Dependence of acetazolamide teratogenesis on fetal and not maternal genotypes.

This study assesses the relative contributions of dam and conceptus to sensitivity to acetazolamide teratogenesis in an in vivo mouse model. Reciprocal F1 outcrosses were made between mice resistant to the teratogenic effects of acetazolamide (SWV) and the susceptible C57BL/6JBk line. Female F1 progeny were backcrossed either to C57BL or to SWV males. The F1 outcross resulted in identical fetuses developing in genetically different uterine environments. In the backcrosses, genetically identical hybrid dams were gestating genetically different populations of segregating fetuses. In both F1 outcrosses and backcrosses, as well as in pure-line controls, dams were treated on day 9 of gestation by subcutaneous injection of acetazolamide (ACZM) at a dose level of 1500 mg/kg, or identical volume of the vehicle. The principal abnormality seen was right front limb ectrodactyly. Among pure-line matings, SWV produced no ectrodactyly, but 68% of C57BL showed this defect. Only two cases were seen in outcross of C57BL females to SWV males, and one litter produced three ectrodactylous fetuses in the reciprocal cross. A highly significant increase in ectrodactyly was seen in treated backcrosses to C57BL relative to backcrosses to SWV. Thus, a litter in which most of the genes segregating are from a susceptible line will show major increases in susceptibility, despite being in a heterotic and somewhat resistant uterine environment, whereas the susceptible C57BL uterine environment does not result in significant abnormality in heterozygous resistant fetuses. These results unequivocally demonstrate that it is the genotype of the conceptus, and not the genotype of the dam, nor heterosis, that determines sensitivity to acetazolamide teratogenesis. They further suggest that this genetic approach may be useful in determining the relative contribution of dam and conceptus for other teratogens.     

Topiramate modulates pH of hippocampal CA3 neurons by combined effects on carbonic anhydrase and Cl-/HCO3- exchange

Topiramate (TPM) is an anticonvulsant whose impact on firing activity and intracellular pH (pHi) regulation of CA3 neurons was investigated. Using the 4-aminopyridine-treated hippocampal slice model bathed in bicarbonate-buffered solution, TPM (25-50 microm) reduced the frequency of epileptiform bursts and action potentials without affecting membrane potential or input resistance. Inhibitory effects of TPM were reversed by trimethylamine-induced alkalinization. TPM also lowered the steady-state pHi of BCECF-AM-loaded neuronal somata by 0.18+/-0.07 pH units in CO(2)/HCO(3)(-)-buffered solution. Subsequent to an ammonium prepulse, TPM reduced the acidotic peak but clearly slowed pHi recovery. These complex changes were mimicked by the protein phosphatase inhibitor okadaic acid. Alkalosis upon withdrawal of extracellular Cl(-) was augmented by TPM. Furthermore, at decreased pHi due to the absence of extracellular Na(+), TPM reversibly increased pHi. These findings demonstrate that TPM modulates Na(+)-independent Cl(-)/HCO(3)(-) exchange. In the nominal absence of extracellular CO(2)/HCO(3)(-) buffer, both steady-state pHi and firing of epileptiform bursts remained unchanged upon adding TPM. However, pHi recovery subsequent to an ammonium prepulse was slightly increased, as was the case in the presence of the carbonic anhydrase (CA) inhibitor acetazolamide. Thus, a slight reduction of intracellular buffer capacity by TPM may be due to an inhibitory effect on intracellular CA. Together, these findings show that TPM lowers neuronal pHi most likely due to a combined effect on Na(+)-independent Cl(-)/HCO(3)(-) exchange and CA. The apparent decrease of steady-state pHi may contribute to the anticonvulsive property of TPM.     

Cytotoxic mechanisms of Zn2+ and Cd2+ involve Na+/H+ exchanger (NHE) activation by ROS

The signaling mechanism induced by cadmium (Cd) and zinc (Zn) in gill cells of Mytilus galloprovincialis was investigated. Both metals cause an increase in *O2- production, with Cd to be more potent (216 +/- 15%) than Zn (150 +/- 9.5%), in relation to control value (100%). The metals effect was reversed after incubation with the amiloride analogue, EIPA, a selective Na+/H+ exchanger (NHE) inhibitor as well as in the presence of calphostin C, a protein kinase C (PKC) inhibitor. The heavy metals effect on *O2- production was mediated via the interaction of metal ions with alpha1- and beta-adrenergic receptors, as shown after incubation with their respective agonists and antagonists. In addition, both metals caused an increase in intracellular pH (pHi) of gill cells. EIPA together with either metal significantly reduced the effect of each metal treatment on pHi. Incubation of gill cells with the oxidants rotenone, antimycin A and pyruvate caused a significant increase in pHi (delta pHi 0.830, 0.272 and 0.610, respectively), while in the presence of the anti-oxidant N-acetyl cysteine (NAC) a decrease in pHi (delta pHi -0.090) was measured, indicating that change in reactive oxygen species (ROS) production by heavy metals affects NHE activity. When rosiglitazone was incubated together with either heavy metal a decrease in O2- production was observed. Our results show a key role of NHE in the signal transduction pathway induced by Zn and Cd in gill cells, with the involvement of ROS, PKC, adrenergic and PPAR-gamma receptors. In addition, differences between the two metals concerning NHE activation, O2- production and interaction with adrenergic receptors were observed.     

Exacerbation of acetazolamide teratogenesis by amiloride and its analogs active against NA/H exchangers and NA channels

Postaxial forelimb ectrodactyly induced by acetazolamide given on Day 9.5 of murine gestation is thought to be mediated by reduced intracellular pH (pH_i) within the limb bud. Coadministration of amiloride increases the incidence and severity of acetazolamide-induced forelimb malformations and further reduces limb bud pH_i. These findings were hypothesized to be attributable to the action of amiloride as an inhibitor of Na⁺/H⁺ exchangers (NHEs), plasma membrane-localized proteins involved in the maintenance of cellular pH homeosta- sis. Here, we explored this hypothesis further by coadministering with acetazolamide, amiloride, or analogs known to preferentially inhibit NHEs 5-(N-methyl-N-isobutyl)-amiloride, 5-(N, N-hexamethylene)-amiloride, 5-(N, N-dimethyl-amiloride, and 5-(N-ethyl-N-isopropyl)-amiloride or amiloride-sensitive Na⁺ channels (ben- zamil). The Coadministration of either amiloride, benzamil, 5-(N, N-dimethyl)-amiloride, 5-(N-ethyl-N-isopro- pyl)-amiloride, or 5-(N-methyl-N-isobutyl-amiloride all dose responsively increased the frequency and severity of forelimb malformations compared to acetazolamide alone. None of the analogs given alone induced forelimb ectrodactyly. The data are consistent with the original hypothesis that the exacerbation of acetazolamide teratogenesis is due to NHE inhibition. Surprisingly, benzamil was the most potent potentiator of acetazolamide teratogenesis. This result strongly suggests that amiloride-sensitive Na⁺ channels are also present within the murine embryo and are likely to play a role in pH_i homeostasis.     

Dimethylsphingosine increases cytosolic calcium and intracellular pH in human T lymphocytes

N,N-Dimethyl-D-erythro-sphingosine (DMS) is the N-methyl derivative of sphingosine; both are activators of sphingosine-dependent protein kinases. The aim of this work was to study the effect of DMS on cytosolic calcium and intracellular pH (pHi) in human T lymphocytes. The variations of calcium and pH were determined by fluorescence digital imaging using Fura-2-AM and BCECF-AM, respectively. DMS increased both pHi and Ca(2+)-cytoslic in human T lymphocytes. These effects were dose-dependent. This drug induced a fast increase in pHi and a release of calcium from different intracellular calcium pools than thapsigargin. DMS also induced a Ca(2+)-influx different from the store-operated calcium channels, since drug effect was not modified by 30 microM SKF 96365. The influx of calcium induced by DMS was completely blocked by preincubation in the presence of nickel, or lanthanum, while the increase in pHi was no affected. However, the presence of cadmium reduced but does not block Ca(2+)-influx. The inhibition of G-protein by 100 ng/mL pertussis toxin, and the inhibition of tyrosine kinases by genistein significantly reduced the cytosolic calcium increase induced by DMS by an inhibition of both, release of calcium from intracellular pools and influx from extracellular medium. The inhibition of pools emptiness by these drugs was related with the inhibition that they induce in the DMS cytosolic alcalinization. In summary, DMS increases pHi and as consequence releases calcium from intracellular pools, and it increases calcium-influx through a channel different from store-operated channel (SOC). Both cytosolic calcium and pHi increase are modulated by G-proteins and tyrosine kinases.     

Protein kinase C-mediated intracellular alkalinization in rat and rabbit aortic smooth muscle cells

The influence of protein kinase C (C-kinase) activation on intracellular pH (pHi) of cultured rat (RASM) and rabbit (RBASM) aortic smooth muscle cells was studied by employing a pH-sensitive fluorescent-dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The known C-kinase activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) and mezerine as well as the agonist angiotensin II each caused an intracellular alkalinization of approximately 0.1-0.15 pH units in RASM and RBASM cells grown in serum-free conditions. TPA-induced alkalinization was sensitive to the Na+/H+ exchange blockers amiloride and 5-N-ethylisopropyl-amiloride (EIPA). These results suggest that protein kinase C activation leads to intracellular alkalinization in vascular smooth muscle cells and the increase in pHi might play an important role in receptor-coupled arterial contraction.     

Effect of intracellular pH on cytosolic free [Ca2+] in human epidermoid A-431 cells.

This study characterizes the correlation between intracellular pH (pHi) and the cytosolic free Ca2+ concentration ([Ca2+]i) in suspended and adherent human epidermoid A-431 cells. Using the fluorescent dyes 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester (BCECF) and fura-2, the resting pHi and [Ca2+]i in suspended cells were 7.23 +/- 0.03 and 209 +/- 30 nM; those in adherent cells were 7.28 +/- 0.02 and 87 +/- 5 nM. Removal of external Ca2+ did not change the resting pHi but reduced the resting [Ca2+]i, indicating the resting level of [Ca2+]i is in part maintained by an influx of Ca2+ from the external medium. When both suspended and adherent cells were acidified or alkalinized, resting [Ca2+]i was altered. An intracellular acidification induced a fall in [Ca2+]i, and a rise in pHi induced a rise in [Ca2+]i. These changes in [Ca2+]i were correlated with an uptake of 45Ca2+ from the external medium, whereas no Ca2+ efflux occurred. The alteration in [Ca2+]i induced by modification of pHi was abolished in the absence of external Ca2+ or by adding 2 mM CoCl2, LaCl3, and attenuated by the addition of 2 mM MnCl2 to the bathing medium. It was insensitive to the voltage-gated Ca2+ channel blockers nifedipine or verapamil (1 mM). CoCl2, LaCl3, and MnCl2 each induced changes in pHi and [Ca2+]i but verapamil and nifedipine did not. Because CoCl2, LaCl3, and MnCl2 are also known to block Na+/Ca2+ exchange, intracellular Na+ ([Na+]i) was measured by flame photometry in acidified or alkalinized cells. In either case no change in [Na+]i was observed. Furthermore, treatment with amiloride (100 microM), a blocker of the Na+/Ca2+ exchanger, did not inhibit the pH-induced changes in [Ca2+]i. 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (100 microM), a Ca2+ chelator, induced a decrease in pHi as well as a reduction of [Ca2+]i, also supporting the direct relation between pHi and [Ca2+]i. 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)ocytl ester HCl (TMB-8) (100 microM), a known blocker of intracellular Ca2+ mobilization, did not change the resting pHi and [Ca2+]i in normal cells or cells acidified or alkalinized. This observation, taken together with data from cells incubated in the absence of external Ca2+, suggests intracellular Ca2+ pools are not involved in changes in [Ca2+]i that result from a modification of pHi. Resting pHi and [Ca2+]i in cells treated with either 8-bromo-dibutyryl cAMP (1 mM) or forskolin (150 microM) are not changed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular pH of cardiac muscle after administration of potassium-magnesium-aspartate (author's transl)]

27 Sprague-Dawley rats were anesthetized with pentobarbital. Artificial ventilation was given by a Starling pump respirator via a tracheal tube. Intracellular pH of cardiac muscle was determined by means of the indirect procedure of measuring the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO) in intra- and extracellular spaces (DMO-method). 12 rats were injected i.p. with 0.33 mval potassium-magnesium-aspartate/100 g body weight and 15 rats served as controls. Using a regression analysis, the following relationships were obtained: 1. animals injected with potassium-magnesium-aspartate pHa = --0.75 log PaCO2 + 8.535, PHi = --0.30 log PaCO2 + 7.509, pHi = 0.41 pHa + 4.036; 2. control animals pHa = --0.59 log PaCO2 + 8.308, pHi = --0.27 log PaCO2 + 7.381, pHi = 0.47 pHa + 3.503. At a pCO2 of 40 torr a pHa of 7.33 (7.36) and a pHi of 7.03 (6.95) was obtained. At an arterial pH of 7.40 the pHi was 7.07 (6.98). The results of the control group are written in brackets. -- The experiments demonstrate an increase in the intracellular buffer bases after administration of potassium-magnesium-aspartate. This effect can be recognized by a concomitant increase in the intracellular pH of 0.09 compared to the control group.     

Levetiracetam inhibits Na+-dependent Cl-/HCO3- exchange of adult hippocampal CA3 neurons from guinea-pigs

The novel anticonvulsant levetiracetam (LEV) was tested for effects on bioelectric activity and intracellular pH (pHi) regulation of hippocampal CA3 neurons from adult guinea-pigs. In 4-aminopyridine-treated slices, LEV (10-100 microm) reduced the frequency of action potentials and epileptiform bursts of CA3 neurons by 30-55%, while the shape of these potentials remained largely unchanged. Suppressive effects were reversed by an increase of pHi with trimethylamine (TMA). Using BCECF-AM-loaded slices, we found that LEV (10-50 microm) reversibly lowered neuronal steady-state pHi by 0.19 +/- 0.07 pH units in the presence of extracellular CO2/HCO3- buffer. In the nominal absence of extracellular CO2/HCO3- or in Na+-free CO2/HCO3(-)-buffered solution, LEV had no effect on steady-state pHi. Recovery of pHi subsequent to ammonium prepulses remained unchanged in the absence of CO2/HCO3- buffer, but was significantly reduced by LEV in the presence of CO2/HCO3- buffer. These findings show that LEV inhibits HCO3(-)-dependent acid extrusion, but has no effect on Na+/H+ exchange. LEV did not affect Na+-independent Cl-/HCO3- exchange because intracellular alkalosis upon withdrawal of extracellular Cl- remained unchanged. These data show that LEV at clinically relevant concentrations inhibits Na+-dependent Cl-/HCO3- exchange, lowers neuronal pHi, and thereby may contribute to its anticonvulsive activity.     

Effects of trimetazidine on pHi regulation in the rat isolated ventricular myocyte.

1. We have examined the effects of trimetazidine (TMZ) on intracellular pH (pHi) regulation in rat isolated ventricular myocytes. pHi was recorded ratiometrically by use of the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphtorhodafluor). 2. Following an intracellular acid load (induced by 10 mM NH4Cl removal), pHi recovery in HEPES-buffered Tyrode solution was significantly slowed down upon application of 0.3 mM TMZ only when myocytes were pretreated for 5 h 30 min (slowing by approximately 50%; P < 0.01). This effect of TMZ on pHi recovery was shown to be not only time- but also dose-dependent with a large, quickly reversible, effect obtained with 1 mM TMZ applied for 2-3 h (slowing by approximately 64%; P < 0.001). This slowing of pHi recovery was also associated with a decrease of the NH4+ removal-induced acidification. 3. Relationship between intracellular intrinsic buffering power (beta i) and pHi was assessed in absence or presence of TMZ (0.3 mM or 1 mM). As expected, beta i increased roughly linearly with a decrease in pHi in all cases. However, both concentrations of TMZ significantly increased beta i (by approximately 55 and 65% at pHi 7.1, respectively). 4. When Na+/H+ exchange was inhibited by dimethyl amiloride (DMA; 40 microM), trimetazidine (1 mM) did not change the H+ flux estimated at pHi 7.1 (0.31 +/- 0.03 mequiv l-1 min-1, n = 5, control, versus 0.30 +/- 0.025 mequiv l-1 min-1, n = 5, TMZ), ruling out any effect of TMZ on background acid loading. 5. Acid efflux carried by Na+/H+ exchange was significantly decreased only when myocytes were pretreated with 1 mM TMZ, for 2-3 h (JeH = 2.86 +/- 0.38 mequiv l-1 min-1, n = 26, control, versus 1.66 +/- 0.26 mequiv l-1 min-1, n = 10, TMZ, estimated at pHi 7.1; P < 0.05). 6. In conclusion, the present work demonstrates that, following an intracellular acid load in HEPES-buffered medium, trimetazidine slows down pHi recovery in rat isolated ventricular myocytes, primarily through an increase of beta i. An effect on Na+/H+ exchange is also detected but only after long-term incubation of the myocytes with TMZ.     

心肌肥厚对κ阿片受体介导的信号通路受损

目的：分离正常及慢性缺氧性右心肥厚的心室肌细胞,观察细胞内[Ca^2 ]i及细胞内pHi对心肌κ－阿片受体激动后的反应。方法：以fura-2和BCECF分别为[Ca^2 ]i和pHi的指示剂,用光谱荧光法测定电刺激引起的细胞内[Ca^2 ]i瞬变及pHi。结果：κ阿片受体选择性激动剂U50,488H可降低电刺激引起的[Ca^2 ]i瞬变和增加pHi,该作用是由蛋白激酶C（PKC）所介导。在肥厚的心室肌细胞,U50,488H的上述作用显著减弱。与此相对应,PKC的激动剂PMA引起的[Ca^2 ]i瞬变降低和pHi的增加作用在肥厚的心室肌细胞亦消失。用NH4C1法观察Na^ -H^ 交我器的功能显示其在肥厚的心室肌细胞无明显改变。结论：心肌肥厚时κ－阿片受体介导的信号通路受损,受损部位发生在PKC与效应器之间。     

Electrochemical behavior and square wave voltammetric determination of doxorubicin hydrochloride

The electrochemical behavior of doxorubicin hydrochloride was investigated by cyclic voltammetry (CV) and square wave voltammetry (SWV). From CV and SWV studies of doxorubicin hydrochloride in the acetate buffers of various pH values, it was found that protons were involved in the reduction of the antibiotic at the H+/e- ratio at one (deltaEp/pH = -53 approximately -61 mV at 23 degrees C), proposing the electrochemical reduction of the quinone moiety in its anthraquinone aglycone. Its electrochemical behavior was pseudo-reversible in the acetate buffer of pH 3.5 by exhibiting the well-defined single cathodic and anodic waves and the ratio of Ip(a)/Ip(c) at approximately one over the scan rates of 10 approximately 100 mV/s. Fast and sensitive SWV showing a single peak of doxorubicin has been applied for its quantitative analysis using an acetate buffer of pH 3.5. A linearity was obtained when the peak currents (Ip) were plotted against concentrations of doxorubicin in the range of 5.0 x 10(-7) M approximately 1.0 x 10(-5) M with a detection limit of 1.0 x 10(-7) M.     

Non-invasive single cell pH measurements in the isolated perfused pancreas

1. A new method was developed for non-invasive investigations of intracellular pH (pHi) regulation in different cell types of the isolated perfused pancreas using a confocal laser scanning technique. 2. After removal of the rat pancreas the coeliac artery was cannulated and the splenic segment of the pancreas was perfused with dextran (5%)-Ringer solution at a constant flow rate of 2 mL/min. In a temperature-controlled (37 degrees C) chamber, pH regulation was studied using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein (BCECF) with a confocal microscope (MRC-600; Bio-Rad, Hercules, CA, USA). 3. Image analysis permitted the identification and comparison of different cell types with a pHi of 7.26+/-0.1 in acinar cells and of 7.02+/-0.1 in endothelial cells. Increasing PCO2 from 5 to 20% resulted in a rapid decrease in pHi. Omission of sodium from the perfusate resulted in a smooth decline in pHi. Both decreases were found to be fully reversible. Increasing PCO2 under sodium-free conditions also resulted in a drop of pHi that was, however, not fully reversible, suggesting involvement of the Na+/H+ exchanger in the regulation of pHi in the intact organ. 4. The above method completely preserves tissue integrity and, therefore, allows the study of pH regulation in different cell types of the pancreas simultaneously and without interference with their functional arrangement. The technique should be of specific value to investigate experimental disease states of the pancreas.     

Verapamil和Mn~（2＋）对缺氧及缺氧复氧心肌细胞内pH的影响

采用荧光探针ＢＣＥＣＦ／ＡＭ结合计算机图像处理技术测定不同时间的缺氧和缺氧复氧单心肌细胞内ｐＨ的变化以及Ｃａ２＋通道阻滞剂Ｖｅｒａｐａｍｉｌ和Ｎａ＋－Ｃａ２＋交换抑制剂Ｍｎ２＋对其的影响。结果显示随着缺氧时间的延长，细胞内ｐＨ也逐渐降低。复氧开始４０ｍｉｎ内，细胞内ｐＨ并未恢复正常。Ｖｅｒａｐａｍｉｌ能减轻缺氧细胞内酸化程度并使其接近正常水平（Ｐ＞０．０５），却未能减轻缺氧复氧细胞内的酸化。无论是缺氧或缺氧复氧心肌细胞，Ｍｎ２＋均未能减轻细胞内的酸化程度。本实验结果提示缺氧和缺氧复氧时细胞内酸化途径并非完全一致。ＶｅｒａＰａｍｉｌ抑制缺氧细胞内ｐＨ下降是其保护缺氧心肌作用的机制之一。     

Influence of cytoplasmic pH on the aggregation and Ca2+ mobilization in rabbit platelets.

The aggregability of rabbit platelets was studied under various cytoplasmic pHs (pHi). Nigericin, a K+/H+ ionophore, which can induce a decrease in pHi, at 2-10 microM in 2 min incubation reduced both platelet aggregation and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) stimulated with thrombin or U46619. The reduced aggregability recovered 10 min after incubation with nigericin in parallel with an increase in pHi. In contrast, when pHi was increased by simultaneous addition of NH4Cl, methylamine or monensin, aggregation in response to a low concentration of thrombin, U46619, arachidonic acid or A23187 was enhanced significantly. The enhancing effect of NH4Cl was lowered by prolonged incubation with NH4Cl, by which the increased pHi was improved concomitantly. Indomethacin, an inhibitor of cyclooxygenase, failed to inhibit the enhancement of aggregation by NH4Cl under stimulation with U46619. In addition, treatment with NH4Cl enhanced an increase in [Ca2+]i in response to U46619 in a concentration-dependent manner, although the treatment by NH4Cl alone did not affect [Ca2+]i. When pHi was artificially altered during the ranges of 6.6-7.4 by treatment with nigericin in K+-rich medium, aggregation by low concentrations of thrombin was dependent on the pHi. These data indicate that pHi is an important factor for platelet activation including intracellular Ca2+ mobilization and aggregation.     

心肌肥厚时κ阿片受体介导的信号通路受损(英文)

目的:分离正常及慢性缺氧性有心肥厚的心室肌细胞,观察细胞内[Ca~(2 )]_i及细胞内pH_i对心肌к-阿片受体激动后的反应。方法:以fura-2和BCECF分别为[Ca~(2 )]_i和pH_i的指示剂,用光谱荧光法测定电刺激引起的细胞内[Ca~(2 )]_i瞬变及pH_i。结果:к阿片受体选择性激动剂U50,488H可降低电刺激引起的[Ca~(2 )]_i瞬变和增加pH_i,该作用是由蛋白激酶C(PKC)所介导。在肥厚的心室肌细胞,U50,488H的上述作用显著减弱。与此相对应,PKC的激动剂PMA引起的[Ca~(2 )]_i瞬变降低和pH_i的增加作用在肥厚的心室肌细胞亦消失。用NH_4Cl法观察Na~ -H~ 交换器的功能显示其在肥厚的心室肌细胞无明显改变。结论:心肌肥厚时к-阿片受体介导的信号通路受损,受损部位发生在PKC与效应器之间。     

Alpha 1-adrenoceptor stimulation increases intracellular pH and Ca2+ in cardiomyocytes through Na+/H+ and Na+/Ca2+ exchange

The effects of alpha 1-adrenergic stimulation on intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were investigated in isolated rat cardiomyocytes with fluorescence dyes, BCECF and fura-2, respectively. In the presence of 5 or 25 mM HCO3- norepinephrine (NE) increased pHi in a dose-dependent manner. Intracellular alkalinization was inhibited by prazosin and phentolamine but not by yohimbine. NE-induced alkalinization was inhibited in the presence of a Na+/H+ exchange inhibitor (5-(N,N-hexamethylene) amiloride (HMA)), a C kinase inhibitor (H-7) or a calmodulin inhibitor (W-7), or in the absence of extracellular Na+. NE also increased [Ca2+]i following the pHi increase, which was abolished in the absence of extracellular Na+ or Ca2+. This Ca2+ influx was inhibited by HMA but not by diltiazem (10(-5) M). Thus, we conclude that alpha 1-adrenergic stimulation enhances Na+/H+ exchange by activation of C kinase, thereby allowing intracellular alkalinization, and that subsequent activation of Na+/Ca2+ exchange increases Ca2+ influx.     

细胞内pH对胆碱能受体介导的Kir3.1/3.4电流的调节

目的　研究细胞内pH对胆碱能受体介导的Kir3 1/3 4电流的调节作用。方法　应用AzideNa、KHCO3 和通过灌流直接降低细胞内 pH ,用双电极电压钳和膜片钳方法观察在蛙卵细胞中表达的Kir3 1/ 3 4钾离子通道电流的变化和M受体激活对Kir3 1/ 3 4电流调节的变化。结果　细胞内 pH降低能抑制Kir3 1/ 3 4的电流 ;Kir3 1/ 3 4对细胞内pH的敏感性介于另外两种Kir通道Kir2 1和Kir2 3之间 ,即这三种通道对细胞内 pH的敏感性依次为Kir2 3>Kir3 1/ 3 4 >Kir2 1;细胞内pH降低能够减弱M1受体激活对Kir3 1/ 3 4的抑制作用 ,加强M2 受体激活Kir3 1/ 3 4电流后的去敏作用。结论　在维持细胞静息电位方面起重要作用的Kir3 1/ 3 4通道在细胞内pH降低的情况下基础电流和M受体激活调节电流均发生了变化 ,这些变化在缺血缺氧引起的细胞 (如心肌细胞 )兴奋性改变中可能有重要的生理学意义     

Effects of lactate on intracellular pH and hypercontracture during simulated ischemia and reperfusion in cardiac ventricular myocytes of the guinea pig.

Effects of lactate on changes in intracellular pH (pHi) and contractility during simulated ischemia and reperfusion were examined in single myocytes of the guinea pig cardiac ventricle. The conditions of simulated ischemia were produced by the exchange of perfusion medium from the standard one oxygenated with 95% O2-5% CO2 gas (pH 7.4) to one containing no glucose, 8 mM K+, and 0-30 mM sodium-D,L-lactate and was gassed with 90% argon - 10% CO2 (pH 6.6). The pHi was decreased by the simulated ischemia from approx. 7.3 to approx. 6.9 regardless of lactate concentration, while the rate of pHi decrease was increased by lactate in a concentration-dependent manner. The contraction induced by electrical stimulation disappeared faster in the presence of lactate. The incidence of irreversible hypercontracture of myocytes was significantly reduced by 20-30 mM lactate. The overshoot of pHi to approx. 7.7 and excess contractions were induced by withdrawal of lactate during the reperfusion, but not observed when lactate was continuously present. The recovery of normal contractility during reperfusion was facilitated by lactate. It can be concluded that lactate added to or removed from the perfusion medium increases the rate of pHi change under the simulated ischemia and reperfusion, respectively, and that the continuous presence of lactate reduces cell injury under these conditions.     

Decrease in internal H+ and positive inotropic effect of heptaminol hydrochloride: a 31P n.m.r. spectroscopy study in rat isolated heart.

1. The cardiotonic effect of heptaminol hydrochloride (Hept-a-myl, Delalande) was studied using 31P-nuclear magnetic resonance (n.m.r.) spectroscopy and left ventricular pressure (LVP) measurements in rat isolated hearts. The possibility of this effect being mediated by an intracellular realkalinisation was tested. 2. Isolated hearts were perfused at 10 ml min-1 by the Langendorff method with Krebs-Henseleit solution at 37 degrees C and stimulated at 5 Hz. Mechanical activity was measured as variations of left ventricular pressure (LVP). 31P-n.m.r. spectra were recorded every 2 min. Changes in cardiac adenosine triphosphate (ATP), phosphocreatine (PCr) and inorganic phosphate (Pi) were followed and intracellular pH (pHi) was estimated from the chemical shift of Pi. 3. The effects of heptaminol were tested in different conditions: normoxia, moderate ischaemia, severe ischaemia, and moderate ischaemia in the presence of amiloride or guanidinium chloride as inhibitors of the Na-H exchange. 4. In normoxia, heptaminol induced a cyclic increase of systolic LVP, associated with an increase in Pi. No significant effect on pHi was observed. In changing from normoxia to moderate ischaemia, PCr and systolic LVP decreased; a mild intracellular acidification (pHi 6.96) was obtained. Heptaminol induced a restoration of pHi and increased LVP. In severe ischaemia, the realkalinization effect and the restoration of LVP induced by heptaminol were no longer observed. During moderate ischaemia, Na-H exchange inhibitors decreased pHi and LVP. Heptaminol applied in the presence of these inhibitors was unable to restore pHi and LVP.(ABSTRACT TRUNCATED AT 250 WORDS)