The present study was aimed at re-evaluating the mechanism by which partial hepatectomy (PH) and the resulting mitogenic stimulation act to positively modulate the action of a chemical hepatocarcinogen applied 24 hours after surgery. Using as a marker the immunocytochemical expression of glutathione S-transferase placental form (GST-P), we compared the early incidence of single altered hepatocytes and the time-course of appearance and growth of subsequently developed liver neoplasia in diethylnitrosamine (DENA) treated sham-operated or partially hepatectomized animals. The data showed that, compared to sham-operation, partial hepatectomy increases the frequency of early GST-P positive single hepatocytes appearing 72 h after treatment with DENA (50 mg/kg b.w.), indicating a positive (co-carcinogenic) effect of PH on the frequency of the initiation event. At 2 months after this treatment, foci of altered hepatocytes were about 5 times more numerous in PH-pretreated than in sham-operated animals and their average volume about four times greater. The latter difference persisted for up to 10 months after carcinogen treatment, indicating that the lesions had grown at similar rates in the meantime. The simplest hypothesis to account for this difference in size of the lesions is that it is due to the 2-3 waves of normal-like cell division the altered hepatocytes underwent during the very first days after removal of part of the liver mass, rather than to true promotion or to an intrinsic difference in proliferative phenotype of the initiated cells. The increased size and higher frequency of the lesions in PH-pretreated rats concur to increase the population of cells at risk towards subsequent events implied in multistep progression and, by this, to positively modulate the latter. Accordingly, only PH treated rats had developed neoplastic nodules at 10 months and tumours at 15 months. This can therefore be explained without resorting to the currently held view that cell divisions in PH would increase the carcinogenic efficiency of DENA, due to the fact that resulting cell divisions would intervene before full repair of DENA-induced DNA damages.
DMH-induced (25mg/kg/wk) rodent intestinal carcinogenesis was re-examined using histometry under various conditions including modulation of host natural killer (NK) cell activity and mutagen administration. Early lesion was enterocyte hyperplasia all along the intestinal tract. Cell kinetic analysis showed that it was caused by a slightly altered but still functional "initiated" enterocyte population added to the normal population which remained unchanged. Primarily, initiated stem (IS) cells were added which then produced their initiated progeny which still renewed normally. NK cells countered the initiated cells selectively, either inhibiting IS cell proliferation or killing them when activated by lymphokines. Evidence was obtained that the IS cells could further transform into preneoplastic stem (PS) and then neoplastic stem (NS) cells under the influence of promoters and mutagens, respectively. Subsequent transformation of normal stem cells into IS, PS, and NS cells apparently is the basis of carcinogenesis. These produce lesions, hyperplastic, preneoplastic, and neoplastic, respectively, only when and where NK activity is inhibited. Under the influence of normal NK cells, they remain dormant (nonproliferative), compatible with normal life.
The excretory/secretory (ES) products of the nematode parasite Trichostrongylus colubriformis have been found to increase the in vitro proliferation of the epithelial cell line HT29-D4. To assess the specificity of this effect, ES products from other trichostrongyle species were tested on colonic (HT29-D4) and gastric (HGT-1) tumour cell lines. Adult worms of six different nematode species, parasites of the stomach or the small intestine of ruminants, were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h. The conditioned media were then added at different concentrations to the culture medium of the two cell lines. A stimulation of the HT29-D4 cell growth occurred with the ES products of two parasite species of the small intestine, at the concentrations of 0.1 microgram protein/ml (Trichostrongylus vitrinus) and 1.0-5.0 micrograms/ml (Cooperia curticei). Inversely, a decrease in cell number was observed with the ES products of another intestinal species, Nematodirus battus at concentrations of 1.0-5.0 micrograms/ml. With the ES products of the abomasal nematodes, a proliferation of HT29-D4 cells was obtained at 0.25-5.0 micrograms/ml with ES products of Teladorsagia circumcincta but no significant effect was observed for Haemonchus contortus. On the tumoral gastric cell line HGT-1, the ES products from the 6 nematode species gave a similar stimulative effect. These in vitro results suggest that nematode parasite species secrete or excrete component(s) which could affect the epithelial regeneration of the host digestive tract.
Introduction of fecal bacteria into germ-free (GF) Balb/c mice induces class II major histocompatibility complex (MHC) molecules on the small intestinal epithelium. In this study, we elucidated the regulatory mechanisms for the class II MHC molecule induction on the mouse small intestinal epithelium during microbial colonisation of the gut in ex-GF mice. Intraperitoneal injection of interferon-gamma (IFN-gamma) into GF Balb/c mice induced class II MHC expression on the small intestinal epithelial cells. Induction of these molecules was inhibited by peritoneal injection of a monoclonal antibody (mAb) against IFN-gamma on the conventionalisation of GF mice.RNA reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the small intestinal epithelium indicated that the class II transactivator (CIITA), a regulatory factor for the class II MHC gene, and the I-E alpha chain, but not IFN-gamma receptor mRNA, increased during conventionalisation. The induction of class II MHC on the epithelial cells during the conventionalisation of GF C.B-17 scid mice was much lower than that in GF Balb/c mice.Immunocytochemical and RT-PCR analysis showed that both the number of IFN-gamma producing IEL and the level of the IFN-gamma mRNA in gamma delta TCR IEL were very low in the GF state, and gradually increased after microbial colonisation. After in vivo treatment with a mAb against gamma delta TCR, the number of gamma delta TCR-expressing IEL greatly decreased and the expression of class II MHC molecules on the small intestinal epithelium was repressed during the conventionalisation of GF mice. Taken together, these results suggested that gamma delta TCR-bearing IEL modulate class II MHC molecule expression on the small intestinal epithelium through the production of IFN-gamma during microbial colonisation in ex-GF mice.
We previously demonstrated that spreading and clustering of in vitro cultured human keratinocytes are autocrine-induced phenomena, mediated by keratinocyte-secreted soluble factor. In this paper, the effects of this factor on spreading, number of dendrites and cell-cell contacts of the two cellular components of skin, melanocytes and fibroblasts have been studied 24 h after plating cells on uncoated plastic surfaces in MCDB153 serum-free medium or in the same medium conditioned by keratinocytes (KCM). Spreading of melanocytes present in the epidermal cell population remained constant at increasing cell density, while that of keratinocytes showed a statistically significant increase. Moreover, time-course experiments showed that the rate of spreading was faster for melanocytes. At increasing epidermal cell density, a statistically significant increase in number of dendrites and cell-cell contacts of melanocytes was observed. Similar results were obtained when melanocytes were plated both in coculture with keratinocytes (as epidermal cell cultures) or as a pure cell population in keratinocyte conditioned medium (KCM), suggesting that the observed phenomena are due to keratinocyte-secreted soluble factors and not to direct keratinocyte-melanocyte interactions. The addition of nerve growth factor (NGF) to fresh medium or addition of an inactivating anti-NGF monoclonal antibody (alpha D11) to KCM did not affect the number of dendrites or cell-cell contacts of melanocytes. Keratinocyte-secreted soluble factor(s) present in KCM also dramatically influenced morphology and cell-cell contacts of human dermal fibroblasts.
The present study investigates the spatial organisation of epithelial cell proliferation in human small intestinal xenografts, in order that direct comparisons can be made with paediatric small bowel. For this purpose we employed the MIB-1 (Ki-67) monoclonal antibody and [3H]thymidine to analyse the crypt growth fraction and DNA synthesising (S-phase) cells, respectively. The spatial distribution of cycling (MIB-1+) cells was appropriately confined to the xenograft crypts where it closely resembled that of paediatric intestine, both in terms of the labelling index and an ability to form runs of labelled cells, thereby demonstrating synchronous patterns of cell division. In addition, the S-phase representation in xenograft intestine was uniform throughout the crypt proliferation compartment thereby indicating cell-cycle homogeneity. This chimeric model system now provides a new approach to investigate altered proliferative responses of human gut to a number of potentially harmful substances e.g. carcinogens, the assessment of which is not feasible in patients or volunteers.
In vivo, bicarbonate can affect proximal tubule intermediary metabolism, including gluconeogenesis, ammoniagenesis and maintenance of the mitochondrial substrate supply. In vitro, rabbit proximal tubule cells (RPTC) in primary culture revert from gluconeogenesis to glycolysis and their mitochondrial metabolism remains lower than in vivo. To determine whether the bicarbonate buffer system could have an effect on these deregulations, RPTC in primary culture grown in the absence of insulin and glucose in the culture medium were developed either with the standard sodium bicarbonate buffer with 5% CO2 or with a Hepes hydrogen ion buffer in the presence of 0.5% CO2. Duration of the bicarbonate-free cultures was increased until at least day 17 after seeding, compared with day 11 in bicarbonate-buffered cultures. As could be expected, succinate dehydrogenase activity remained stable as a function of time in bicarbonate-free cultures while an early marked decrease of this activity occurred from seeding in cultures developed in the presence of bicarbonate buffer. Compared to bicarbonate-buffered cells, higher phosphoenolpyruvate carboxykinase activity concomitant with lower intracellular lactate dehydrogenase activity was observed in cultures developed in the absence of bicarbonate, which is indicative of closer carbohydrate metabolism orientation to the in vivo situation for RPTC. Immunofluorescence staining of RPTC with monoclonal antibodies directed to neutral endopeptidase (NEP), and dipeptidyl-peptidase IV (DPP II) showed similar extensive labelling with DPP and NEP in both culture conditions. Confocal microscopy analysis of NEP subcellular distribution, showed exclusive targetting of NEP to the apical plasma membranes. In both models, cAMP production was stimulated by parathyroid hormone and unaffected by arginine vasopressin. In conclusion, bicarbonate withdrawal from the culture medium (without changing the pH of the medium) allows a marked improvement of mitochondrial capacity and carbohydrate metabolism pattern without any loss of differentiated properties.
Anchoring fibrils (AFs) are derived from basal keratinocytes, but the kinetics of their formation is unknown, In this study, de novo generation of AFs by cultured human keratinocyte autografts was assessed from 1 week to 6 years postgrafting. Within 2 weeks, AF population densities were equal to those of normal controls and remained normal thereafter. However, AF diameters were narrow compared to controls (P < 0.05) until 3 years postgrafting. The depth of extension of AF-anchoring plaque lattices into the subjacent stroma was normal by 3 weeks but, after 1 month, was typically 1.5- to 2-fold greater than normal. The findings indicate that: (1) basal keratinocytes immediately re-establish a full complement of AFs; (2) once reformed, AF populations remain normal in density over time; (3) nascent AFs are thin and require several years to reach full maturity; and (4) abnormally thick AF lattices may form over time in healed wounds.
The developing renal collecting duct epithelium of neonatal rabbits exhibits 3 different zones. The ampullary tip epithelium acts as an embryonic inducer and is responsible for the generation of all of the nephron anlagen. It pilots the whole microarchitecture of the kidney. In the ampullary neck epithelium multiple cell divisions cause the elongation of the embryonic collecting duct so that the organ can grow. Finally, the cells in the ampullar shaft transdifferentiate into the functional collecting duct epithelium (CD) consisting of Principal (P) and various kinds of Intercalated (IC) cells. It is unknown by which morphogenic mechanisms the ampullar cells develop into the heterogeneously composed collecting duct epithelium. Using both morphological and immunohistochemical methods, we investigated the transdifferentiation patterns leading from the ampullar epithelium to the P and IC cells in the neonatal kidney. An electron microscope analysis of the cortico-medullary course of the developing collecting duct revealed that conspicuous morphological alterations start in the neck of the ampulla. The lumen of the neck region is narrowed to a slit. While most of the cells in the ampullar tip exhibit few, short microvilli, the neck cells bear numerous, extremely long microvilli at their apical cell poles. All of the neck cells exhibit the same cytoplasmic staining pattern and the same number of mitochondria. Farther down in the shaft, clearly recognizable P and IC cells are found. Thus, differentiation into P and IC cells starts with a transitional precursor cell type in the ampullar neck. Perfusion culture experiments with the embryonic collecting duct epithelium made it possible to generate transitional and differentiated cell types for the first time under in vitro conditions. The cultured epithelial cells showed characteristics common to both P and IC cells. Immunohistochemical findings revealed that morphological differentiation starts before the functional properties of P and IC cells can be detected.
The presence of the nematode parasite Trichostrongylus colubriformis in the small intestine is associated with an increase in epithelial renewal. To assess the possible role of excretory/secretory products from the worm on cell proliferation, adult Trichostrongylus colubriformis were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h and the conditioned medium was added to the culture medium of the transformed epithelial cell line HT29-D4. A stimulation of the HT29-D4 cell growth was ascertained at concentrations of 0.25-1.0 micrograms protein/ml using counts of cell numbers, the MTT method and incorporation of tritiated thymidine. An increased incorporation of tritiated thymidine was also observed with the excretory/secretory products from T. colubriformis fourth stage larvae at 1.0 microgram/ml. Dialysis of the medium conditioned by the worms indicated that the molecular weight of the factor is greater than 8000 Daltons in size. Heat treatment, acid hydrolysis and precipitation by trichloracetic acid of the conditioned medium resulted in the disappearance of the proliferative effect while treatment with trypsin partially depleted the stimulative activity. These results suggest that T. colubriformis produce some protein factor which could increase the epithelial regeneration in the host small intestine.
The expression of EGF receptors has been studied on luminal and basal cells of human breast in vitro. Primary cultures of normal adult human breast epithelium were prepared as single cell suspensions containing a mixture of luminal and basal cells. The cells were simultaneously immunolabelled with antibodies recognising EMA (luminal epithelial cells), CALLA/CD10 (basal cells) and the epidermal growth factor receptor (EGFR). Flow cytometric analysis of these triple labelled cells detected low levels of EGFR on both cell types, with proportionally more EGFR on basal cells compared with luminal cells. Separated populations of basal and luminal cells were prepared from single cell suspensions by flow sorting or by immunomagnetic methods and cultured with and without EGF. Increased proliferation was detected in both cell types in the presence of EGF. To determine the localisation of the EGF receptor, purified cell populations were immunolabelled with anti-EGFR antibody and an FITC-labelled second antibody for fluorescence light microscopy and colloidal gold-labelled antibody for scanning electron microscopy (SEM). Low levels of EGFR were detected by indirect immunofluorescence on both cell types with higher levels on basal cells compared with luminal cells. The detailed subcellular distribution of the receptor was examined by SEM, with gold-labelling of EGFR detected using a field emission scanning electron microscope with a YAG crystal backscattered electron detector. Both luminal and basal cells expressed EGFR over the upper surface of individual cells when these were growing in isolation, but when cells formed part of a confluent island, levels of EGFR on the upper surface of cells were obviously reduced. Observations made by SEM on cells at the edges of such confluent islands showed that cultured basal cells expressed much higher levels of EGFR on their basal, as compared with their upper surfaces.
We have previously shown that the 180-kD bullous pemphigoid antigen (BPAII), which is a transmembrane collagenous protein of hemidesmosomes, is distributed at adhesion sites on glass coverslips on the basal membrane forming a concentric ring, or arch pattern, in a human squamous cell carcinoma cell line (DJM-1), when studied by immunofluorescence microscopy using monoclonal antibodies to BPA II. This concentric ring/arch pattern of ''footsteps'' of BPA II has been shown to be collapsed in association with a transient activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, therefore, the effects of TPA on the phosphorylation of BPA II was examined. DJM-1 cells, which were metabolically labelled with [(32)Pi], were lysed and the extracts were subjected to immunoprecipitation with anti-BPAII and anti-230 kDa bullous pemphigoid antigen (BPAI) monoclonal antibodies. The results showed that only BPA II, but not BPA I, was phosphorylated at serine residues before TPA treatment. After TPA treatment phosphorylation was prominently increased so as to generate a 190 kDa-phosphorylated peptide. This 190-kDa peptide was reacted with anti-BPA II monoclonal antibodies by immunoblotting, and it was not detected when cells were pretreated with a specific protein kinase C inhibitor (H7) before TPA treatment, suggesting that the 190 kDa peptide is phosphorylated BPAII with TPA. Prolonged treatment with TPA abolished both of 180- and 190-kDa BPA II from Triton X-100-soluble fractions. These findings suggest that the BPA II, but not BPA I, is a substrate of protein kinase C, and the generation of 190-kDa-phosphorylated BPA II has a key role in the TPA-induced collapse of the assembly of BPA II on the basal plasma membrane, probably, at hemidesmosomes.
A technique is described for the reproducible primary culture of colonic epithelium from adult mice. A collagenase-dispase digestion technique (adapted form Evans et al. 1992) is used to release the epithelium, followed by differential sedimentation to produce a high purity crypt preparation with maintained structural integrity and minimal mesenchymal contamination. The crypt units attach to collagen coated plastic within 24 h and the epithelial cells quickly begin to migrate outwards producing a monolayer surrounding the attached crypts. Electron microscopy revealed that the migrating epithelial cells possessed both desmosomes and microvilli. Proliferation in the colony supports the outward migration of cells until the migratory cells of adjacent colonies connect and a confluent monolayer begins to form. Proliferation is routinely maintained for 10 days (although cultures have now been maintained without subculturing for 35 days) and is demonstrated by increased cell numbers in spite of continuous cell loss into the culture media. Culture growth is enhanced by increasing concentrations of fetal calf and mouse serum and EGF but does not appear to respond significantly to added transferrin. Growth is also stimulated by a murine small intestinal extract thought to contain a potentially novel growth factor or cocktail of factors. This culture model has considerable potential for studies on growth factor control of this carcinoma susceptible tissue and its differentiated function as well as studies into the mechanisms of carinogenesis.
In this morphological study the (ultra)structural changes that lead to contraction of detached cultured epithelium were investigated. Keratinocytes, isolated from human skin and oral mucosa, were grown to form stratified cell sheets. The multilayers were examined with light and electron microscopy before, during and after detachment from the culture vessel. Attached epithelium had a stretched morphology with flattened cells and nuclei. Evidence is provided that after enzymatical detachment with dispase (1) basal cells became columnar by contraction of actin bundles in the basal cortex, which was accompanied by blebbing of the basal cell membrane; (2) in all cell layers cytokeratin bundles contracted resulting in displacement of desmosomes and a spherical shape of the cells and nuclei. By slow dispase-detachment at 4 degrees C or by quick mechanical detachment, shrinkage of the sheet was partly suppressed but contraction of cytokeratin and related events occurred indicating that these were the result of the spontaneous reassembly of the intermediate filament system. The results suggested that the shape and ultrastructure of all cells in an epithelial multilayer are dependent on the interaction of the basal cells with the underlying extracellular matrix.
We have investigated expression of pemphigus vulgaris (PV) antigen, desmoglein 3 (Dsg3), and pemphigus foliaceus (PF) antigen, desmoglein 1 (Dsg1), in various tissues. Immunofluorescence studies suggested that Dsg1 and Dsg3 are preferentially expressed in the upper and lower epidermis, respectively. With immunoblotting of human epidermal extracts, all PV sera reacted with Dsg3 but not with Dsg1. By contrast, only half of PF sera reacted with Dsg1 but none reacted with Dsg3. These results confirm the distinct antibody specificity between PV and PF sera. They also suggest that PV sera contain antibodies against linear epitopes present even on the denatured antigen, while some PF sera contain antibodies only against conformational epitopes present on the native antigen. With immunoblotting of bovine desmosome preparations, certain PV sera and an anti-Dsg monoclonal antibody reacted with both Dsg1 and Dsg3. Affinity-purification of these PV antibodies suggested that the simultaneous reactivity with Dsg1 and Dsg3 was produced by two different subsets of antibodies and not by cross-reactivity of single antibodies. This study indicates that pemphigus serum is a useful probe with which we learn various aspects of keratinocyte biology.
Lactating mammary epithelial cells synthesize large quantities of milk proteins, which they secrete vectorially at the apical membrane into the alveolar lumen of the gland. Recent work suggested that mammary protein secretion is not wholly constitutive, but may also occur in part through a regulated secretory pathway. This study used mouse mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix to compare the proportions of basally and apically-directed proteins secreted constitutively or in a regulated manner. On EHS matrix, mammary cells formed mammospheres, multicellular structures enshrouded in matrix material, within which they became polarised, formed tight intercellular junctions, and secreted milk proteins vectorially. Protein secreted basolaterally was collected in culture medium, whereas apically-secreted milk proteins accumulated in a closed lumen within the mammosphere, and were recovered by EGTA treatment of the cells in situ. Protein secretion was measured by following the release of radiolabelled protein after pulse-labelling with [35S]-methionine. Basolateral and apical secretion of [35S]-protein appeared complete within 1 h of pulse-labelling, consistent with immediate secretion through constitutive secretory pathways. However, addition of the calcium ionophore ionomycin induced a second wave of secretion in both directions. Ca(2+)-stimulated secretion occurred within 15 min of ionomycin addition, doubled the extent of basolateral and apical secretion, but did not change the populations of proteins secreted. Ionomycin treatment did not affect mammosphere morphology or mammary cell ultrastructure. The results suggest that lactating mammary epithelial cells secrete proteins apically and basolaterally by two pathways, one a Ca(2+)-independent constitutive pathway, the other a regulated pathway stimulated by elevation of intracellular Ca2+.
Cultured intestinal epithelial monolayers serve as models for mechanistic studies of intestinal inflammation. One crucial aspect of epithelial function modulated by inflammation is permeability. Indices of permeability typically obtained are transepithelial resistance or more formalized assays using Ussing chambers modified for cultured monolayers. Such Ussing chamber systems are inconvenient for screening assays and their geometry precludes evaluation of important inflammatory responses such as transepithelial migration of neutrophils. Here we use a novel flux assay to investigate the kinetics of fluorescein-labelled dextran (FD) molecules across intestinal epithelial cell lines grown on permeable membrane supports. Our results show that we could consistently demonstrate picomolar flux of FD in a range of sizes (3-70 kD) across T84 epithelial monolayers. Such FD flux was time- and dose-dependent and flux increased exponentially with decreasing transepithelial resistance. Exposure of intestinal epithelia to mucosal-derived lymphocytes or to soluble lymphocyte products (interferon-gamma, interleukin-4 or interleukin-13) increased FD flux in a dose-dependent fashion. Finally, studies of neutrophil transepithelial migration revealed qualitative and quantitative differences in FD flux depending on FD size. We conclude that in vitro transepithelial FD flux may be a useful tool to study aspects of intestinal permeability in health and in disease.
The asymmetric distribution of the Na,K-ATPase in the plasma membrane of epithelial cells is essential to the establishment of the transepithelial Na+ gradient that supports the vectorial transport of ions and solutes. To investigate the changes that occur during the development of polarity, we have characterized Na,K-ATPase expression and activity in two epithelial cell culture lines, Madin Darby canine kidney (MDCK) and Caco-2 cells. RNA and immunoblot analysis of both cell lines demonstrate that only the alpha 1 and beta 1 isoforms are expressed in nonpolarized and polarized cultures. Interestingly, alpha 1 and beta 1 message increases in MDCK cells with the development of polarity, yet there is little change in the amount of protein for either subunit. In contrast, alpha 1 and beta 1 polypeptide expression increases in Caco-2 cells with the development of polarity, even though the amount of both transcripts decreases. The lack of correlation between the changes that occur at the level of the message and protein suggest that appropriate expression is mediated in part by a combination of transcriptional and translational events. Furthermore, while there was a slight decrease in activity in polarized MDCK cells, there was a 1.9 fold increase in Na,K-ATPase activity in the polarized Caco-2 cells as compared to nonpolarized cells. These results demonstrate that the regulation of Na,K-ATPase alpha 1 and beta 1 isoform expression is mediated by a combination of transcriptional and translational events during the development of polarity in both cell lines.
It has been reported that keratinocytes possess phospholipase C (PLC)-mediated signal transduction system(s), that can be triggered by histamine, bradykinin, thrombin, platelet-activating factor (PAF), and epidermal growth factor (EGF)/transforming growth factor-alpha (TGF-alpha). Since the activation of PLC results in release of 1,2-diacylglycerol (DAG), the physiologic activator of protein kinase C (PKC) that modulates the epidermal adenylate cyclase, we investigated the effects of these PLC activating chemicals on the adenylate cyclase responses of dispase-separated normal pig epidermis. Among these chemicals and factors only histamine decreased the successive histamine-induced cyclic AMP accumulation and increased forskolin-, and cholera toxin-induced AMP accumulations. These effects were similar to those of PKC activators. However, in contrast to the PKC-activator-induced partial and receptor-non-specific desensitization, the histamine-induced desensitization was completely-inducible and specific to the histamine receptor system, and was not affected by the PKC inhibitor, H-7. Similar modulation of the epidermal adenylate cyclase was induced by other adenylate cyclase stimulators (epinephrine, adenosine and prostaglandin E2), but not by bradykinin, thrombin, PAF, or EGF. The combined addition of bradykinin, thrombin, PAF and EGF to the culture medium had no effect on the adenylate cyclase responses, either. Thus no evidence for receptor-agonist dependent PLC-induced modulation of the adenylate cyclase was obtained in the normal pig epidermis. Although keratinocytes might contain PLC-mediated signal transduction systems, that are triggered by histamine, bradykinin, thrombin, PAF, and EGF/TGF-alpha, none of the activators singly or in combination appear to activate PKC sufficiently for the modulation of adenylate cyclase responses of the normal pig epidermis.
Topical application of retinoic acid (RA) induces skin irritation, increased epidermal DNA synthesis and hyperplasia by an unknown mechanism, possibly in common with other hyperplasiogens. To further characterize this regeneration-like effect of RA, the cell cycle traverse in hairless mouse epidermis in vivo after application of a single dose of 100 nmol RA was investigated. Two BrdUrd labelled basal cell cohorts, one exposed to RA during the S phase, the other stimulated by RA into S phase 16 h earlier, were followed for 30 h in their progression through the cell cycle. The basal cells were isolated and prepared for bivariate BrdUrd/DNA flow cytometry analysis (FCM). Both cohorts showed induced proliferation of cells stimulated from G(0)/G(1) into S phase as shown by increase in the S phase fraction, but without reduction in cell cycle time as expected for regenerative growth. This implies that RA stimulates proliferation differently than other hyperplasiogens, and hence may induce irritation and growth stimulation through another mechanism.
