Intracellular Ca antagonist TMB-8 blocks catecholamine secretion evoked by caffeine and acetylcholine from perfused cat adrenal glands in the absence of extracellular Ca

Unlike acetylcholine, caffeine was much more effective in releasing catecholamine in the absence of extracellular Ca²⁺ than in its presence in perfused cat adrenal glands. The intracellular Ca²⁺ antagonist, TMB-8 (10⁻⁴ M), inhibited reversibly the catecholamine secretion evoked by caffeine (40 mM) and that induced by acetylcholine (10⁻⁴ M) in the presence of hexamethonium (10⁻³ M) during perfusion with Ca²⁺-free Locke solution containing EGTA (10⁻⁵ M). These results support our view that muscarinic receptor activation causes catecholamine secretion by mobilizing Ca²⁺ from an intracellular pool just as caffeine does.     

A simple method for monitoring changes in cell height using fluorescent microbeads and an Ussing-type chamber for the inverted microscope

In this study, we report two developments for studies of ion transport in cultured epithelial cells. First, a convenient method is presented for measuring apparent cell height using fluorescent microbeads as high-contrast landmarks of the apical and basal cell surfaces. The apparent cell height is then used as an indicator to monitor the time course of changes in cell volume in response to osmotic perturbations. Second, an Ussing-type chamber design for the inverted fluorescence microscope is presented, which allows determination of transepithelial electrical properties. Using these two methods, we obtained simultaneous measurements of cell height and transepithelial electrical parameters for cultured renal (A6) epithelium. Cell height was measured by alternately focusing the microscope between microbeads marking the apical and basal surfaces. The distance between these two surfaces was measured electrically from the voltage output of a potentiometer that was mechanically coupled to the fine-focusing knob of the microscope. Following decreases in the bathing solution osmolality, the cell height and transepithelial Na⁺ transport rate (measured as short-circuit current, I _SC) increased. The increase in cell height preceded changes in I _SC by several minutes, suggesting a lack of direct linkage between changes in cell volume and transepithelial Na⁺ transport. Both the fluorescent microbead cell height method and the Ussing-type chamber can be used in conjunction with patch-clamp techniques, intracellular microelectrode impalements, or fluorescent probes of intracellular composition. Therefore, this system may be advantageous for studies of epithelial cell volume and channel regulation.     

Removal of optic tectum prolongs the cell body reaction to axotomy in goldfish retinal ganglion cells

Injury to the optic axons of goldfish elicits dramatic changes in the cell bodies of the neurons from which these axons arise, the retinal ganglion cells. The changes include a large increase in cell size and in synthesis and axonal transport of protein. The cells begin to return to normal about 3 weeks after the injury, when the axons invade the contralateral (homotopic) lobe of the optic tectum, and recovery is essentially complete by 8-10 weeks after the lesion. However, if the homotopic lobe of the tectum was removed at the time of nerve crush, we found that the cell body reaction was greatly prolonged. The cells remained enlarged, and [3H]proline incorporation and fast axonal transport of protein remained elevated, until at least 10-12 weeks after nerve crush, although by this time most of the regenerating axons had probably regained their normal length and many had entered the remaining ipsilateral (heterotopic) lobe of the tectum. The cells showed partial recovery by the latest time tested, 26 weeks after nerve crush, when the projections from the two eyes had segregated into separate bands in the heterotopic tectal lobe.     

N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine (DPPE), a chemopotentiating and cytoprotective agent in clinical trials: interaction with histamine at cytochrome P450 3A4 and other isozymes that metabolize antineoplastic drugs

Purpose: N,N-diethyl-2-[4-(phenylmethyl)phe- noxy]ethanamine · HCl (DPPE), an intracellular histamine (HA) antagonist with chemopotentiating and cytoprotective properties, is currently in phase 2 and 3 clinical trials in breast and prostate cancer. DPPE modulates growth at in vitro concentrations that antagonize HA binding to cytochromes P450 in rat liver microsomes. HA inhibits P450 metabolism of some drugs. Recent in vitro studies in human colon cancer cells have linked DPPE enhancement of paclitaxel, doxorubicin and vinblastine cytotoxicity to inhibition of the P-glycoprotein (P-gp) pump. Many substrates of P-gp are also substrates of CYP3A4, a P450 isozyme that metabolizes a variety of antineoplastic agents and is highly expressed in some malignant tissues. Therefore, we assessed whether (a) DPPE and HA interact at CYP3A4 and other P450 human isozymes, and (b) DPPE inhibits the catalytic activity of CYP3A4. Methods: Using spectral analysis, we measured DPPE and HA binding to insect microsomes that express human P450 isozymes 1A1, 2B6, 2D6 or 3A4. Employing thin-layer chromatography, we assessed the metabolism of DPPE by each isozyme and DPPE inhibition of testosterone metabolism by CYP3A4 and by rat liver microsomes. Results: (1) DPPE evoked “type I” (substrate site binding) absorbance-difference spectra with CYP2D6 (K_s=4.1 ± 0.4 μM), CYP3A4 (K_s= 31 ±15 μM) and CYP1A1 (K_s=40 ± 9 μM), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1; no metabolism occurred with CYP2B6. (3) HA evoked “type II” (heme iron binding) absorbance-difference spectra with all four isozymes, with K_s values in the range 80–600 μM. DPPE inhibited HA (600 μM) binding to CYP2D6 (IC₅₀=4 μM, 95% CI=1.8–8.9 μM) and CYP1A1 (IC₅₀=135 μM: 95% CI=100–177 μM), but stimulated HA (500 and 1000 μM) binding to CYP3A4 (EC₅₀=155 μM, 95% CI=104–231 μM). DPPE did not affect HA binding to CYP2B6. (4) DPPE inhibited the metabolism of testosterone by CYP3A4. The concentration/effect curve was biphasic: DPPE inhibited metabolism by 30% at the first site (IC₅₀=3 μM, 95% CI=0.5–25.5 μM), and an additional 70% inhibition occurred at the second site (IC₅₀=350 μM, 95% CI=215–570 μM). A similar result was observed with rat liver microsomes. Conclusion: DPPE is a substrate for CYP3A4, CYP2D6 and CYP1A1, but not CYP2B6. DPPE inhibits testosterone metabolism by interacting at two sites on CYP3A4, the first correlating with its K_s value to bind the substrate site and the second, with its EC₅₀ value to enhance HA binding to the heme iron. We postulate that (1) the inhibitory effect of DPPE on CYP3A4 activity is mediated directly at the substrate site and indirectly by its enhancement of the binding of HA to the heme moiety; (2) in tumor cells that express high constitutive levels of CYP3A4, potentiation of chemotherapy cytotoxicity by DPPE results, in part, from inhibition of CYP3A4-mediated metabolism and P-gp-mediated efflux of antineoplastic drugs; (3) in normal cells that express low constitutive levels of the isozyme, cytoprotection by DPPE results, in part, from induction of CYP3A4 and P-gp, resulting in an increase both in metabolism and efflux of antineoplastic drugs. Received: 26 April 1999 / Accepted: 3 September 1999     

皮瓣血运观察记录单在皮瓣手术患者中的应用效果

目的探讨皮瓣血运观察记录单在皮瓣手术患者中的应用效果。方法选取2018年6月至2019年5月我院应用皮瓣血运观察记录单的48例皮瓣手术患者作为观察组,选取2017年6月至2018年5月我院未应用皮瓣血运记录单的48例皮瓣手术患者作为对照组。比较两组的护理效果。结果观察组静脉及动脉血管危象检出率、护理满意度高于对照组,护理不良事件总发生率低于对照组(P<0.05)。观察组的护理工作效率评分及护理差错评分均高于对照组(P<0.05)。结论皮瓣血运观察记录单在皮瓣手术患者中的应用效果显著,不仅能提高静脉与动脉血管危象检出率,而且还能够提高护理满意度,减少护理不良事件的发生。     

Effects of parathyroid hormone and calcitonin on Na+, Cl−, K+, Mg2+ and Ca2+ transport in cortical and medullary thick ascending limbs of mouse kidney

The effect of parathyroid hormone (PTH) on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ transport was investigated in isolated perfused cortical thick ascending limbs (cTAL) and that of human calcitonin (hCT) was tested in both cortical and medullary thick ascending limbs (mTAL) of the mouse nephron. The transepithelial ion net fluxes (J _x) were determined by electron probe analysis of the perfused and collected fluids. Simultaneously, the transepithelial voltage (PD_te) and resistance (R _te) were recorded. In cTAL segments, PTH and hCT significantly stimulated the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺. hCT generated a net K⁺ secretion towards the lumen and PTH tended to exert the same effect. Neither PD_te nor R _te were significantly altered by either PTH or hCT. However, in the post-experimental period a significant decrease in PD_te was noted. Time control experiments carried out under similar conditions revealed a significant decrease in PD_te with time, which could have masked the hormonal response. In mTAL segments, Mg²⁺ and Ca²⁺ transport was close to zero. hCT did not exert any detectable effect on either PD_te or J _Cl ^–, J _Na ⁺ J _K ⁺, J _Mg ²⁺ and J _Ca ²⁺ in these segments. In conclusion, our data demonstrate that PTH and hCT stimulate NaCl reabsorption as well as Mg²⁺ and Ca²⁺ reabsorption in the cTAL segment of the mouse. These data are in agreement with and extend data obtained in vivo in the rat.     

HCO 3 − -dependent ACh-activated Na+ influx in sheep parotid secretory endpieces

In the present study we used the Na⁺-sensitive fluorescent dye SBFI and optical measurement of endpiece volume to investigate the transport of Na⁺ in sheep parotid secretory cells. Sheep parotid endpiece cells bathed in a HCO ₃ ^– -free Cl^–-rich solution had a resting intracellular Na⁺ concentration ([Na⁺]_i) of 17±2 mmol/l (n=39). Exposure of the cells to a 2-min pulse of acetylcholine (ACh) (3×10^–7 mol/l) in a HCO ₃ ^– -free bathing solution produced no change in [Na⁺]_i or in cell volume. Changing from a Cl^–-containing HCO ₃ ^– -free bath solution to a Cl^– solution containing 25 mmol/l HCO ₃ ^– caused the endpieces to swell by 8±2 % (n=11) and the [Na⁺]_i to increase by 10±2 mmol/l (n=14). Subsequent exposure of the cells to ACh led to shrinkage of the cells by 12±2 % from the volume in the HCO ₃ ^– -containing solution prior to ACh exposure, with the maximum decrease occurring after 29±7 s (n=9). This shrinkage was accompanied by a rapid and transient increase in [Na⁺]_i, the [Na⁺]_i reaching a peak at 70±5 mmol/l above the unstimulated level (n=9). Substitution of gluconate for Cl^– did not significantly alter the effects of HCO ₃ ^– on unstimulated [Na⁺]_i or endpiece volume, nor did it significantly inhibit the effects of ACh on these two parameters when HCO ₃ ^– was present. Addition of 200 mol/l dihydrogen-4,4-diisothiocyanatostilbene-2,2-disulfonic acid (H₂-DIDS) to the gluconate/HCO ₃ ^– solution significantly reduced the peak of the ACh-induced increase in [Na⁺]_i to 34±10 mmol/l (n=4), but did not have any significant effect on the magnitude of the ACh-induced shrinkage. At 500 mol/l, H₂-DIDS abolished the ACh-induced increase in [Na⁺]_i and also significantly reduced the shrinkage due to ACh. Finally, we found that the rate of endpiece shrinkage following ACh stimulation did not depend on the presence of Cl^–.We interpret these results as indicating that sheep parotid secretory cells do not contain significant Na⁺-K⁺-2Cl^– co-transport activity and do not actively accumulate Cl^–. Rather, the mechanism of spontaneous basal secretion by these cells, in the presence of extracellular HCO ₃ ^– , is based on the accumulation of HCO ₃ ^– by the Na⁺-H⁺ exchanger. During ACh stimulation, the concentration of HCO ₃ ^– in the cytosol is also maintained by the operation of a H₂-DIDS-sensitive Na⁺-HCO ₃ ^– co-transporter. HCO ₃ ^– efflux across the apical membrane occurs via a HCO ₃ ^– conductance pathway rather than by the coupled operation of a Cl^– channel and a Cl^–-HCO ₃ ^– exchanger.     

核苷酸转运体表达与吉西他滨治疗胰腺癌患者临床疗效相关性的研究进展

目的 探讨核苷酸转运体表达与吉西他滨抗胰腺癌临床疗效相关性的研究进展。方法 通过查阅国内外文献,对相关研究归纳、总结进行综述。结果 吉西他滨是胰腺癌化疗的一线药物,较大个体化差异和有效率偏低是目前临床应用的挑战,核苷酸转运体是吉西他滨摄取入细胞的内吞转运体,它的蛋白或mRNA表达可能与接受吉西他滨治疗胰腺癌患者的临床疗效和不良反应相关,但尚未达成共识。结论 核苷酸转运体表达与吉西他滨抗胰腺癌临床疗效的相关性还需要进一步研究,为吉西他滨在胰腺癌中实现临床个体化治疗提供参考依据。     

Atrial natriuretic factor (ANF) does not affect ion transport in human intestine but does in porcine intestine

The aim of this study was to test whether atrial natriuretic factor (ANF) exerts any effect on human intestinal ion transport, and the porcine intestine was used as a positive control of ANF's effects. Tissues from human proximal (n = 6) and distal (n = 6) colons, and from distal ileum (n = 6) were mounted in Ussing chambers, and short circuit current (I_sc) was measured subsequent to serosal application of ANF (10^--⁶ m), 8–Br-cyclic guanosine monophosphate (8–Br-cGMP) (10^--⁴ m), and theophylline (10^--² m). ANF did not affect I_sc whereas 8–Br-cGMP increased I_sc by 28 (8–53), 16 (3–36), and 16 (5–41) μA cm^-2 in the distal colon (DC), proximal colon (PC) and distal ileum (DI), respectively. Likewise, transepithelial potential difference (PD) became more negative by 5.0 (0.6–8.9), 2.5 (0.4–4.0) and 0.9 (0.3–2.3) mV in DC, PC, and DI, respectively, subsequent to addition of 8–Br-cGMP. I_sc and PD were further increased by theophylline. Additional radio-isotope flux studies in human colon revealed that ANF did not affect electroneutral sodium and chloride transport either. For comparison, ANF (10^--⁶ m) was administered to large intestinal tissues from young pigs in which ANF induced a significant increase in I_sc which was comparable to the 8–Br-cGMP response in humans. The porcine I_sc response was partly inhibited by chloride-free solution on the serosal side, by serosal application of bumetanide (10^--⁴ m) and BaCl₂ (10^--³ m), and mucosal application of the chloride-channel blocker diphenylamine-2–carboxylate (DPC) (10^--³ m). Mucosal amiloride (10^--⁵ m) pre-treatment reduced baseline I_sc but did not affect the porcine intestinal I_sc response to ANF. In vitro radio-autography demonstrated specific binding sites for ANF in porcine distal colon, whereas no apparent labelling was observed in human distal colon. These findings suggest that the lack of effect of ANF on sodium and chloride transport in human distal ileum and colon is probably due to lack of ANF receptors. In the porcine intestine, however, the I_S0 response induced by ANF seems to involve stimulation of electrogenic chloride secretion, whereas electrogenic sodium absorption seems unaffected.     

Surface immunoglobulins mediate efficient transport of antigen to lysosomal compartments resulting in enhanced specific antigen presentation by B cells

A BCL₁ immunoglobulin (Ig) transfectant, expressing wild-type surface (s)IgM with the TEPC-15 idiotype (T15-Id) and anti-phosphorylcholine (PC) specificity, was previously shown to present PC-conjugated hen egg-white lysozyme (PC-HEL) to a HEL-specific T cell hybridoma at a lower antigen (Ag) concentration than that required for native HEL. Two variant Ig transfectants, expressing T15-Id sIgM with substitutions either in the entire spacer, transmembrane (TM) domain and cytoplasmic tail (B186 variant) or in the NH₂-terminal third of TM domain only (TM2 variant), failed to display this sIgM-mediated, enhanced presentation of PC-HEL at low concentrations. However, prolonged treatment with anti-T15-Id monoclonal antibody (mAb) led to a reduction of surface expression of the T15-Id sIgM in the wild-type and TM2 variant, but not in the B186 variant sIgM transfectants. Treatment with anti-T15-Id mAb also resulted in an increased intracellular accumulation of T15-Id sIgM in the wild-type transfectant, but not in the B186 variant. Subcellular fractionation analysis revealed that the ligands bound to the T15-Id sIgM are not efficiently transported to the dense lysosomal compartments in both B186 and TM2 transfectants, as compared to the wild-type sIgM transfectant. A significant increase in tyrosine phosphorylation after cross-linking of the T15-Id sIgM was observed only in the wild-type sIgM transfectant. These results suggest that, while the NH₂-terminal third of the TM region is not involved in the process responsible for the ligand-induced reduction of surface expression of sIgM, it appears to be essential for subsequent transport of sIgM/ligand complexes to the lysosomal compartments, as well as efficient activation of tyrosine kinases. These results strongly suggest that sIg-mediated enhancement of specific antigen presentation reflects the ability of sIg to efficiently transport antigen to the lysosomal compartments, and possibly the activation of protein tyrosine kinases.