Chloride transport in primary cultures of rabbit colonocytes at different stages of development

BACKGROUND & AIMS: Ontogeny of colonic Cl- transport and its regulation has been characterized inadequately. The aim of this report was to study developmental changes in Cl- transport in primary cultures of rabbit distal colonocytes. METHODS: Colonocytes from newborn (7-9 days old), weanling (25-28 days old), and adult (6 months old) rabbits were cultured for 24 hours on a collagen IV matrix, and Cl- transport was measured using the fluoroprobe 6-methoxyquinolyl acetoethyl ester. RESULTS: Cl- permeabilities were dependent on [Cl-]o with maximal rates (in millimoles per liter per second) at [Cl-]o = 75 mmol/L (newborns; 0.15 +/- 0.04; weanlings; 0.2 +/- 0.02; and adults, 0.32 +/- 0.06). Influx was inhibited significantly by the Cl- channel (50 mumol/L diphenylamine-2-carboxylate) and the Na(+)-K(+)- 2Cl- cotransport (10 mumol/L furosemide) inhibitors. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues, prostaglandin E1 (1 mumol/L), forskolin (1 mumol/L), and 8-bromo-cAMP (100 mumol/L), and the protein kinase C activator, phorbol 12-13 dibutyrate (1 mumol/L), increased Cl- influx significantly in all groups with adults showing greatest stimulation. However, taurodeoxycholate (0.025-1 mmol/L) had an effect only in the adult and the guanosine 3',5'-cyclic monophosphate (cGMP) activators STa and 8-bromo-cGMP had no effect. CONCLUSIONS: Rabbit distal colonocytes possess inhibitor-sensitive Cl- permeabilities even in neonates. However, the ontogeny of their regulation depends on the secretagogue-signaling pathway.     

Hormonal regulation of the estrogen receptor in primary cultures of hepatocytes from female rats

Estrogen treatment affects the hepatic synthesis and/or secretion of several proteins involved in clinically important pathological processes such as atherosclerosis, hypertension, and thrombosis. The endocrine regulation of the estrogen receptor (ER) concentration in primary cultures of rat hepatocytes was studied. Human growth hormone (hGH) and dexamethasone (DEX) in combination increased ER concentration 6-fold and ER mRNA levels 2.5-fold. These effects were not significantly different from those observed after treatment with the purely somatogenic bovine growth hormone (GH) in combination with DEX. Treatment with the lactogen ovine prolactin in the presence or absence of DEX did not significantly affect ER or ER mRNA concentrations. Triiodothyronine treatment at the most effective concentration (50 nM) increased ER and ER mRNA levels twofold. Medium supplementation with estradiol (0.1 nM) throughout the experiment did not affect the response to treatment with hGH and DEX. Treatment with high concentrations of ethinylestradiol in combination with hGH and DEX, however, increased the ER level twice as much as hGH and DEX without addition of estradiol or ethinylestradiol, whereas the ER mRNA concentration was the same in both the GH+DEX group and GH+ DEX+ (estradiol or ethinylestradiol) groups. These data indicate the importance of GH in combination with glucocorticoids for the maintenance of ER concentrations in the rat liver. Thyroid hormones may be of some, although minor importance, whereas the data suggest that prolactin is not directly involved in hepatic ER regulation.     

Inhibition of peroxisomal degradation by 3-methyladenine (3MA) in primary cultures of rat hepatocytes

BACKGROUND: A significant reduction in peroxisomes has been demonstrated in primary cultures of rat hepatocytes. This report demonstrates that 3-methyladenine (3MA), a potent inhibitor of autophagy, inhibits this effect. METHODS: Hepatocytes from male Wistar rats were isolated by a two-step in situ perfusion technique using collagenase and were cultured in Williams E medium. After a 2-hr attachment period (day 0 of culture), the cells were treated with 200 microM bezafibrate (BF), a peroxisome proliferator, and 5 mM 3MA for 3 days. The cells in the culture dish were fixed in situ, stained for catalase, and embedded in Poly/Bed 812. The number and size of peroxisomes in electron micrographs were analyzed morphometrically. RESULTS: After 3 days of culture, the number of peroxisomes had decreased to 30% of the day 0 level. However, the day 0 level was maintained by treatment with 3MA. In BF-treated cells, many autophagosomes were observed, and peroxisomes had proliferated significantly, although they did not exceed the day 0 level. In cells treated with a combination of 3MA and BF, the number and size of peroxisomes had increased remarkably. CONCLUSIONS: These results suggest that 3MA is effective in maintaining both the number and size of peroxisomes in the course of primary cultures of rat hepatocytes. Suppression of peroxisome proliferation by treatment with BF may be regulated by autophagic/lysosomal degradation.     

Changes in the thermal denaturation profiles of DNA from different developmental stages of the newt Triturus vulgaris

The melting profiles of DNA samples from the early gastrula and early neurula of Triturus vulgaris are essentially the same, whereas DNA from mid to late gastrula possesses higher Tm values and shows a deviation from the regular sigmoidal shape at temperatures above Tm. The plot on normal probability paper indicates a second DNA fraction which melts at higher temperatures and, consequently, it has a higher GC-content than the bulk of DNA. These facts confirm our idea that differential DNA replication occurs during gastrulation.     

Long-term decline of atmospheric and marine pollution by polycyclic aromatic hydrocarbons (PAHS) in Germany

Concentrations of benzo[a]pyrene and benzo[e]pyrene in the atmosphere as markers for the class of PAHs decreased by about 70% within one decade in clean air as well as in industrially polluted areas of Germany when measured with passive samplers (spruce sprouts, poplar and beech leaves). The same trend has been found for East-Germany during 1991-1995. Mussels (Mytilus edulis) were found to accumulate PAHs from the aquatic environment and exhibited a seasonal periodicity of the PAH concentration. After an initial decline from 1985 to 1990, the PAH concentration remained constant until 1995 in the North Sea area investigated.     

Gas chromatographic-mass spectrometric investigations on the formation of phenolic and aromatic hydrocarbons in food (author''s transl)

Experience from arthroscopy in 224 patients with knee complaints is reported. The common method for arthroscopy is compared with a new modification developed during the study. The new method includes introduction of a 5 mm arthroscope through the patellar tendon at the level of the joint line and the use of hooks to test the menisci and the ligaments. The experience from this method shows that it gives an expanded field of vision and that technical failures are less frequent. No complications occurred. It is stated that arthroscopy is of great value in the diagnosis of knee injuries. However, a high diagnostic accuracy can only be reached by an experienced operator using a strictly standardized method.     

Binding and degradation of lipoprotein(a) and LDL by primary cultures of human hepatocytes. Comparison with cultured human monocyte-macrophages and fibroblasts

Although lipoprotein(a) (Lp[a]) has structural similarities to low-density lipoprotein (LDL) that include the presence of apolipoprotein B100, there is some disagreement over the strength of its interaction with the LDL receptor and its cellular catabolism by the LDL receptor-mediated pathway. To clarify this subject we evaluated LDL receptor-mediated binding and degradation of Lp(a) and LDL in three human cell lines. The binding of 50 nmol/L Lp(a) at 37 degrees C to the LDL receptor of primary hepatocytes, macrophages, and fibroblasts was only 10%, 29%, and 29% of the respective value obtained with 50 nmol/L LDL. Analysis of 4 degrees C binding curves indicated that Lp(a) and LDL had equal affinities for the LDL receptor of fibroblasts, whereas maximal binding of Lp(a) was remarkably lower than that of LDL. LDL receptor-mediated degradation of 50 nmol/L Lp(a) in hepatocytes, macrophages, and fibroblasts was only 17%, 22%, and 26%, respectively, of the value obtained with 50 nmol/L LDL and varied greatly among the cells in that it was lowest in hepatocytes, an order of magnitude greater in macrophages, and two orders of magnitude greater in fibroblasts. In contrast, the nonspecific degradation rate of Lp(a) was similar to that of LDL in each of the three tested cell lines. However, the proportion of the degradation of Lp(a) that was nonspecific varied greatly, being 76%, 58%, and 33% in hepatocytes, macrophages, and fibroblasts, respectively. These studies indicate that not only is Lp(a) recognized by the LDL receptor but also that, in fibroblasts, Lp(a) and LDL have equal affinities for the LDL receptor, although Lp(a) has a much lower receptor occupancy than LDL. Additionally, they show that there are great cellular differences in the LDL receptor-mediated degradation of Lp(a). If these results can be extrapolated in vivo, where normal LDL levels are 40- to 50-fold higher than those of Lp(a), it would be unlikely that the hepatic LDL receptor is significantly involved in the degradation of Lp(a).     

Interactions of transthyretin (TTR) and retinol-binding protein (RBP) in the uptake of retinol by primary rat hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP-transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP-TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [3H]retinol-RBP or the [3H]retinol-RBP-TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [3H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP-TTR complex was nearly twofold greater than that from RBP alone. The uptake of [3H]retinol from protein-bound retinol was inhibited by an excess of either retinol-RBP or retinol-RBP-TTR complex. Moreover, retinol uptake through the RBP-TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP-TTR complex into both PC and NPC. The uptake of [3H]retinol (2 microM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

Environmental and biological monitoring of polycyclic aromatic hydrocarbons (PAHs) in coke plants and other workplaces

Primary aldosteronism remains a diagnostic challenge. Certain immunoassay techniques, simplified diagnostic testing, and the introduction of sensitive imaging techniques have facilitated the diagnosis, but obstacles that remain include a lack of optimal screening methods, low sensitivity and specificity of current diagnostic tests, and a growing number of etiological subgroups. A rational approach to the diagnosis of primary aldosteronism is described, as is the differentiation of the surgically correctable lesion (adenoma) from the other etiological subgroups.     

Generation, identification, and evaluation of expressed sequence tags from different developmental stages of the Asian blood fluke Schistosoma japonicum

Here we report 658 expressed sequence tags (ESTs) generated from the 5'-termini of clones randomly selected from directional cDNA libraries constructed from mRNAs from three developmental stages of Schistosoma japonicum. Putative identifications were assigned to 46. 2% of the ESTs; 6.4% were previously known from S. japonicum, 5.6% were previously known from S. mansoni, 34.2% were known from other organisms, and the remaining 53.8% may represent S. japonicum-specific genes. These 658 ESTs appeared to be derived from 457 unique genes, which together represent 2 to 3% of the 15,000 to 20,000 genes predicted to occur in the schistosome genome.     

Benzo(a)pyrene-albumin adducts in humans exposed to polycyclic aromatic hydrocarbons in an industrial area of Poland

Antiarrhythmic drugs administered intravenously run the risk of producing a hemodynamic collapse even when used by expert and well trained hands. The arrhythmias in the focal point of a preexcitation syndrome constitute a very special situation in which extreme caution must be used when using intravenous drugs, because the conduction through accessory channels can vary, depending on multiple factors. We describe a case of a patient with an accessory atrioventricular pathway and orthodromic tachycardia who developed cardiac arrest by wide QRS tachycardia after receiving intravenous amiodarone.     

Electron microscopic studies of antlerogenic cells from five developmental stages during pedicle and early antler formation in red deer (Cervus elaphus)

Previous studies using light microscopy have revealed that histogenesis of deer pedicle and antler has four ossification stages. The first of these stages is the development of the permanent pedicle. Initial development of the pedicle is from the cellular layer cells of the antlerogenic periosteum and these cells have been termed initial antlerogenic cells (IACs). Apart from the IACs, it has also been shown that the cellular layer cells of the apical periosteum/perichondrium, the peripheral periosteum of pedicles or antlers, and the marginal periosteum surrounding the pedicles are also capable of either partially or fully generating a pedicle or an antler. Therefore, these cells can all be considered antlerogenic cells and called apical antlerogenic cells (AACs), peripheral antlerogenic cells (PACs), and marginal antlerogenic cells (MACs), respectively. The aim of this study was to examine the ultrastructure of these antlerogenic cells, and to determine whether there were ultrastructural correlates with the changes of these antlerogenic cells and ossification stages. The ultrastructure of each type of antlerogenic cells was systematically examined using transmission electron microscopy, at each stage of pedicle and first antler growth. At the first ossification stage, the IACs were spindle-shaped and inactive. The most obvious feature was the presence of abundant intracellular glycogen. The MACs were similar to the IACs. During the early second stage, most of the AACs changed in appearance from preosteoblasts to prechondroblasts. Much less heterochromatin was found in the AACs than in the IACs. The most striking attribute of the AACs was the existence of intracellular collagen fibers. The MACs showed abnormal dilation of the rough endoplasmic reticulum (RER). During the late second stage, the majority of the AACs were prechondroblasts. AAC nucleoli were clearly discernible and the cisternae of the RER were arranged in parallel. The MACs contained a greater proportion of abnormally-dilated RER. During the third stage, the AACs were all prechondroblasts. The Golgi apparatus in these cells was well developed. Many free ribosomes in rosettes were scattered in the cytoplasm. Most cytoplasm of the majority of the MACs was occupied by abnormally-dilated RER (the lumen of the RER was extremely dilated and appeared electron-lucent). During the fourth stage, the AACs were similar to their counterparts from the third stage, but the boundaries of some AACs were ill-defined. Some MACs were found to be undergoing apoptosis. The PACs were becoming less and less active from distal to proximal along the shaft of the antler. It is a novel finding that antlerogenic cells change in appearance and subcellular content from preosteoblasts to prechondroblasts prior to the transition from intramembranous to endochondral ossification during pedicle formation. Therefore, the differentiation process from antlerogenic cells to chondroblasts is a matter of maturation from prechondroblasts to chondroblasts. The fact that the antlerogenic cells are rich in glycogen makes them more like embryonic cells. The local membrane deficiency of some AACs at the fourth stage and the presence of mature collagen fibrils within the AACs may reflect the unusually high demand for collagen fibrils during the period of rapid antler growth.     

Regulation of cytochrome P450 2B1/2 mRNAs by Kepone (chlordecone) and potent estrogens in primary cultures of adult rat hepatocytes on Matrigel

We previously reported that when primary cultures of rat hepatocytes were treated with phenobarbital (PB) or one of several organochlorine pesticides, including Mirex, there was co-induction of cytochrome P450 2B1 and 2B2 mRNAs and immunoreactive proteins, whereas Kepone selectively induced 2B2 (Kocarek et al. (1991) Mol. Pharmacol. 40, 203-210). Indeed, Kepone treatment actively suppressed induction of 2B1 and 2B2 mRNAs in hepatocytes cotreated with phenobarbital. Because Kepone differs chemically from Mirex only in the replacement of 2 chlorine atoms with a ketone group, which exists in aqueous solution as a gem-diol and appears to confer weak estrogenic properties, we treated hepatocyte cultures with one of 3 potent estrogens, beta-estradiol, 17 alpha-ethinylestradiol or diethylstilbestrol. Treatment with each of these estrogens induced 2B1 and 2B2 mRNA only at very high doses (10(-4) M). Beta-Estradiol (10(-4) M) treatment also induced 2B1/2 mRNA in hepatocyte cultures prepared from a prepubescent female rat. The anti-estrogen tamoxifen failed to reverse 2B1/2 mRNA induction following beta-estradiol or Kepone treatment of adult hepatocyte cultures. High doses of beta-estradiol or 17 alpha-ethinylestradiol failed to induce 2B1/2 mRNA in treated rats. We also examined the effects of chloral hydrate, a simple gem-diol, on 2B1/2 mRNA induction in the hepatocyte cultures. Treatment with chloral hydrate (3 x 10(-3) M), like Kepone (10(-5) M), suppressed 2B1/2 mRNA induction following phenobarbital (10(-4) M) treatment, while Kepone alcohol (10(-5) M), which is not a gem-diol, produced less suppression. Our results suggest that selective induction by Kepone of 2B2 is unlikely related to its effects as a weak classical estrogen, while the ability of Kepone to suppress induction of 2B1 and 2B2 by PB may be related to its properties as a gem-diol.     

In vitro toxicity of zamifenacin (UK-76,654) and metabolites in primary hepatocyte cultures

1. We compared the sensitivities of primary hepatocytes from rat, dog and monkey to zamifenacin and two major metabolites, the methylenedioxy ring-opened catechol, UK-80,178 and its methylated product, UK-82,201. Toxicity was determined both via neutral red uptake and enzyme leakage data. 2. Canine hepatocytes were most sensitive to the cytotoxic effects of zamifenacin during 24-h exposure. Significant decreases in medium concentrations of zamifenacin in the presence of primary hepatocytes verified cellular uptake during the initial 2-h incubation. All three cell types were much more sensitive to UK-82,201 than to the catechol metabolite or parent drug. 3. The rapid onset of cytotoxicity indicated by elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers in the medium after UK-82,201 exposure, the delayed but substantial cytotoxic response to the parent drug which was suggestive of biotransformation to a reactive moiety, in vivo and in vitro drug metabolism results and subacute toxicology data suggest that dog may more effectively transform zamifenacin into UK-82,201, which is relatively hepatotoxic. 4. Because the catechol was generally less toxic than the O-methylated product, species that eliminate zamifenacin primarily as the catechol or its conjugate may be less affected by the potential hepatotoxicity of the methylated product. Our studies show that dog is the most sensitive species due to metabolism of the common catechol metabolite. The low incidence of potential hepatotoxicity in the clinic points to rare but important differences in the metabolism of Zamifencin. We conclude that the findings in dog were not predictive of subsequent effects in man.     

An animal experimental study on the retention rate of polycyclic aromatic hydrocarbons in the lung and in the subcutaneous tissue (author's transl)

The retention rate of five polyaromatic hydrocarbons (PAH) (benzo(a)pyrene, dibenz(ah)anthracene, chrysene, pyrene, benz(a) anthracene) was studied after intratracheal instillation into syrian hamsters and subcutanous injection into mice. For subcutanous injection the PAH were solved in 0.5 ml tricaprylin, for intratracheal instillation a suspension in NaCl was used. The results were the following: 1. With respect to the retention rate of the five PAH the largest difference was found comparing the application modes. The ratio of the averaged half-time-values for the intratracheal test to the subcutanous test is about 1:35. 2. The retention rates for each polycyclic hydrocarbon differs significantly. An interrelation of PAH after application of a PAH-mixture was not detectable. 3. The retention rates determined in the lung and in the subcutanous tissue do not give a constant ratio concerning each PAH. Thus DBahA is retained in the lung as well as in the subcutanous tissue definitely longer than BaP. Chrysene and benz(a)anthracene behave - respecting the retention rate - in the subcutanous test like BaP. In lung tissue, however, the different behaviour becomes obvious: while benz(a)anthracene is eliminated most rapidly, chrysene can be detected for a relative long time.