Effects of gestational or neonatal treatment with alpha-difluoromethylornithine on ornithine decarboxylase and polyamines in developing rat brain and on adult rat neurochemistry

Pregnant rats were treated for five consecutive days during gestation with s.c. injections of the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO). Treatment beginning at gestational days 13 or 14 was effective in inhibiting ODC and altering polyamine levels, and resulted in relatively small decreases in body and forebrain weight, but not in significant differences in adult neurochemistry. Neonatal rats were treated with DFMO from postnatal day 0 (PD 0) to PD 24. In addition to some somatic effects (decreased body weight, delayed eyelid opening and delayed fur growth) the postnatal treatment resulted in a permanent decrease in brain weight, which was mainly due to a dramatic decrease in cerebellar size. During treatment, and 3 days after the end of it, the levels of putrescine and spermidine, but not those of spermine, were consistently lower in the cerebellum and forebrain of DFMO-treated rats than in controls. On the other hand, ODC appeared strongly inhibited only during the first phase of the treatment and showed recovery, and also rebound of the activity, during the second part of the treatment. A screening of neurochemical markers related to cholinergic, GABAergic and glutamatergic neurons, as well to astrocytes and oligodendrocytes was performed in several brain regions (cerebellum, olfactory bulbs, cortex, striaturn, hippocampus) of some of these rats once they became adults. Significant alterations for all the parameters tested, with the exception of the marker for the glutamatergic transmission, were measured in the undersized cerebellum of the neonatally DFMO-treated rats. A shorter neonatal treatment with DFMO (from PD 1 to 6) resulted, in the adult, in decreased cerebellar size and in neurochemical alterations, both very similar to those occurring after the prolonged treatment. In the other brain regions a few minor differences were noticed. The present results show that: (1) the brain polyamine system is differently regulated in foetuses with respect to newborns; (2) the effects of chronic ODC blockade are different on prenatally or postnatally proliferating neurons, due either to a lower sensitivity of gestation ally proliferating neurons or to a subsequent recovery; and (3) chronic postnatal ODC inhibition has a strong effect on proliferating neurons, but little effect on further maturation of postmitotic neurons.     

Elevated hepatocyte-specific functions in fetal rat hepatocytes co-cultured with adult rat hepatocytes

Fetal hepatocytes (FHEPs) are a potential source of highly proliferative transplantable cells but express low levels of liver-specific functions. We hypothesized that the microenvironment of adult hepatocytes (AHEPs) may upregulate these functions. Primary FHEPs were seeded on top of collagen-sandwiched AHEPs directly or separated by a porous transwell membrane insert. In direct co-cultures, albumin (ALB) secretion, urea synthesis, and cytochrome P450 (CytP450) activity were all approximately 2 times as high as the sum of the corresponding monocultures. Using a transwell porous insert led to similar results, suggesting a major role for soluble factors. When AHEPs and FHEPs were separated after co-culture, they both initially showed significantly higher ALB secretion than control monocultures, whereas urea synthesis was significantly lower for the FHEPs only. Functions of previously co-cultured FHEPs normalized over the course of a week, but AHEP function remained high even after separation. In conclusion, co-culturing AHEPs with FHEPs increases expression of liver-specific functions in both cell types. The effect on FHEPs, but not AHEPs, was reversible. Unraveling the underlying mechanisms and optimizing this phenomenon will be useful in making fetal liver cells a potential cell source for hepatic tissue-engineering applications.     

Hormonal regulation of mannan-binding lectin synthesis in hepatocytes

Activation of the complement system via the plasma protein mannan-binding lectin (MBL) provides a first line of defence against infections. The plasma level of MBL is, in part, determined genetically, but may also be influenced by different hormones in vivo. Here we study the hormonal regulation of MBL synthesis from the human hepatocyte cell line HuH-7. Cells were exposed to medium with growth hormone (GH), hydrocortisone, insulin-like growth factor (IGF)-1, insulin, interleukin (IL)-6 or thyroid hormones (T3 or T4). After 3 days the concentration of MBL in the culture supernatants was determined and the amount of mRNA for MBL was measured, relative to mRNA for beta2 microglobulin. GH, IL-6, T3 and T4 significantly increased MBL synthesis in a dose-dependent manner, while hydrocortisone, insulin and IGF-1 had no effect. T3 caused a fourfold increase at 1 nM of T3 (P < 0.001) and at 100 nM of T3 the production was increased more than eightfold. The effect of T4 was less potent, reaching an eightfold increase at 1 microM of T4 (P < 0.001). GH augmented the production of MBL threefold at a concentration of 100 ng/ml (P = 0.018) with no further effect up to 10 microg/ml, whereas IL-6 caused only a very weak increase in MBL production. MBL mRNA levels were stable during the first 24 h of T3 stimulation but increased significantly between 24 and 48 h. The results suggest that MBL synthesis in humans may be increased by thyroid hormone and GH, whereas it does not exhibit a classical IL-6-dependent response.     

Preparation of primary monolayer cultures of adult rat hepatocytes

Summary We describe in detail the technique of hepatocyte isolation and establishment of primary cell cultures of adult rat hepatocytes. These cultures contain hormonally responsive hepatocytes that retain many adult characteristics under completely serum-free conditions. The cells retain a normal morphology and do not exhibit fetal characteristics during a 4 d culture period.     

长期培养的大鼠原代肝细胞功能和形态学观察

目的：研究大鼠原代肝细胞长期体外培养后功能和形态的变化。 方法： 采用两步胶原酶原位灌流法分离大鼠肝细胞，并用Percoll分离液进行密度梯度离心进一步纯化肝细胞，采用0.4%台盼蓝染色观察细胞活力。然后将细胞接种于HepatoZYME-SFM培养基中培养，定期收集肝细胞培养液上清检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶（AST）、白蛋白、尿素氮的水平。采用乙氧基试卤灵O-脱乙基酶活性（EROD）方法检测肝细胞P450的CYPⅠA1功能。 结果： 新鲜分离的大鼠肝细胞总数（2-3）×108cells/whole liver，Percoll分离液纯化后活力和纯度在90%以上。HepatoZYME-SFM培养下肝细胞生长良好并保持正常形态。AST、ALT水平在培养3 d后下降显著，6-9 d后趋于相对稳定的低水平。白蛋白的分泌功能、尿素合成能力在18 d内维持在较高水平。可在3-6 d检测到CYPⅠA1酶活性。 结论： Percoll液纯化新分离肝细胞可提高其活率和纯度，HepatoZYME-SFM培养条件下肝细胞可有效保持良好形态结构和一定的生物合成代谢能力，适合肝细胞的体外长期培养和功能研究。     

Comparative study of proliferation of suckling and adult rat hepatocytes in primary culture in response to growth-stimulating factors

The replicative responses of suckling and adult rat hepatocytes in primary culture to growth-stimulating factors were compared. By addition of L-proline alone, the [3H]-thymidine labeling of suckling rat hepatocytes was dramatically enhanced, but that of adult ones was not. Epidermal growth factor (EGF), insulin, triiodothyronine (T3) and glucagon also enhanced the labeling of suckling rat hepatocytes regardless of the presence or the absence of L-proline. On the other hand, in the absence of L-proline, only EGF enhanced the labeling of adult rat hepatocytes, and, in the presence of L-proline, insulin as well as EGF enhanced the labeling. In the presence of growth factors and L-proline, the number of suckling rat hepatocytes increased up to about 143%, whereas that of adult rat hepatocytes hardly increased. Thus, a remarkable difference in replicative responses to growth factors and L-proline was observed between suckling and adult rat hepatocytes in primary culture.     

Different factors possibly involved in post-translational regulation of ornithine decarboxylase activity

Present results concern a microsome-bound enzymatic system which has been recognized as responsible for the rapid inactivation in vitro of ornithine decarboxylase (ODC). Two different models have been investigated: a) rat liver after a single thioacetamide administration, and b) the 3924 A Morris hepatoma. In both these models we observed variations in the microsome-bound ODC-inactivating capacity. In parallel, changes in ODC properties were observed. The possibility of a causal relationship between the two events is discussed. The actual role of the microsome-bound ODC-inactivating system, in ODC activity regulation in vivo cannot be established, but it remains as a fairly plausible working hypothesis.     

Tryptophan-induced stimulation of hepatic ornithine decarboxylase activity in the rat

The effect of a single tube feeding of l-tryptophan on hepatic ornithine decarboxylase (ODC) activity in rats was investigated. The levels of ODC activity in the livers of control and experimental rats were assayed in vitro by measuring the release of ¹⁴CO₂ from DL-[1-¹⁴C]ornithine. Single tube feedings of varying levels of l-tryptophan ($\text{2.5–30 mg}\text{100 g}$ body wt) to overnight-fasted rats 1 hr before sacrifice exhibited increases in the hepatic ODC activities. l-Tryptophan ($\text{30 mg}\text{100 g}$ body wt) tube fed to overnight-fasted rats $\text{1}\text{6}$ to 12 hr before sacrifice induced hepatic ODC activities which were significantly elevated beginning at 1 hr and peaking at 2 hr (6.5-fold increase over controls). In vitro [¹⁴C]leucine incorporation into protein using hepatic microsomes of tryptophan-treated rats was significantly increased at 1 hr in comparison with that of controls. The tryptophan-induced stimulation of hepatic ODC activity was not affected by prior adrenalectomy but was abolished by pretreatment with cycloheximide. These studies demonstrate that a single feeding of l-tryptophan can significantly enhance in the rat the activity of ODC, a key enzyme in the biosynthesis of polyamines.     

Rhythm of ornithine decarboxylase activity in rat parotid gland slices

In rat parotid gland slices incubated in vitro ornithine decarboxylase activity and the rate of protein synthesis were determined. A circahoralian rhythm of enzyme activity preceding inphase fluctuations in the range of protein synthesis was found. These results indicate a role of ornithine decarboxylase in maintenance of the circahoralian rhythm of the rate of protein synthesis in parotid gland cells.Laboratory of Cytology, Institute of Developmental Biology, Academy of Sciences of the USSR. Department of Biochemistry, Central Research Laboratory, Attached to the 4th Main Board, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 12, pp. 726–728, December, 1978.     

Selection of medium for serum-free primary culture of adult rat hepatocytes

To select a suitable medium for serum-free primary culture of adult rat hepatocytes, ten commercially-available synthetic media were compared for their ability to maintain the cells under serum-free and serum-supplemented conditions with special reference to attachment, survival and albumin secretion. It was found that Williams' medium E and DM-160 medium were the best among the ten media for maintaining hepatocytes under serum-free conditions in primary culture.     

Semi-automatic counting of connexin 32s immunolocalized in cultured fetal rat hepatocytes using image processing

Connexin 32s (Cx32s) were immunolocalized in fetal rat hepatocytes and their distribution was determined qualitatively. We used image analysis using a quantitative index (QI) of Cx32 (QI Cx32) defined as the area of Cx32s/number of cells in cultured fetal rat hepatocytes. Hepatocytes from livers of fetal rats were separated by collagenase digestion and low centrifugation on gestational day 17. Cells were cultured for 3 days in dexamethasone (DEX)-supplemented medium (Dex0). The medium was replaced with fresh medium and cells were continuously cultured for 3 days with DEX or epidermal growth factor supplemented medium (Dex3 or EGF3). After culture termination, cells were fixed and stained using the fluorescein-labeled antibody method for Cx32s and diaminophenylindole staining for nuclei. Thirty pairs of histological images for Cx32s and nuclei, 180 images in total, were obtained from each condition. The QI Cx32 significantly increased from 284.1 ± 102.0 (mean and SD, n = 26) of Dex0 to 428.9 ± 101.0 of Dex3 (n = 28) (P < 0.05, Kruskal-Wallis test, then Steel-Dwass test). The increase of QI Cx32 was compatible with the morphological observations. The image analysis processing time after preparation for 180 images was reduced from 8 h needed for manual operations to 1 min using ImageJ software with our macro routine.     

Hormonal effects on the surfactant protein B (SP-B) mRNA in cultured fetal rat lung

Glucocorticoids, triiodothyronine (T3), and cyclic adenosine monophosphate (cAMP) have been shown previously to modulate phosphatidylcholine and surfactant protein A (SP-A) synthesis in fetal rat lung explant cultures. In this report, we have examined the hormonal regulation of the rat surfactant protein B (SP-B) mRNA to determine whether SP-B expression is coordinately regulated with the surfactant phospholipids or with SP-A. Dexamethasone (1 to 200 nM) and cAMP (200 microM) had a stimulatory effect on SP-B mRNA levels, whereas T3 tended to inhibit the accumulation of SP-B mRNA. In combination experiments, treatment with dibutyryl-cAMP (200 microM) and dexamethasone (100 nM) resulted in about a 22-fold increase, whereas dexamethasone or dibutyryl-cAMP alone produced 18- and 2-fold increases, respectively. When the cAMP analogue 8-bromo-cAMP (200 microM) was used in combination with dexamethasone, there was no significant difference between the combined effect and that of dexamethasone alone. T3 treatment, however, resulted in a significant reduction of the dexamethasone-induced stimulation from about a 22-fold to a 14-fold increase. Tissue in situ hybridization showed that dexamethasone stimulated the levels of SP-B mRNA in cells from both the alveolar and bronchiolar epithelium. These data indicate that there are differences in the hormonal regulation of the components of surfactant, suggesting that they are independently regulated.     

Effect of Garcinia hydroxybiflavanonols on protein synthesis in primary cultured rat hepatocytes

Garcinia kola Heckel (Guttiferae) known as Aki-ilu (meaning bitter kola) is a large economic tree indigenous to the southern part of Nigeria. Its seeds have been of interest to researchers because of its use in the treatment of various disease conditions in Nigerian traditional medicine. The effect of the Garcinia hydroxybiflavanonols GB1 and GB2 on the rate of protein synthesis in primary cultured hepatocytes from rat livers was studied in vitro. This work provides further information on the mechanism of action of the liver active principles of G. kola nuts. A dose-dependent stimulatory effect of the drugs on the rate of protein synthesis in rat hepatocytes was found. Both GB1 and GB2 showed significant stimulatory effect on liver cell regeneration. These findings demonstrate that the reported antihepatotoxic activity of the Garcinia hydroxybiflavanonols could be explained, at least in part, by their stimulatory effect on the protein synthetic apparatus. GB1 increased the rate of protein synthesis about thrice as much as silibinin, a flavonolignan with proven antihepatotoxic activity, and suggests that this compound could be a potential novel antihepatotoxic agent.     

A dual-functional fibrous scaffold enhances P450 activity of cultured primary rat hepatocytes

We have designed a novel dual-functional electrospun fibrous scaffold comprising two fiber mesh layers that were modified differently to induce two separate biological responses from hepatocytes. The first fiber layer was galactosylated on the surface to mediate hepatocyte attachment, while the second layer was loaded with 3-methylcholanthrene (3-Mc) to enhance cytochrome P450 activity of hepatocytes. Primary rat hepatocytes cultured on the galactosylated fibrous scaffolds loaded with different concentrations of 3-Mc were compared for their cell attachment efficiency, albumin secretion activity and cytochrome P450-dependent 7-ethoxycoumarin O-deethylase activity. This hybrid fibrous scaffold mediated hepatocyte attachment with slightly lower efficiency (76+/-2.3%) than a single-layer galactosylated fibrous scaffold (84+/-3.5%). More importantly, the cytochrome P450 activity of the hepatocytes cultured on the hybrid scaffold correlated well with the 3-Mc loading level. The results also showed that transfer of 3-Mc to hepatocytes through direct cell-fiber contact was the dominant transport route, with the induced cytochrome P450 activity being 1.9- to 4.8-fold higher than that of transfer of 3-Mc to hepatocytes via dissolution from fibers to medium. This study demonstrates the feasibility of creating multi-functional fibrous scaffolds that serve both as an adhesive substrate and as a delivery vehicle for bioactive molecules.     

Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts

Gap junctions were assayed during re-differentiation of adult rat cardiomyocytes in long-term culture to gain insight into the processes of remodeling. Double immunostaining allowed the localization of connexins Cx40, Cx43, and Cx45 between myocytes and demonstrated co-expression and co-localization in individual cells and gap junction plaques, respectively. Immunoblots showed differential time-dependent changes in connexin expression and phosphorylation. The total amount of connexins and the ratio of phosphorylated/non-phosphorylated isoforms gradually increased during the re-establishment of intercellular communication. Dual voltage-clamp studies showed the involvement of several types of gap junction channels. Multichannel currents yielded diverse spectra of g(j,inst)=f( V(j)) and g(j,ss)=f( V(j)) relationships ( g(j,inst): instantaneous gap junction conductance; g(j,ss): conductance at steady state; V(j): transjunctional voltage), indicative of homotypic and heterotypic channels. Single-channel currents revealed two prominent conductances reflecting gamma(j,main) and gamma(j,residual). The histograms of gamma(j,main) showed four discrete peaks (41-44, 59-61, 70-76, and 100-107 pS) attributable to a combination of Cx45-Cx45, Cx40-Cx45 and Cx43-Cx45 channels (1st peak), Cx43-Cx43 and Cx40-Cx43 channels (2nd peak), Cx43-Cx43 channels (3rd peak) and Cx40-Cx40 and Cx40-Cx43 channels (4th peak). However, the presence of heteromeric channels cannot be excluded. The data are consistent with an up-regulation of Cx45 and Cx43 during re-differentiation.     

Dissociation of estrogen-induced uterine growth and ornithine decarboxylase activity in the postnatal rat

Estrogens are teratogens and developmental carcinogens in several species. We have used uterine growth to quantitate the potency of three estrogens [estradiol (E2), diethylstilbestrol (DES), ethynylestradiol (EE2)] during four postnatal periods (days 1-5, 10-14, 20-24, and 60-64) in the rat. Alphafetoprotein (AFP), present at high levels in neonatal serum, is thought to regulate estrogen bioavailability. Association constants for DES and EE2 were 2.7% and 4.9% of that for E2 binding to AFP, determined in a batch Sephadex equilibrium binding assay. On days 1-5, DES and EE2 were about 80-fold more potent than E2 in increasing uterine weight. As AFP levels fell, potency differences between E2 and the synthetic estrogens decreased. In the adult, which essentially lacks AFP, the three estrogens were nearly equipotent. These data are consistent with AFP regulation of estrogen potency. On days 10-14, uterine growth was less sensitive than at other ages to all three estrogens, perhaps related to uterine differentiation and/or the high endogenous serum E2 levels reported at this age. However, when we examined another uterine estrogen response, ornithine decarboxylase (ODC) induction at 6 h following estrogen injection, all three hormones were about equipotent in both neonatal and adult animals. This apparently AFP-independent event shows dissociation of ODC induction and uterine growth, which could be due to separate mechanisms for hormone entry to target tissue or subsequent intracellular events.