Relationship between Para-aminohippurate Secretion and Cellular Morphology in Rabbit Proximal Tubules

Previous studies in the mammalian proximal tubule have suggested that para-aminohippurate (PAH) secretion is ~threefold greater in the straight segment, or pars recta, than in the convoluted segment, or pars convoluta. However, the possibility that the site of maximal PAH secretion might be related better to particular tubule segments as identified by cell type had not been explored. In addition, the presence or absence of differences in PAH secretion between morphologically identical regions of superficial (SF) vs. juxtamedullary (JM) proximal tubules has not been examined. These issues were studied using a combination of histologic methods and measurement of [³H]PAH secretion in isolated perfused tubules. Measurements of microdissected SF and JM proximal tubules from young and adult rabbits revealed that SF proximal tubules were slightly but significantly longer than JM tubules ([young rabbits: SF, 8.69±SE 0.14 mm vs. JM, 7.97±SE 0.13 mm; P < 0.01] [adult rabbits: SF, 10.61±SE 0.28 mm; JM, 9.17±SE 0.19 mm; P < 0.001]). Light and electron microscopy revealed three sequential segments (S₁, S₂, and S₃) along the length of SF and JM proximal tubules as defined by cell type. PAH secretion was measured in each of these three segments by the isolated perfused tubule technique. Net PAH secretion in fmol/mm per min in SF proximal tubules was: S₁, 281±SE 21; S₂, 1,508±SE 104; S₃, 318±SE 46. Corresponding values in JM proximal tubules were 353±SE 31, 1,391±SE 72, and 188±SE 23. Net PAH secretion did not differ between comparable segments of SF and JM proximal tubules. It is concluded that differences in PAH secretion along the proximal tubule correlate best with cell type rather than the arbitrary division of the proximal tubule into pars convoluta and pars recta according to its external configuration. Evidence of functional heterogeneity between comparable segments of SF and JM proximal tubules was not observed.     

The Mechanism of Bicarbonate Secretion in Rabbit Ileum Exposed to Choleragen

BICARBONATE MAY BE SECRETED INTO THE INTESTINAL LUMEN IN CHOLERA BECAUSE: HCO(3) (-) ions are transported, or because OH(-) ions accumulate and react with dissolved CO(2) to form HCO(3) (-). If HCO(3) (-) ions are transported into the lumen from the interstitial fluid, lumenal P(CO2) should increase (HCO(3) (-) right harpoon over left harpoon OH(-) + CO(2)); if OH(-) accumulates, P(CO2) should diminish. Net movement of H(2)O, and HCO(3) (-), and changes in pH and P(CO2) in lumenal fluid were studied in adjacent segments of rabbit ileum in vivo, one of which was exposed to choleragen. 4 h after exposure, segments were drained and infused with gassed Krebs-Henseleit solution whose P(CO2) exceeded arterial P(CO2). After 45 min, fluid was collected anaerobically from control and cholera segments. Among 13 cholera segments, lumenal P(CO2) diminished by a mean of 8.4 torr and was less than femoral arterial blood in six instances. In the paired control segments, mean P(CO2) increased by 4.4 torr, and was always greater than arterial P(CO2). Dilution could not account for the low P(CO2) in cholera segments because in hypertonic solutions that caused water to move into the lumen, the P(CO2) did not differ from control values obtained with isotonic solutions. The results suggest that OH(-) accumulation (by addition of OH(-) or removal of H(+)) causes HCO(3) (-) secretion in cholera. This does not result from secretion of some other base (e.g., HPO(4) (-)), because HCO(3) (-) accounts for most of the base in the lumenal fluid. The P(CO2) changes suggest that OH(-) reacts with CO(2) at the cell-lumen interface, but reaction at the cell-interstitial fluid interface cannot be excluded.     

Study of Chloride Transport Across the Rabbit Cortical Collecting Tubule

Recent micropuncture studies have suggested that the collecting tubule may be involved in the regulation of extracellular fluid volume. The present studies were designed to evaluate chloride transport across the in vitro-perfused rabbit cortical collecting tubule inasmuch as chloride ion would ultimately affect extracellular fluid volume. The tubules were perfused and bathed with artificial solutions simulating ultrafiltrate. Four groups of studies were conducted. In groups one and two, tubules from rabbits not receiving desoxycorticosterone (DOCA) were compared to tubules from rabbits which had received DOCA (5 mg/day) for 1 wk. In groups three and four, tubules were obtained only from rabbits not receiving DOCA. In group one, sequential bidirectional chloride fluxes were measured. The ratio of chloride efflux to influx was 0.99±0.04 in tubules obtained from rabbits not receiving DOCA whereas it was 1.28±0.09 in tubules obtained from rabbits receiving DOCA, suggesting stimulation of net chloride flux under these conditions. In group 2, chemical chloride concentration and osmolality of the collected fluid were measured. Neither the chemical chloride concentration nor the osmolality of the collected fluid decreased significantly below their respective perfusion fluid values in tubules from non-DOCA-treated rabbits but there was a significant decrease in the chemical chloride concentration (10-42 meq/liter) and osmolality (10-42 mosmol/kg H₂O of the collected fluid in tubules from DOCA-treated rabbits. In group three, unidirectional chloride permeabilities from lumen-to-bath were determined during the passage of current down the perfusion pipette. The alterations of the average lumen potential, −35±4 and +28±2 mV, did not influence unidirectional chloride movement suggesting that the cortical collecting tubule is quite impermeable to chloride. In group four, unidirectional chloride permeability from lumen-to-bath was measured before and after substitution of NaCH₃SO₄ for sodium chloride in the bath. Replacement of chloride by CH₃SO₄ reversibly decreased the apparent chloride permeability from 2.41±0.50 to 0.69±0.08 (×10⁻⁵ cm/s) demonstrating that ³⁶Cl permeability is dependent on the chemical concentration of chloride.     

Dietary Modulation of Active Potassium Secretion in the Cortical Collecting Tubule of Adrenalectomized Rabbits

Addisonian patients can maintain potassium homeostasis despite the absence of mineralocorticoid. The present in vitro microperfusion studies examine what role the cortical collecting tubule might play in this process. All studies were performed on tubules harvested from adrenalectomized rabbits, which were maintained on 0.15 M NaCl drinking water and dexamethasone 50 μg/d. Perfusion and bath solutions were symmetrical Ringer's bicarbonate with [K] of 5 meq/liter. Initial studies on cortical collecting tubules from adrenalectomized animals ingesting a high potassium chow (9 meq K/kg body wt) demonstrated net potassium secretion against an electrochemical gradient (mean collected fluid [K] 16.5±2.6 meq/liter with an observed transepithelial voltage of −6.3±4.1 mV; predicted voltage for passive distribution of potassium being −28.2 mV). To examine whether this active potassium secretion could be modulated by dietary potassium, independent of mineralocorticoid, two diets identical in all respects except for potassium content were formulated. Potassium secretion was compared in cortical collecting tubules harvested from adrenalectomized animals on low (0.1 meq K) and high (10 meq K) potassium intake.     

Characteristics of Volume Reabsorption in Rabbit Superficial and Juxtamedullary Proximal Convoluted Tubules

Segments of superficial and juxtamedullary proximal convoluted tubules of the rabbit were perfused in vitro to examine the mechanisms responsible for net volume reabsorption. The very early postglomerular segments were not studied. Fluid reabsorptive rates and transepithelial potential differences were compared under various conditions: (a) with perfusate that simulated glomerular filtrate; (b) with perfusate that lacked glucose, amino acids, and acetate and that had HCO(3) and Cl concentrations of 5 and 140 mM, respectively; (c) with perfusate that lacked glucose, amino acids, and acetate but with 20 meq of NaHCO(3) replaced with 20 meq of Na cyclamate; (d) with the same perfusate as in b but in the presence of ouabain in the bath; (e) with ultrafiltrate of rabbit serum titrated with HCl to final HCO(3) and Cl concentrations of 2 and 134 mM, respectively. Tubules were perfused with this titrated ultrafiltrate at 37 degrees C, 21 degrees C, and in the presence of 0.1 mM ouabain in the bath. Bath fluid in all experiments was regular rabbit serum. Under conditions a and b superficial proximal convoluted tubule (SFPCT) and juxtamedullary proximal convoluted tubule (JMPCT) behaved similarly with the exception that SFPCT exhibited a lumen-positive and JMPCT a lumen-negative electrical potential under condition b. However, under condition c SFPCT failed to exhibit net volume reabsorption, whereas reabsorption in JMPCT continued unchanged. Ouabain did not affect volume reabsorption in SFPCT under condition d, whereas neither ouabain nor hypothermia affected SFPCT under condition e. In contrast, ouabain and hypothermia totally inhibited volume reabsorption in JMPCT under conditions d and e. These studies document heterogeneous mechanisms responsible for volume reabsorption in the major portions of SFPCT and JMPCT with passive forces predominating in SFPCT and active forces in JMPCT.     

In Vitro Uptake of Gentamicin by Rat Renal Cortical Tissue

The mechanism of gentamicin uptake in vitro by renal cortical slices of rat kidney was investigated. The cortical-slice-uptake ratio of gentamicin concentration in 1.0 g of tissue water to that of 1.0 ml of incubation medium (SW/M) was 1.44 ± 0.04. The uptake of gentamicin was inhibited by 2 × 10⁻⁵ M dinitrophenol (SW/M = 1.03 ± 0.04) and by anoxia (SW/M = 1.01 ± 0.04). The results indicate that aerobic phosphorylation is required to transport gentamicin into the cells. The uptake of p-aminohippurate and tetraethylammonium chloride by renal cortical slices was not affected by gentamicin.     

危重病肺分泌物不同收集法病原学检测的研究

目的 探讨建立人工气道患者肺分泌物的收集方法。方法 分别采用常规法(A)、1ml注射器法(B)和无菌玻璃小瓶法(C)收集肺分泌物,对其病原学检测的敏感性、可靠性进行比较。结果 B、C两法与A法相比,在病原学检出率方面差异有显著意义(P＜O．05),B、C两法无差异(P＞O．1),但在可操作性方面C法更优于B法。结论 无菌玻璃小瓶法是一种简便、快捷、可靠的收集肺分泌物进行病原学检测的方法。     

Glycoprotein Synthesis and Secretion by Mucosal Biopsies of Rabbit Colon and Human Rectum

Elucidation of mechanisms involved in the control of colonic production of mucus requires direct examination of glycoprotein synthesis and secretion by colonic mucosa. In the past, the limited viability of intestinal mucosa in vitro has hampered such investigations. When maintained in an organ culture system, mucosal biopsies of rabbit colon and human rectum remained viable for 24 h as documented by morphologic appearance and a steady rate of protein synthesis and secretion. These biopsies also incorporated (14)C-labeled glucosamine into tissue glycoproteins and secreted labeled glycoproteins at a steady rate for 24 h. Glucosamine was predominantly incorporated into macromolecules that were ultimately secreted, in contrast to leucine, which was predominantly incorporated into tissue macromolecules. When studied by autoradiography, cultured rabbit colonic biopsies synthesized and secreted glycoproteins in vitro at cellular sites and over a time-course similar to those previously described for the intestine of intact animals. Acetylcholine consistently stimulated secretion of labeled glycoproteins but did not alter glycoprotein synthesis. In contrast, cycloheximide inhibited glycoprotein synthesis but had no effect on the secretion of newly synthesized glycoproteins. Rectal biopsies from patients with active ulcerative colitis incorporated increased amounts of [(14)C]glucosamine into glycoproteins during organ culture and secreted labeled glycoproteins more rapidly into the incubation medium when compared to biopsies obtained from healthy volunteers These findings indicate that organ culture provides a useful means of directly examining the synthesis and secretion of glycoproteins by healthy and diseased colonic mucosa.     

Response of the Distal Tubule and Cortical Collecting Duct to Vasopressin in the Rat

Renal micropuncture observations in the rat suggest that the entire "distal tubule" (defined by the micropuncturist as that portion of the renal tubule extending between the macula densa and its first junction with another (renal tubule) may be responsive to vasopressin. However, this portion of the renal tubule contains two segments that are morphologically dissimilar. The "early" distal tubule is lined by epithelium characteristic of the distal convoluted tubule, while the "late" distal tubule is lined by epithelium characteristic of the cortical collecting duct. Thus, the present study was initiated to identify the most proximal site of action of vasopressin in the distal renal tubule. A water diuresis was established in rats with hereditary hypothalamic diabetes insipidus. In one-half of the animals the diuresis was interupted by an i.v. infusion of exogenous vasopressin. Morphological preservation of the kidneys was initiated after induction of vasopressin-induced antidiuresis or during maximum water diuresis. Cell swelling and dilatation of intercellular spaces, morphological findings indicative of vasopressin responsiveness, were observed in the cortical collecting duct including the late segment of the distal tubule, a segment that has also been described by morphologists as the initial collecting tubule. Morphological evidence of vasopressin-responsiveness was not observed in the early distal tubule (distal convoluted tubule). Additional morphological studies in Wistar, Long-Evans, and Sprague-Dawley rats demonstrated a marked difference in the random availability of distal convoluted tubules versus initial collecting tubules potentially available for micropuncture just beneath the renal capsule. The results suggest that hypotonic tubular fluid entering the early distal tubule (distal convoluted tubule) remains hypotonic to plasma until it enters the late distal tubule (initial collecting tubule) and that vasopressin-induced osmotic equilibration is a function of the latter segment alone. The findings emphasize the importance of morphological characterization of those segments of the renal tubule that are subjected to physiological investigation.     

可注射性纳米材料与兔成骨细胞的生物相容性

目的:将可注射性纳米材料与兔成骨细胞复合构建可注射性组织工程骨,观察其体外实验的生物相容性.方法:取16周龄、体质量约1.5 kg的新西兰大耳白兔双股骨大转子部和胫骨干骺端骨髓4～5 mL,将传至第三代的骨髓基质干细胞以成骨条件培养基定向诱导培养,获取成骨细胞.将成骨细胞与可注射性纳米材料体外复合培养,用噻唑蓝法测定细胞活性,检测碱性磷酸酶(ALP)活性,并行激光共聚焦显微镜及扫描电镜观察.结果:可注射纳米材料组的成骨细胞保持正常的分裂增殖速度.可注射纳米材料组ALP活性值为2.135±0.085,对照组为2.141±0.107,两者差异无统计学意义(P＞0.05).激光共聚焦显微镜及扫描电镜可见,成骨细胞在可注射性纳米材料上可良好地增殖、生长,细胞的活性未受到材料的影响.结论:可注射性纳米人工骨具有良好的细胞相容性,可作为可注射性骨组织工程载体材料.     

超声辐照对离体兔全血黏度的影响及安全性评价

目的 探讨超声辐照对离体兔全血黏度的影响及安全性,为临床应用超声辐照改善血液流变状态提供实验基础.方法 20只健康新西兰大白兔,每只取全血50 ml,随机分为5组,1组为对照组,其余4组均以频率0.87 MHz的超声波连续辐照,声强及辐照时间分别为2 W/cm2×1 min、3 W/cm2×1 min、4 W/cm2×1 min、4 W/cm2×3 min,随后对5组血样行血细胞计数,血黏度检测及白细胞吞墨试验.结果 3 W/cm2组与对照组比较血细胞计数及粒细胞吞墨率无差异,低切全血表观黏度红细胞聚集指数分别下降7.76%,6.24% (P＜0.05);4 W/cm2×1 min组与对照组比较血小板下降8.24% (P＜0.05),粒细胞吞墨率下降明显,血黏度进一步下降(P＜0.05);2 W/cm2组及4 W/cm2×3 min组与对照组比较血黏度无显著差异.结论超声辐照能改善离体兔全血的流变特性,选择3 W/cm2×1 min超声辐照,可在不影响血细胞形态及功能的前提下改善离体血液流变状态.     

体外冲击波对兔膝软骨细胞增殖的影响

目的体外观察体外冲击波(ESW)对软骨细胞增殖的影响。方法酶法消化获得正常兔膝软骨细胞,体外分别施加0、1×105、1.5×105、2×105、2.5×105、3×105、3.5×105 Pa ESW 0、300、600、900 次。倒置显微镜观察,HE 染色观察,CCK-8 染色筛选ESW促进软骨细胞增殖最佳压强和次数。结果1.5×105 Pa、600 次ESW干预后,软骨细胞增殖活性达最高;随压强、次数升高,软骨细胞增殖活性明显下降。结论软骨细胞是对ESW应力敏感细胞。ESW在一定压强和次数下显著促进软骨细胞增殖。     

Continuous Observation of Rabbit Preimplantation Embryos In Vitro by Using a Culture Device Connected to a Microscope

We developed a compact culture device that maintains developing embryos in vitro under constant temperature and CO₂ concentration. Using this device, we cultured rabbit embryos from the pronuclear stage to the hatched blastocyst stage and recorded their development digitally for 7 d. Recorded images were converted to a movie, and the developmental movement of individual embryos was analyzed. With this culture system, we can observe embryonic development in a suitable environment continuously for several days; similar long-term observation is not possible in the conventional system. The proportion of embryos that developed from the pronuclear stage to the blastocyst stage was the same in the new system (73.1%; 38 of 52) as in the conventional (control) system (77.6%; 38 of 49). Compaction of embryos occurred from the 8-cell to the morula stage at 32.5 ± 0.71 h after insemination. The time of blastocyst formation (77.2 ± 3.2 h after insemination) varied somewhat between embryos. Average hatching time was 98.7 ± 4.4 h after mating. Therefore, the cleavage, blastomere movement, and hatching processes of blastocysts can be followed clearly and recorded by using this new culture system.Reproductive characteristics of the rabbit, such as easy manipulation of ovulation and the formation of blastocysts with far more inner cell mass cells than those of mice, make the rabbit an excellent model for the study of embryology, developmental biology, and genetic engineering.²⁶ The preimplantation rabbit embryo undergoes a rapid series of cell divisions resulting in a blastocyst with 128 cells by day 3 after fertilization.^1,5,11 Rabbit 1-cell embryos have successfully been cultured to blastocysts in a variety of complex media, including largely defined conditions.^4,12,13 Embryonic cells are sensitive to environmental changes: during in vitro culture of mammalian embryos, even a slight change in the culture conditions has lasting effects on the offspring.²¹ In addition to the medium composition, the gas phase and temperature of the medium are important factors influencing embryo development.Continuous observation of developing embryos in vitro is difficult. Heat stress during the critical stage of early embryo development increases the incidence of early embryonic death.²³ Therefore, a suitable atmospheric environment is necessary for normal embryonic growth in vitro.^12,14,23 An effective gaseous environment for culturing rabbit zygotes in synthetic medium is 10% CO₂ combined with 5% O₂.⁶ In conventional culture systems, cells and tissues are maintained in culture dishes, which are not airtight, in an incubator filled with an appropriate mixture of O₂ and CO₂. However, when the culture dish is removed from the incubator for examination of embryos, the CO₂ in the embryos’ environment is lost rapidly, thus increasing the pH of the medium, an effect that is likely to be detrimental to embryos. Continuous observation or recording of embryo growth requires maintaining a constant CO₂ concentration of the culture medium under the microscope.Here we describe a small airtight chamber that fits over the stage of the microscope and provides CO₂ gas perfusion. The incoming gas is humidified and warmed to the desired temperature before being passed over the cells. This apparatus, combined with a computerized digital image analysis system, enabled us to record the cell movement of rabbit preimplantation embryos continuously for 7 d. The device can be used for examination of virtually any type of cell over extended periods of time. Therefore, preimplantation embryos that are suitable for embryo transfer can be selected by close examination of the developmental pattern of the embryos.     

兔干细胞的体外培养和碱性成纤维细胞生长因子对其代谢的影响

目的探索体外培养的骨髓间充质干细胞（BMSCs）和脂肪干细胞（ASCs）变化，以及碱性成纤维细胞生长因子（bF—GF）对其代谢的影响。方法分别用DMEM、DMEM／F12（2：1）、a—MEM培养兔BMSCs和ASCs。将体外培养的第3代兔BMSCs和ASCs各分为2组后培养到第3代。A组：软骨诱导培养基（CM）＋ASCs，B组：CM＋ASCs＋bFGF5ng／ml，C组：BMSCs＋CM，D组：BMSCs＋CM＋bFGF5ng／ml。倒置显微镜观察细胞形态学变化，测定^35SO4^2-摄入量及羟脯氨酸浓度。结果a—MEM培养基比DMEM、DMEM／F12（2：1）制备的BMSCs和ASCs所需时间缩短。单层培养的BMSCs和ASCs增殖较快，贴壁较牢且生成细胞集落，但经CM诱导后，细胞增殖较慢，贴壁不牢，且细胞没有集落生成。诱导后的BMSCs和ASCs的形状趋向于类圆形，B组和D的细胞数量比A组和C组的细胞多，B组、D组分别比A组、C组表达羟脯氨酸增加、^35SO4^2-摄入量增加，而D组羟脯氨酸表达和^35SO4^2-摄入量与B组无显著性差异。结论添加了bFGF的CM诱导液诱导的兔干细胞生长较好，代谢增加。     

Abnormal Secretion of Insulin and Glucagon by the In Vitro Perfused Pancreas of the Genetically Diabetic Chinese Hamster

Hereditary insulin-deficient diabetes mellitus occurs in certain sublines of nonobese Chinese hamsters. Several characteristics of this syndrome are similar to those seen in insulin-deficient human diabetics. Therefore, to characterize pancreatic islet function, dynamic insulin and glucagon release from normal and nonketotic diabetic hamster pancreases in response to glucose (300 mg/100 ml) and theophylline (10 mM), infused singly and together, was studied in vitro.20-min glucose infusions of normal hamster pancreases caused biphasic insulin release, consisting of a rapid first peak and a gradually rising second phase, similar to that reported for man in vivo. Both phases were significantly reduced in the diabetic pancreases. Theophylline alone stimulated similar nonphasic insulin release in both the normal and the diabetic pancreases. Glucose and theophylline together caused greater insulin release than either stimulant alone in both normals and diabetics; however, the diabetic response was still subnormal.Glucose suppressed glucagon release from normal pancreases; suppression was significantly impaired in diabetics. Theophylline stimulated nonphasic glucagon release in both the normals and diabetics. Glucose partially suppressed the theophylline-stimulated release in both groups.Insulin/glucagon molar ratios of the diabetics were consistently subnormal, although individual hormone levels often overlapped into the normal range.IN SUMMARY, THE PANCREASES OF GENETICALLY DIABETIC CHINESE HAMSTERS PERFUSED IN VITRO SHOWED: (a) decreased first and second phase insulin release in response to glucose-containing stimuli-only partially ameliorated by theophylline-, and (b) impaired suppression of glucagon in response to glucose, resulting in (c) a decreased insulin/glucagon molar ratio. These data support the suggestion that both alpha and beta cells of diabetic pancreases may be insensitive to glucose.     

Focal Cortical Blood Flow Activation Is Regulated by Intrinsic Cortical Cholinergic Neurons

We evaluated the cholinergic mechanism underlying focal cortical vascular response to neuronal activation, using positron emission tomography for use on animals to measure cerebral blood flow and glucose metabolism activation upon vibrotactile stimulation in cats. Bromopyruvate, which blocks acetylcholine synthesis through inhibition of the production of acetyl CoA, was injected into the cerebral cortex and basal forebrain as well as the sphenopalatine ganglion, all of which have been confirmed to supply cholinergic terminals to the cerebral cortex. Although glucose metabolism was preserved, indicating that the neuronal activities were enhanced, cerebral blood flow increase during cortical neuronal activation was abolished by bromopyruvate injection into only the cerebral cortex and not other cholinergic systems. We conclude that the cholinergic intrinsic neurons control the focal cerebral blood flow increase in response to neuronal activation.