Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.

UNLABELLED: We investigated, in isolated bile duct units (IBDU) and cholangiocytes isolated from normal rat liver, the occurrence of acetylcholine (ACh) receptors, and the role and mechanisms of ACh in the regulation of the Cl-/HCO3- exchanger activity. The Cl-/HCO3- exchanger activity was evaluated measuring changes in intracellular pH induced by acute Cl- removal/readmission. M3 subtype ACh receptors were detected in IBDU and isolated cholangiocytes by immunofluorescence, immunoelectron microscopy, and reverse transcriptase PCR. M1 subtype ACh receptor mRNA was not detected by reverse transcriptase PCR and M2 subtype was negative by immunofluorescence. ACh (10 microM) showed no effect on the basal activity of the Cl-/HCO3- exchanger. When IBDU were exposed to ACh plus secretin, ACh significantly (P < 0.03) increased the maximal rate of alkalinization after Cl- removal and the maximal rate of recovery after Cl- readmission compared with secretin alone (50 nM), indicating that ACh potentiates the stimulatory effect of secretin on the Cl-/HCO3- exchanger activity. This effect of ACh was blocked by the M3 ACh receptor antagonist, 4-diphenyl-acetoxy-N-(2-chloroethyl)-piperidine (40 nM), by the intracellular Ca2+ chelator, 1,2-bis (2-Aminophenoxy)- ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (50 microM), but not by the protein kinase C antagonist, staurosporine (0.1 microM). Intracellular cAMP levels, in isolated rat cholangiocytes, were unaffected by ACh alone, but were markedly higher after exposure to secretin plus ACh compared with secretin alone (P < 0.01). The ACh-induced potentiation of the secretin effect on both intracellular cAMP levels and the Cl-/HCO3- exchanger activity was individually abolished by two calcineurin inhibitors, FK-506 and cyclosporin A (100 nM). CONCLUSIONS: M3 ACh receptors are markedly and diffusively represented in rat cholangiocytes. ACh did not influence the basal activity of the Cl-/HCO3- exchanger, but enhanced the stimulation by secretin of this anion exchanger by a Ca2+-dependent, protein kinase C-insensitive pathway that potentiates the secretin stimulation of adenylyl cyclase. Calcineurin most likely mediates the cross-talk between the calcium and adenylyl cyclase pathways. Since secretin targets cholangiocytes during parasympathetic predominance, coordinated regulation of Cl-/HCO3- exchanger by secretin (cAMP) and ACh (Ca2+) could play a major role in the regulation of ductal bicarbonate excretion in bile just when the bicarbonate requirement in the intestine is maximal.

Intraoperative radiation therapy as adjuvant treatment in locally advanced stage tumours involving the middle ear: a hypothesis-generating retrospective study.

The objective of this study was to evaluate the safety, effectiveness and functional outcomes of intraoperative radiotherapy (IORT) followed by intensity-modulated radiation therapy (IMRT) in locally advanced stage tumours involving the middle ear. Data on 13 consecutive patients treated for malignant tumor of external auditory canal involving the middle ear were retrospectively reviewed. Median follow-up was 33 months (range 6-133). Five (38%) patients were stage III and 8 (62%) were Stage IV according to the University of Pittsburgh staging system. Lateral temporal bone resection (LTBR) was performed in all cases. LTBR was associated with parotidectomy in 5 (38%) cases, and with neck dissection and parotidectomy in 6 (46%) cases. No patients had gross residual tumour. Surgical treatment was followed by IORT (12 Gy) and IMRT (50 Gy). Adjuvant chemotherapy was used in 4 (30%) cases. Preoperative and postoperative audiometric tests were performed to assess hearing loss. 5-year local-control (LC), 5-year distant-metastasis (DM), 5-year disease-free-survival (DFS) and 5-year overall-survival (OS) were calculated with Kaplan-Meyer method. Significant changes in bone conduction were reported after treatment. Partial flap necrosis was the only early complication observed in three (23%) cases, while meningeal fistula was seen in one (7.6%) case as a late complication. The 5-year LC-rate was 68%. The 5-year DM-rate was 90%. The 5-year DFS-rate was 61%. The 5-year OS-rate was 69%. IORT followed by IMRT for the treatment of advanced external auditory canal and middle ear tumours seems to be safe. No intraoperative death was reported. IORT may reduce the postoperative irradiation of remnant tissue obtaining the same full dose on the tumour bed. No complications of the residual external ear were observed. Detriment of neurosensory hearing may be expected. Future studies are required to confirm the benefit of this procedure in the ear.

The role of proton therapy in the treatment of large irradiation volumes: a comparative planning study of pancreatic and biliary tumors.

PURPOSE: The purpose of this study was to examine the potential benefit of proton therapy for abdominal tumors. Extensive comparative planning was conducted investigating the most up-to-date photon and proton irradiation technologies. METHODS AND MATERIALS: A number of rival plans were generated for four patients: two inoperable pancreatic tumors, one inoperable and one postoperative biliary duct tumor. The dose prescription goal for these large targets was 50 Gy, followed by a boost dose up to 20 Gy to a smaller planning target volume (PTV). Photon plans were developed using "forward" planning of coplanar and noncoplanar conformal fields and "inverse" planning of intensity-modulated (IM) fields. Proton planning was simulated as administered using the so called spot-scanning technique. Plans were evaluated on the basis of normal tissues' dose-volume constraints (Emami B, Lyman J, Brown A, et al. Tolerance of normal tissue to therapeutic irradiation. Int J Radiat Oncol Biol Phys 1990;21:109-122) and coverage of treatment volumes with prescribed doses. RESULTS: For all cases, none of the forward calculated photon plans was able to deliver 50 Gy to large PTVs at the same time respecting the dose-volume constraints on all critical organs. Nine evenly spaced IM fields achieved or nearly achieved all maximum dose constraints to critical structures for two out of three inoperable patients. IM plans also obtained good results for the postoperative patient, even though the dose to the liver was very close to the maximum allowed. In all cases, photon irradiation of large PTV1s to 50 Gy followed by a 20 Gy boost entailed a risk very close to or higher than 5% for serious complications to the kidneys, liver, or bowel. Simple arrangements of 2, 3, and 4 proton fields obtained better dose conformation to the target, allowing the delivery of planned doses including the boost to all patients, without excessive risk of morbidity. Dose homogeneity inside the targets was also superior with protons. CONCLUSION: For the irradiation of large PTVs located in the abdominal cavity, where multiple, parallel structured organs surround the target volumes, proton therapy, delivered with a sophisticated isocentric technique, has the potential to achieve superior dose distributions compared with state-of-the-art photon irradiation techniques. IM photon plans obtain better results in the postoperative case, because the reduced volume lessens the effect of the unavoidable increase of integral dose to surrounding tissues.

Primary biliary cirrhosis: modalities of injury and death in biliary epithelium.

BACKGROUND/AIM: Despite the number of studies on primary biliary cirrhosis, contrasting data remain concerning modalities of cholangiocyte death. Liver biopsies obtained from 40 patients with anti mitochondrial antibody-positive primary biliary cirrhosis, at various stages of the disease, were examined, and special attention was paid to the expression of subcellular damage and evidence of apoptosis. METHODS: Liver sections were stained with haematoxylin/eosin or Sirius red. Ductular mass was evaluated on sections after cytokeratin 7 staining. Apoptosis was evaluated on haematoxylin/eosin stained material or after processing for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling assay. In 16 patients, part of the biopsy was processed for electron microscopy. Twenty histologically normal liver biopsies were used for control purposes. RESULTS: According to Scheuer's classification, 29 patients were classified as stage I-II, and 11 as stage III-IV. A strong staining of bile ducts was evident after immunohistochemistry for cytokeratin 7, often associated with ductular metaplasia in lobular zone 1. Cytokeratin 7-positive cells occupied 3.0+/-1.3% of liver mass as compared to 0.25+/-0.03% in controls. Ductular metaplasia accounted for 1.4+/-0.07% of all cytokeratin 7-positive cells. Regardless of staging, apoptotic bodies were seen only exceptionally in epithelial wall of bile ducts, whereas cholangiocyte damage leading to extensive lytic necrosis appeared responsible for most of the bile duct mass loss, as also confirmed by ultrastructural studies. A few terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling-positive nuclei were occasionally associated with the inflammatory infiltrate and evidence of apoptosis in hepatocytes was frequent, especially in zone 1. CONCLUSION: Regardless of staging, lytic necrosis and not apoptosis accounts for most of the bile duct loss in primary biliary cirrhosis. Furthermore, ductular metaplasia appears as a late event with highly variable pattern being observed between patients.

[L-sulpiride versus metoclopramide in functional dyspepsia: a randomized double-blind study]. / L-sulpiride contro metoclopramide nella dispepsia funzionale: studio randomizzato in doppio cieco.

Fifty patients suffering from functional dyspepsia have been treated in a double-blind study either with 1-sulpiride (75 mg die per os) or with metoclopramide (30 mg die per os) for 30 days. The frequency and severity of the symptoms in the two patient groups were similar. The administration of either drug was followed by a reduction of the symptoms, but 1-sulpiride was more effective on nausea, headache, pyrosis, epigastric pain, and showed an earlier effect than metoclopramide in inducing total regression of symptoms.

Organ preservation in the management of pancreatic cancer.

Over a 5-year period, 6 patients with potentially resectable (cT2) pancreatic adenocarcinoma underwent exclusive radiotherapy. Surgery was ruled out because of the patients' age (> 75 years) in 5 cases and for contraindication for anesthesia in the remaining one. Four patients underwent intraluminal brachytherapy (50 Gy) with linear 192Ir sources; the remaining two underwent concomitant radiochemotherapy (39.6 and 50.4 Gy respectively) followed by a boost dose (50 and 20 Gy respectively) of intraluminal brachytherapy. All patients completed the treatment without relevant acute side-effects. One patient undergoing combined treatment showed gastric ulcer 8 months after treatment. Two patients showed local progression of the disease and two distant metastases. In one of the two patients with local progression, digestive by-pass was required. All patients died. Median survival was of 14 months. Actuarial survival at 1, 2 and 3 years was 66%, 33% and 16% respectively. No patient showed pain during follow-up. The fairly good quality of life observed in these patients undergoing conservative treatment and the rate of survival, comparable with that of surgical series, suggest that irradiation should be considered in those patients in whom radical surgery in contraindicated.

Radiotherapy, local control and survival in brain tumors.

High grade glial brain tumors and brain metastases are a complex subject with still unsatisfactory therapeutic results for the frequent absence of early and precise diagnosis as well as for the limited therapeutic interval between the tumor and presumed healthy tissues. The therapeutic problems of cellular hypoxia, the rapid recovery of sublethal damage for neoplastic cells, the rapid regrowth, have led to a number of efforts to deliver the dose of radiotherapy in various associations. The constant technological trend to increase the high dose gradient to the peripheral tumor, is reported.

Activity and safety of bevacizumab plus fotemustine for recurrent malignant gliomas.

BACKGROUND: No established chemotherapeutic regimen exists for the treatment of recurrent malignant gliomas (rMGs). Herein, we report the activity and safety results of the bevacizumab (B) plus fotemustine (FTM) combination for the treatment of rMGs. PATIENTS AND METHODS: An induction phase consisted of B 10 mg/kg days 1, 15 plus FTM 65 mg/m(2) days 1, 8, and 15. Nonprogressive patients entered the maintenance phase with B 10 mg/kg plus FTM 75 mg/m(2) every 3 weeks. The primary endpoint was response rate; secondary endpoints included safety, progression free survival (PFS), and overall survival (OS). RESULTS: Twenty-six patients affected by recurrent MGs (50% glioblastoma) were enrolled. Eight partial responses (31%) were observed. Median PFS and OS were 4 (95% C.I.: 2.8-5.1) and 6 months (95% C.I.: 4.2-7.8), respectively. Responses were significantly associated with both improved PFS and OS (P = 0.002 and P = 0.001, resp.). Treatment adverse events were mostly mild to moderate in intensity. Bevacizumab-related adverse events included grade 3 venous thromboembolic event (8%), grade 2 epistaxis (4%), hypertension (8%), and gastrointestinal perforation (4%). CONCLUSIONS: Bevacizumab plus FTM showed activity and good tolerability in pretreated MGs. Further investigations are needed in order to verify the benefits deriving from the addition of B to a cytotoxic in this clinical setting of patients.

Anatomical and dose changes of gross tumour volume and parotid glands for head and neck cancer patients during intensity-modulated radiotherapy: effect on the probability of xerostomia incidence.

AIMS: To quantify the changes in dose as well as in the prediction of parotid gland toxicity due to anatomical changes during therapy of head and neck cancer patients. MATERIALS AND METHODS: Fifteen patients with advanced locoregional head and neck cancer, with no evidence of distant metastasis, were enrolled in a prospective study. All patients were treated with intensity-modulated radiotherapy. Multiple computed tomography scans were repeated at the end of each treatment week. The original treatment plans were copied to the per-treatment scans to create hybrid plans. The normal tissue complication probability (NTCP) was calculated assuming the end point to be grade ≥3 xerostomia according to the Radiation Therapy Oncology Group late toxicity scale. RESULTS: The gross tumour volume dose coverage was slightly affected by the anatomical changes, whereas the mean dose (D(mean)) to the parotids changed from 26.1 ± 6.0 to 27.4 ± 7.4 Gy, with a mean increase of 0.22 Gy/treatment week. Consequently, the mean NTCP increased from 0.15 ± 0.06 to 0.18 ± 0.10, primarily due to a few patients exhibiting a marked increase. The absolute gross tumour volume shrinkage and the percentage parotids shrinkage were the best independent predictors for the NTCP variations. CONCLUSIONS: On average, the increase in the parotids D(mean) as well as in NTCP during treatment is limited, and the observed variations were strongly patient-dependent.

The significance of apoptosis in the liver.

Apoptosis is a form of cell death which occurs in normal as well as in pathological tissues. We provide a description of the morphological changes during apoptosis and an overview of the role of apoptosis dysregulation in the pathogenesis of non-neoplastic liver diseases.

Morphological features and modulation of the intrahepatic biliary epithelium.

Much has been learned in the past few years concerning the morphology and function of the intrahepatic biliary epithelium. Immunohistochemistry, together with ultrastructural studies has allowed a better identification or the smallest branches of the biliary tree and of subcellular components (cytoskeleton, specializations of the cell membrane, specific receptors). Modulation of the biliary epithelium in response to physiological or pathological stimuli has renewed the interest concerning the existence of facultative stem cells in the liver. More information is needed however concerning the mechanisms of cell loss in vanishing bile duct syndromes.

Effect of a xanthine analog on human hepatocellular carcinoma cells (Alexander) in culture and in xenografts in SCID mice.

Hepatocellular carcinoma (HCC) frequently overexpresses the MDR1 gene and is resistant to drugs transported by the multidrug-resistance efflux pump. A xanthine analog, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (CT-2584,CTI), is cytotoxic to many tumors in culture and was four times more effective than verapamil in inhibiting Rhodamine 123 secretion in MDR1-overexpressing Chinese hamster ovary cells. However, studies using PRF/PLC/5 (Alexander) cells revealed that CT-2584 is cytotoxic by another mechanism not involving inhibition of MDR1 function. Alexander cells have integrated the hepatitis B surface antigen (HBsAg) gene and quantitatively secrete HBsAg. The parent cell line, Alex 0, has low MDR1 expression and is drug-sensitive, whereas a derived line, Alex 0.5, is drug-resistant and overexpresses MDR1 100 times. Both cell lines were similarly killed within 24 or 48 hours by CT-2584. Freshly isolated rat and human hepatocytes were considerably more resistant to killing by CT-2584. In vivo, CT-2584 significantly reduced tumor growth in SCID mice bearing Alex 0 or 0.5 xenografts as determined by serial measurements of HBsAg. Hepatic parenchyma was normal, whereas apoptosis and cellular loss were observed in xenografts. The xenograft model is useful for testing pharmacological therapy of HCC.

Regulation of intracellular pH in isolated periportal and perivenular rat hepatocytes.

BACKGROUND: Liver acinus shows a well-known metabolic zonation. The aim of this study was to investigate intracellular pH (pHi) regulation in isolated periportal (PP) and perivenular (PV) rat hepatocytes. METHODS: 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein was used as pH-sensitive dye. Hepatocyte subconfluent monolayers were acid-loaded by exposure to 20 mmol/L NH4Cl and alkali-loaded by reducing external CO2 and HCO3- at an external pH of 7.4. RESULTS: In the presence of HCO3-/CO2, (1) baseline pHi was higher in PP (7.25 +/- 0.018) than in PV hepatocytes (7.20 +/- 0.013) (P < 0.05); (2) pHi recovery from an acid load was 40% higher in PP than in PV hepatocytes (P < 0.02) and was inhibited by amiloride by 36% in PV and 7% in PP hepatocytes; (3) DIDS inhibited amiloride-independent pHi recovery from an acid load by 65% in PP and 52% in PV cells. In the absence of HCO3-/CO2, baseline pHi and pHi recovery from an acid load were not significantly different in PP and PV hepatocytes. pHi recovery from an alkali load was 30% higher in PV than in PP cells (P < 0.02). CONCLUSIONS: Our data suggest that isolated PP rat hepatocytes show higher activity for Na(+)-HCO3- cotransport, whereas PV cells show greater activity for Cl-/HCO3- exchanger.

Brefeldin A inhibits the transcytotic vesicular transport of horseradish peroxidase in intrahepatic bile ductules isolated from rat liver.

The fungal metabolite Brefeldin A (BFA) has become a valuable tool to address mechanisms of membrane transport in eukaryotic cells. The aim of the study was to investigate the action of BFA on the endocytic and transcytotic pathways in the biliary epithelium. Intrahepatic bile ductules were isolated from rat liver by collagenase digestion and mechanical separation of biliary tree from parenchymal tissue. Tissue remnants were first incubated in L-15 culture medium in absence or presence of BFA (10 or 20 mumol/L) or a BFA-inactive analog (B-36, 10 or 20 mumol/L) for 20 minutes at 37 degrees C. They were then exposed to horseradish peroxidase (HRP) (10 mg/mL) for 3 minutes at 37 degrees C and finally prepared for electron microscopy immediately (time 0) or after further 5, 10, 15, 20, 60, or 120 minutes' incubation in HRP-free medium with or without BFA. In control cells, HRP was predominantly found in regularly shaped, spherical vesicles. In the presence of BFA but not of its analog, HRP was retained in a prominent tubular juxtanuclear network. Part of this network was labeled for thiamine pyrophosphatase (TPP), a Golgi enzyme marker. A morphometric analysis of HRP-containing structures was performed to quantify the intracellular distribution of HRP. In presence of BFA, the volume density (VD = % area) of HRP-containing structures in the basolateral region was not significantly different with respect to control cells at 0 (1.08 +/- 0.11 vs. 1.32 +/- 0.11) or 5 minutes, respectively (1.33 +/- 0.19 vs. 1.40 +/- 0.13). On the contrary, VD or HRP-containing structures in the apical region at 15 minutes decreased from 1.95 +/- 0.19 in control cells to 1.12 +/- 0.20 (P < .02) in BFA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical analysis of S-phase cells in normal human and rat liver by PC10 monoclonal antibody.

The expression of proliferating cell nuclear antigen (PCNA) was examined in normal human and rat liver fixed in either formaldehyde or methanol, and was compared with the incorporation of bromodeoxyuridine (BrdU) in S-phase cells. Codistribution of PCNA and BrdU was assessed in rat liver by double immunohistochemical staining using PC10 and anti-BrdU monoclonal antibodies to identify labelled nuclei of parenchymal and sinusoidal cells. In formaldehyde-fixed human biopsies (n = 13) PCNA-labelling index (PCNA LI) was 0.43 +/- 0.24% (mean +/- SEM) for hepatocytes and 0.09 +/- 0.03% for sinusoidal cells. A great interspecimen variability was observed and a preferential lobular distribution was not evident. In methanol-fixed human liver (n = 8) the immunostaining was strong. PCNA LI was 0.05 +/- 0.01% for hepatocytes and 0.14 +/- 0.01% for sinusoidal cells. 75% of labelled hepatocytes and 60% of labelled sinusoidal cells were found in acinar zone 1. In formaldehyde-fixed rat liver (n = 10) a weak nuclear staining and a great interspecimen variability were evident. LI was 0.13 +/- 0.07% for hepatocytes and 0.40 +/- 0.21% for sinusoidal cells without preferential acinar distribution. In methanol-fixed rat liver (n = 10), PCNA LI was 0.14 +/- 0.02% for hepatocytes and 0.40 +/- 0.04% for sinusoidal cells. 64% of labelled hepatocytes and 50% of labelled sinusoidal cells were found in zone 1. Only on methanol-fixed material did double immunohistochemistry show an almost complete overlap of BrdU and PCNA labelling. The PCNA LIs and the zonal distribution of labelled nuclei as obtained in methanol-fixed material are in keeping with previous reports using 3H-thymidine (3H-Thy) incorporation, suggesting that PCNA immunostaining represents a valid alternative to 3H-Thy.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic ethanol feeding increases apoptosis and cell proliferation in rat liver.

The present study was conducted to evaluate if the increased rate of apoptosis previously reported in the liver of ethanol-treated rats was accompanied by increased cell renewal. A quantitative analysis of apoptosis was performed in rats fed an ethanol-containing liquid diet for 5 weeks. S-phase cells were demonstrated by immunohistochemistry, using the Bromodeoxyuridine/anti-Bromodeoxyuridine method. In ethanol-fed rats apoptosis was five times greater than in pair-fed controls. Bromodeoxyuridine-labelled hepatocytes increased from 0.07 +/- 0.009% in controls to 0.17 +/- 0.013% (p < 0.001) and Bromodeoxyuridine-labelled lipocytes (desmin-positive sinusoidal cells) increased from 3.43 +/- 0.28% to 6.60 +/- 1.04% (p < 0.001). The lobular distribution of labelled cells was modified with a shift towards the perivenular areas. The results of this study suggest that the replacement of liver cells lost by ethanol-induced apoptosis is not impaired in intact (non-operated) animals. The impaired regeneration following partial hepatectomy reported in ethanol-fed rats is possibly due to differences in the extent of parenchymal loss, to altered relationships between hepatocytes and blood supply and to the modalities of regeneration involved.

Regulation of intracellular pH in periportal and perivenular hepatocytes isolated from ethanol-treated rats.

The aim of this study was to gain information on intracellular pH (pHi) regulation in periportal (PP) and perivenular (PV) hepatocytes isolated from rats pair-fed liquid diets with either ethanol (T rats) or isocaloric carbohydrates (C rats). pHi was analyzed by the pH-sensitive dye BCECF in perfused subconfluent hepatocyte monolayers. Cells were acid-loaded by pulse exposure to NH4Cl and were alkali-loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHout. In cells from C rats: (a) steady-state pHi was higher in PP than in PV hepatocytes in the presence, but not in the absence, of bicarbonate; (b) pHi recovery from an acid load was 35% higher in PP than in PV cells in the presence of HCO3-, whereas it was similar in HCO3(-)-free experiments; and, on the contrary, (c) pHi recovery from an alkaline load was 30% higher in PV than in PP cells. In cells from T rats: (a) steady-state pHi was always lower than in cells isolated from pair-fed animals; (b) steady-state pHi was similar in PP and PV hepatocytes either in the presence or absence of bicarbonate in the perfusate; (c) pHi recovery from an acid load was not significantly different in PP and PV cells either in the presence of HCO3- or in HCO3(-)-free experiments; and (d) pHi recovery from an alkaline load was similar in PP and PV cells. Our data suggest that chronic ethanol treatment selectively modifies pHi by affecting the activity of ion transport mechanisms regulating pHi in PP and PV hepatocytes isolated from rat liver.

A morphometric study of the epithelium lining the rat intrahepatic biliary tree.

BACKGROUND/AIMS: Morphological and functional heterogeneity of intrahepatic bile duct epithelial cells has been suggested in situ and in isolated cholangiocytes. The aim of this study was to evaluate if: (a) bile ducts, when isolated, maintain morphometric parameters similar to ducts in situ, (b) cellular organelles show heterogeneity in ducts of different size, and (c) some features permit different classes of bile ducts to be distinguished. METHODS: Studies in situ were conducted on normal liver processed for light or electron microscopy. Data were also obtained from preparations of intrahepatic biliary tree isolated from rat liver. The whole biliary tree was cut at different levels to obtain bile ducts of different diameter. The diameter of ducts, the number of lining cells, the size and the area of individual cells, the nucleo/cytoplasmic ratio, the volume density of mitochondria, endoplasmic reticulum, Golgi complex and lysosomes have been evaluated. RESULTS: The diameter of intrahepatic bile ducts ranged from 5 to 100 micrograms and the area of lining cells ranged from 8 to 100 micrograms2. A highly significant linear relationship existed between duct diameter and bile duct epithelial cell area (r = 0.97, p < 0.001) or number of lining cells (r = 0.96, p < 0.001). The volume density of mitochondria ranged from 7.58 +/- 2.0% of cytoplasmic volume in the smallest isolated bile ducts to 8.50 +/- 2.7% in the largest (p = NS). The volume density of lysosomes was low and was not significantly different in ducts of different size. Rough endoplasmic reticulum was inconspicuous in the smallest ducts and increased only slightly in the largest. The inverse relationship between the nucleo/cytoplasmic ratio and duct diameter was striking (r = -0.78, p < 0.001). All morphometric data were equivalent if bile ducts were evaluated in situ or in isolated fragments. Taken together, the data allowed bile ducts to be classified into 3 classes: < 10, 10-50, and > 50 micrograms in diameter. DISCUSSION: Our data show that (a) isolated bile ducts maintain morphometric characteristics similar to the tissue in situ, (b) a low grade of morphological heterogeneity is evident for intracellular organelles in ducts of different diameter and (c) the diameter of ducts, the number of lining cells and especially the nucleo/cytoplasmic ratio may indicate the origin of fragments examined where functional studies are being considered.

Evidence that plasma membrane fluidity of isolated hepatocytes is modified by exposure to microtubule-depolymerizing drugs.

The role of microtubules on membrane fluidity has been investigated on freshly isolated whole rat hepatocytes prepared by the perfusion method and exposed either to the microtubule-depolymerizing drugs colchicine and vincristine or to beta-lumicolchicine, a colchicine analog deprived of biological activity. Exposure of hepatocytes to 6.3 microM colchicine or to 3.0 microM vincristine led to a significant decrease of membrane fluidity as measured by fluorescence polarization of trimethylammoniodiphenylhexatriene (TMA-DPH). No changes were observed in cells exposed to 10.0 microM beta-lumicolchicine. These observations support the hypothesis that the microtubular system plays a role in the modulations of physico-chemical properties of the plasma membrane.

Immunohistochemical identification of proliferating cells following dimethylnitrosamine-induced liver injury.

The present study is concerned with changes in the number and localization of S-phase cells in the liver of rats exposed to dimethylnitrosamine (DMN). S-phase cells were detected by immunohistochemistry after injection of bromodeoxyuridine (BrdU) and exposure of paraffin sections of liver tissue to the antibody anti-BrdU. With respect to controls, the number of S-phase cells increased four to fivefold in DMN-treated animals in the first week of treatment and remained significantly higher thereafter, in association with the formation of septa. At all times, the labelling index was higher in littoral cells than in hepatocytes. No labelling was observed in biliary cells. This behaviour is different from that reported in other situations, for instance in regeneration after partial hepatectomy, which suggests that besides hepatocytes and littoral cells replacement, an involvement of the latter cell line in the inflammatory reaction, synthesis of extracellular matrix components and formation of septa may account for this particular pattern.