometric tension recordings.

Key results:

SK_Ca3 and IK_Ca protein was localized in the endothelium. Bradykinin, but not NS309 or CyPPA increased endothelial cell calcium. Pre-incubation with NS309 or CyPPA enhanced bradykinin relaxation without changing endothelial cell calcium. This enhanced relaxation was abolished by blocking SK_Ca channels with apamin. In the presence of NS309 or CyPPA, mainly inhibition of NO synthase with asymmetric dimethylarginine, but also inhibition of cyclooxygenase with indomethacin, reduced bradykinin relaxation. Bradykinin relaxation was completely abolished by NO synthase and cyclooxygenase inhibition together with a NO scavenger, oxyhaemoglobin.

Conclusions and implications:

In porcine retinal arterioles, bradykinin increases endothelial cell calcium leading to activation of SK_Ca and IK_Ca channels. Without altering endothelial cell calcium, NS309 and CyPPA open SK_Ca channels that enhance NO-mediated bradykinin relaxations. These results imply that opening SK_Ca channels improves endothelium-dependent relaxation and makes this channel a potential target for treatments aimed at restoring retinal blood flow.     

Regulation of oxygen consumption in perfused rat liver: decrease by α-sympathetic nerve stimulation and increase by the α-agonist phenylephrine

In livers perfused with Krebs-Henseleit bicarbonate buffer containing bovine red cells, 5 mM glucose and 2 mM lactate, electrical stimulation round the hepatic artery and the portal vein caused via -receptors a decrease in oxygen consumption and portal flow, an increase in glucose output and a switch from lactate uptake to output.In livers perfused with erythrocyte- and substrate-free buffer both in a volume- or pressure-constant system stimulation of the liver nerves resulted in similar changes. Infusion of the -agonist phenylephrine mimicked the metabolic and hemodynamic nerve effects, but led to an increase in oxygen uptake. The converse effects of -sympathetic nerve stimulation and -agonist infusion on oxygen consumption indicate either a different mode of action or a complex mechanism with opposing metabolic and hemodynamic components.     

HOE642对大鼠停跳心脏供体心肌细胞凋亡的影响

目的研究HOE642对热缺血死亡大鼠颈部异位停跳心脏供体心肌细胞凋亡的作用。方法取清洁级雄性Sprague-Dawley大鼠112只,随机分成7组,每组16只,各有8只分别作为移植受体和供体。具体如下:C组为正常心脏对照组;S10、S30、S45组分别为心脏停搏后10、30、45 min取心组;SH10、SH30、SH45组分别为心脏停搏后10、30、45 min取心并用HOE642灌注组。对6组实验组大鼠进行热缺血死亡处理,取得供体后,用STH-1液对S10、S30、S45组供体灌注30 min,加20μmol/L的HOE642对SH10、SH30、SH45组供体灌注30 min,再用颈部Cuff法进行异位心脏移植。S10、SH10、S30、SH30组于移植后48 h取供心,S45、SH45组于移植术后取供心。脱氧核糖核苷酸末端转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法检测供体心肌细胞凋亡,免疫组织化学法检测Bcl-2、Bax、Caspase-3蛋白的表达。结果大鼠腹主动脉横断放血后9~11 min,平均(10.11±0.59)min死亡。S10、S30组的凋亡心肌细胞数显著高于SH10、SH30组(P<0.05);S10、S30组的Bcl-2表达水平显著低于SH10、SH30组(P<0.05),而Bax、Caspase-3表达水平显著高于SH10、SH30组(P<0.05)。结论热缺血死亡大鼠颈部异位心脏移植模型是进行停跳心脏供体的心肌保护研究的理想模型;HOE642能抑制热缺血死亡后(30 min内)大鼠心脏心肌细胞凋亡的发生。     

Sera from different age cohorts in Belgium show limited cross-neutralization between the mumps vaccine and outbreak strains

ObjectivesMumps used to affect children between 2 and 15 years old. The mumps–measles–rubella (MMR) vaccine is available, with vaccine coverage rate of about 85% after two vaccine doses. Recently new mumps outbreaks have emerged in highly vaccinated populations; the causes for these new outbreaks are yet unknown. We tested if a difference in seroneutralizing capacity against the vaccine and wild-type viruses existed and if waning immunity could be detected.MethodsIn this study, 570 serum samples (age group 2–3 years (n = 96), 8–9 years (n = 95), 13–14 years (n = 94), 18–20 years (n = 96), 24–26 years (n = 92) and 50 + years (n = 97)) in Belgium were tested in the rapid fluorescent foci inhibition test for their neutralizing capacity against the vaccine and wild-type viruses.ResultsNeutralizing antibodies against the vaccine strain were present in 84% (81/97) of the 2–3-year, 74% (70/95) of the 8–9-year, 81% (76/94) of the 13–14-year, 76% (73/96) of the 18–20-year, 67% (62/92) of the 24–26-year and 77% (75/97) of the 50+-year age group serum samples. For all age groups, only about half of these serum samples were also positive for the wild-type virus. The geometric mean titres for the vaccine and wild-type virus for all younger age groups, except for 24–26 years, were significantly different, demonstrating poor in vitro cross-neutralization.ConclusionsA possible contribution of antigenic differences between the genotype A and G mumps virus as well as other immune factors, in addition to lower-than-optimal vaccination coverage and waning immunity, could explain the poor in vitro cross-neutralization and should be further studied.     

不同方法取出根管内塑化物的临床观察

目的:比较G钻、Hero642机用镍钛锉及显微超声技术三种方法取出根管内塑化物的效果。方法:结合手用根管锉,分别采用三种方法对186个根管进行再通畅治疗,通过X线、根尖定位仪对取出根管内塑化物的效果进行评价。结果:G钻组完全再通41个,Hero642镍钛组完全再通34个,显微超声组44个。结论:显微超声技术在三种塑化根管再通畅治疗中有较好的疗效,G钻在临床使用过程中较为方便。