Inositide-dependent phospholipase C signaling mimics insulin in skeletal muscle differentiation by affecting specific regions of the cyclin D3 promoter

Our main goal in this study was to investigate the role of phospholipase C (PLC) beta(1) and PLCgamma(1) in skeletal muscle differentiation and the existence of potential downstream targets of their signaling activity. To examine whether PLC signaling can modulate the expression of cyclin D3, a target of PLCbeta(1) in erythroleukemia cells, we transfected C2C12 cells with expression vectors containing PLCbeta(1) or PLCgamma(1) cDNA and with small interfering RNAs from regions of the PLCbeta(1) or PLCgamma(1) gene and followed myogenic differentiation in this well-established cell system. Intriguingly, overexpressed PLCbeta(1) and PLCgamma(1) were able to mimic insulin induction of both cyclin D3 and muscle differentiation. By knocking down PLCbeta(1) or PLCgamma(1) expression, C2C12 cells almost completely lost the increase in cyclin D3, and the differentiation program was down-regulated. To explore the induction of the cyclin D3 gene promoter during this process, we used a series of 5'-deletions of the 1.68-kb promoter linked to a reporter gene and noted a 5-fold augmentation of promoter activity upon insulin stimulation. These constructs were also cotransfected with PLCbeta(1) or PLCgamma(1) cDNAs and small interfering RNAs, respectively. Our data indicate that PLCbeta(1) or PLCgamma(1) signaling is capable of acting like insulin in regard to both the myogenic differentiation program and cyclin D3 up-regulation. Taken together, this is the first study that hints at cyclin D3 as a target of PLCbeta(1) and PLCgamma(1) during myogenic differentiation in vitro and implies that up-regulation of these enzymes is sufficient to mimic the actions of insulin in this process.     

Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa.

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.     

Immunolocalization of estrogen receptor alpha and beta in gastric epithelium and enteric neurons

A sexual dimorphism in gastric acid secretion has been known for many years, with women secreting less acid ( approximately 40%) than men. The mechanisms mediating this sex difference are unknown, but a role for estrogens is suggested from animal models. Two estrogen receptor (ER) subtypes, ER alpha and ER beta, mediate genomic effects of estrogens, and mRNA for both subtypes has been detected in the rat stomach. The objective of this study was to determine the cellular distribution of ER alpha and ER beta proteins in the rat stomach. ER alpha and ER beta proteins were detected in nuclei of fundic parietal cells and epithelial cells in the progenitor zone. In the antrum, several cells were immunoreactive for ER beta in regions containing stem and neuroendocrine cell types but ER alpha protein was not detected in antral glands. Both ER alpha and ER beta proteins were expressed in enteric neurons within the nucleus and cytoplasm, with specific punctate staining for ER alpha in cell bodies and fibers. These studies are the first to show differences between ER alpha and ER beta proteins in the epithelial cellular distribution in the fundus and antrum and to detect co-expression in enteric neurons. These results suggest that estrogens may inhibit gastric acid secretion via genomic effects in fundic parietal cells through either ER subtype and in antral neuroendocrine cells via ER beta. Moreover, co-expression of ER alpha and ER beta in enteric neurons indicates that estrogenic effects could also be mediated through neurogenic reflexes. Our findings imply that direct regulation of multiple cell types by estrogens may contribute to the modulation of gastric functions that have been recognized during the estrous cycle and between the sexes.     

Anatomical location of enterochromaffin-like (ECL) cells, parietal cells, and chief cells in the stomach demonstrated by immunocytochemistry and electron microscopy

Enterochromaffin-like (ECL) cells are included in the endocrine cells present in the gastric oxyntic mucosa, and have been attracting attention as histamine-secreting cells contributing to gastric secretion. However, the anatomical location of ECL cells in relation to parietal cells and chief cells has not yet been sufficiently investigated. To elucidate this location of ECL cells, we performed an immunocytochemical study using anti-histamine antibody and electron microscopic examination of guinea pig gastric mucosa. ECL cells were located near the basement membranes in the gastric oxyntic region, and were in contact with both chief cells and parietal cells in the same glandular epithelium. The ratio of ECL cells in contact with chief cells was clearly greater than that in contact with parietal cells. An Ω-shaped morphology, indicating emiocytosis, was found in ECL cells by electron microscopy. These findings suggest that ECL cells have a paracrine effect on chief cells and parietal cells, and may have an important physiological role in pepsinogen secretion. Received: April 6, 1998/Accepted: December 18, 1998     

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.     

Cell kinetics of slow renewing cell populations in mice stomach

BACKGROUND: The renewal rates of parietal and chief cells in the gastric mucosa and smooth muscle cells of muscularis propria have not been examined as precisely as superficial epithelial cells. To examine cell renewal of these cells, continuous labeling with tritiated ([3H])-thymidine was performed. METHODS: Mice received 112 repeated injections of [3H]-thymidine at 6-hour intervals for 28 days after birth and were killed immediately thereafter, or 60, 120, 200 or 300 days after the last injection. RESULTS: After continuous labeling, most cells in the stomach were labeled. At 60 days, unlabeled parietal cells in the neck area of the gland and unlabeled chief cells in the middle part of the gland appeared. Thereafter, the area of unlabeled cells expanded downwards to the bottom of the gland. Times required for labeling of total cell populations of parietal and chief cells to half were less than 60 days and more than 200 days, respectively. At 300 days, most parietal cells and about half of the chief cells remained labeled in the bottom of the gland. The labeling index of smooth muscle cells was about 100% for 300 days. CONCLUSIONS: The time required for the newly formed parietal and chief cells to reach the lower end of the gland was more than 300 days. As a total cell population, the renewal rate of parietal cells was more rapid than that of chief cells. However, in terms of the downward migrating cell population, the renewal rate of parietal cells was a little slower than that of chief cells. Smooth muscle cells showed almost no renewal.     

Effects of cyclosporin A on water-immersion stress-induced gastric lesion and gastric secretion in rats

Cyclosporin A is an immunosuppressive agent which is well known as a specific inhibitor of calcineurin (protein phosphatase 2B). In this study, we investigated the effects of cyclosporin A on water-immersion stress-induced gastric ulcer formation and gastric acid secretion in rats. We also examined the localization of calcineurin immunohistochemically. Calcineurin was specifically expressed in gastric parietal cells and chief cells of the gastric mucosa. The intraperitoneal administration of cyclosporin A dose-dependently suppressed the development of gastric mucosal lesions induced by water-immersion stress and inhibited gastric acid secretion, as assessed by pylorus ligation. These results indicated that calcineurin may play an important role in gastric acid secretion. Received: October 14, 1999 / Accepted: January 28, 2000     

Gastric H(+),K(+)-adenosine triphosphatase beta subunit is required for normal function, development, and membrane structure of mouse parietal cells.

BACKGROUND & AIMS: Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H(+),K(+)-adenosine triphosphatase (ATPase), the enzyme primarily responsible for acidification of the gastric lumen. We have produced mice deficient in the H(+),K(+)-ATPase beta subunit to determine the role of the protein in the biosynthesis of this membrane system and the biology of gastric mucosa. METHODS: Mice deficient in the H(+), K(+)-ATPase beta subunit were produced by gene targeting. RESULTS: The stomachs of H(+),K(+)-ATPase beta subunit-deficient mice were achlorhydric. Histological and immunocytochemical analyses with antibodies to the H(+),K(+)-ATPase alpha subunit revealed that parietal cell development during ontogeny was retarded in H(+), K(+)-ATPase beta subunit-deficient mice. In 15-day-old mice, cells with secretory canaliculi were observed in wild-type but not in H(+), K(+)-ATPase beta subunit-deficient mice. Parietal cells of H(+), K(+)-ATPase beta subunit-deficient mice 17 days and older contained an abnormal canaliculus that was dilated and contained fewer and shorter microvilli than normal. In older parietal cells, the abnormal canaliculus was massive (25 micrometer in diameter) and contained few microvilli. We did not observe typical tubulovesicular membranes in any parietal cell from H(+),K(+)-ATPase beta subunit-deficient mice. Histopathologic alterations were only observed in the stomach. CONCLUSIONS: The H(+),K(+)-ATPase beta subunit is required for acid-secretory activity of parietal cells in vivo, normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes.     

Histoenzymatic and ultrastructural findings in the human gastric mucosa during fasting.

In this study the influence of fasting on the structures in the human gastric mucosa was followed using selective histoenzymological and electronmicroscopic method. The gastrobioptical material of 7 healthy volunteers was examined after 24 to 240 hour fasting. During fasting an increased acid phosphatase activity in the chief cells of the human fundal mucosa was observed. The activity of the nonspecific esterase (naphtyl esterase) in the chief cells decreased mainly after 240 hour fasting. In the electronmicroscopic examination of the chief cells during fasting a multiplication of lysosomes and narrowing of the granular endoplasmic reticulum was observed. In some chief cells during fasting an agglomeration of zymogen granules was seen while in others only a few granules were observed. In the parietal and other cells of the human gastric glands a steatosis, which attained excessive values, was seen after 72 and 240 hours of fasting. We believe that this steatosis occurred mainly on the strength fasting lipemia with the possible participation of other factors. The changes in the mitochondrial structure of the parietal and other cells of the human gastric mucosa were only slightly pronounced. "The decreased" activity of dehydrogenase in the steatotic parietal cells under a histochemical examination may represent only findings caused by the agglomeration of a large amount of lipid drops in the steatotic cells. After 72 and 240 hours of fasting the parietal cells contained collapsed, underdeveloped intracellular canaliculi and narrowed tubulovesicular profiles probably related to the decreased HCl production. Generally it can be said that the observed morphological changes are the consequences of the decreased function of human gastric mucosa cells during fasting and that these regressive changes are reversible and they are caused by an insufficiency of nutrition.     

柴胡桂枝汤对大鼠乙酸胃溃疡黏膜表皮生长因子受体的影响

[目的]探讨柴胡桂枝汤(CHGZT)促进泌酸腺再生的作用机制.[方法]对比观察大鼠乙酸胃溃疡形成后3个时间段胃体黏膜、再生黏膜表皮生长因子受体(EGFR)的表达及CHGZT对其的影响.[结果]EGFR表达于胃体黏膜泌酸腺的壁细胞以及再生黏膜CHGZT-溃疡伴随细胞系(UACL)中的去分化或再分化壁细胞;在溃疡形成后的3个时间段,与模型对照组和西咪替丁组相比,CHGZT组溃疡边缘胃体侧正常黏膜以及再生黏膜EGFR的表达均增多,其差异具有统计学意义(均P＜0.01).[结论]CHGZT促进泌酸腺再生,其机制可能与其促进溃疡附近胃体黏膜以及再生黏膜EGFR的表达有关.     

Influence of hydrocortisone on the development of the gastric mucosa in suckling rats. Inhibitory effect on gastrin cell population

The effect of a 6-day hydrocortisone administration on the development of gastric mucosa was investigated in suckling rats. Three doses of hydrocortisone (40, 20 and 5 mg/kg/day) were given during the 2nd week of life. Gastric morphometric parameters, and exocrine parietal cell and endocrine gastrin cell numbers were examined and gastrin content in antral tissue was determined. At the end of hydrocortisone treatment, parietal and chief cells were well differentiated. Their larger size was marked in comparison with controls, which likely explains in itself the significant increase in gastric mucosal height. Only the highest dose significantly increased antral gastrin content over control values. However, hydrocortisone, whatever the dose, reduced the apparent antral gastrin cell population by about 60% (p less than 0.001), without noticeably modifying the numerical density of parietal cells per unit of fundic surface. It is nevertheless likely that the highest dose diminished total parietal cell number since the gastric surface decreased. These results suggest that glucocorticoids affect the development of the gastrin cell population while greatly accelerating the maturation of these cells during the suckling period.     

Calcium, calmodulin, and cyclic adenosine monophosphate modulate prostaglandin E2 release from isolated human gastric mucosal cells

We studied prostaglandin E2 (PGE2) release from isolated cells of the human gastric mucosa. Mucosal cells were enzymatically isolated from biopsy specimens of human fundic mucosa. The results from these crude cell preparations were compared to those obtained in fractions with enriched (65-80%) or depleted parietal cell content (3-7%) which were prepared from gastric mucosa obtained at surgery. PGE2 release in the enriched parietal cell fractions exceeded that from crude or parietal cell depleted preparations 3- and 13-fold, respectively. However, despite this quantitative difference, all preparations responded similarly to the test agents. Newly synthesized PGE2 was not stored intracellularly but was released into the incubation medium. Release increased linearly for 30 min. Addition of the calcium ionophore A23187 enhanced PGE2 release 4- to 5-fold. The effect of A23187 required the presence of extracellular Ca2+ (10(-3) mol/liter). Assuming that A23187 alters Ca2+ flux in gastric cells as it does in other cell systems our data indicate that increased Ca2+ influx enhances PGE2 release. Since calmodulin is of importance for intracellular Ca2+ action, the calmodulin antagonists trifluoperazine and W7 were tested. Both antagonists inhibited PGE2 release by 65-85%, trifluoperazine being slightly more effective. Activation of the adenylate cyclase system by forskolin or direct addition of (Bu)2cAMP, a stable cAMP-analog, also inhibited PGE2 release. We conclude that PGE2 is released from parietal and from nonparietal cells of the human gastric mucosa, although the major quantity is released from the light density fraction that is enriched in parietal cells. In parietal and nonparietal cells Ca2+ is of importance in the regulation of gastric mucosal PGE2 release and calmodulin seems to mediate this intracellular action of Ca2+. cAMP inhibits PGE2-release from gastric cells.     

Long-term examinations of fundic mucosa in gastroduodenal ulcer after SpV and pyloroplasty (author's transl)

Morphological examinations of the fundic mucosa with quantitative analysis of the parietal, chief and mucous neck cells were carried out with duodenal ulcer cases before selective proximal vagotomy and pyloroplasty, and over a period of more than four years after the operation. In the material removed by forceps biopsy from 15 duodenal ulcer cases, an early post-operative parietal cell density reduction of 74.15% was found, and from 5 gastric ulcer cases a reduction of 73.77%. This result occurred over an examination period of four years. The post-operative displacement in the distribution of the parietal cell within the fundic gland was pointed out, as well as the increased post-operative inflammatory indurating changes in the fundic mucosa, which occur only in gastric ulcer cases. The thickness of the mucosa was measured with an ocular micrometer pre- and post-operatively in 8 cases, and a post-operative reduction of about 20% was found. The relationshiop between the degree of post-operative parietal cell reduction and the insulin-stimulated acid-secretion was checked and no significant difference between the absolutely insulin-negative and insulin-positive groups was found post-operatively. To supplement these findings, the reaction of the succinatdehydrogenase activity in the mitochondria of the parietal cell was analysed semiquantitatively and by absorption-photometer. Thus a post-operative reduction of enzyme-activity of 20.45% on average was found in duodenal ulcer cases. In gastric ulcer cases there was no evidence of any reduction.     

A scanning electron microscopic study of normal human oxyntic mucosa using blunt dissection and freeze fracture

Biopsies from the fundic mucosa of healthy volunteers were examined by scanning electron microscopy following blunt dissection and freeze fracture. The mucosal surface exhibited a cobblestone appearance. With increased magnification, microvilli could be demonstrated on the luminal surface of individual surface cells. Blunt dissection of the gastric mucosa revealed tubular gastric pits descending from the surface until they opened into branched tubular gastric glands. The gastric glands are irregular in outline due to the knoblike projections of individual parietal cells. The internal structure of the component cells also was examined with the scanning electron microscope following freeze fracture. Mucous granules were observed in the apical portion of surface epithelial cells. Lumina of parietal cell canaliculi were found to be continuous with the lumen of the gastric glands. Pepsinogen granules could be seen throughout the cytoplasm of chief cells. The blunt dissection-freeze fracture technique utilizing the scanning electron microscope allows, for the first time, a three-dimensional view of human gastric mucosa, including the gastric pits and glands as well as some of the internal architecture of component cells.This work was supported in part by the Medical Research Service of the Veterans Administration and grants from Smith, Kline and French Laboratories, as well as the patient study facilities of the UMMC Clinical Research Center. The technical assistance of Mr. G. E. Jeffrey is gratefully acknowledged.     

Chief cell mass after short-term ranitidine treatment for duodenal ulcer.

The chief cell mass and the parietal cell mass were evaluated in endoscopically obtained biopsy specimens of fundic mucosa from 15 duodenal ulcer patients before and after ranitidine treatment. Patients were given ranitidine, 300 mg/day, for 8 weeks. Chief cell mass and parietal cell mass were expressed respectively by a "zymogenous index" (ZI) and a "parietal index" (PI), obtained by multiplying the number of cells per mm2 by the thickness of the glandular layer. From the results of this study it would appear that, in patients with duodenal ulcer, treatment with ranitidine at a dose of 300 mg/day for 8 weeks results in a significant increase in parietal cell mass and a decrease in chief cess mass.     

Reciprocal modulation of phospholipase Cbeta isoforms: adaptation to chronic morphine

Phosphoinositide turnover and calcium mobilization are fundamental determinants of acute and chronic opioid effects. Phosphoinositide-specific phospholipase C (PLC) are key signaling enzymes that play a pivotal role in mediating opioid modulation of inositol trisphosphate production and cytosolic calcium distribution, substrates for many acute and chronic opioid effects. Notably, phosphorylation of the beta isoforms of PLC, by kinases that are up-regulated after chronic morphine, is a potent modality for their regulation. Direct assessment of PLCbeta1 and PLCbeta3 phosphorylation in the guinea pig longitudinal muscle myenteric plexus tissue revealed substantial alterations after the induction of opioid tolerance. Notably, the direction of this modulation is isoform-specific. Phosphorylation of PLCbeta1 is significantly reduced, whereas that of PLCbeta3 is substantially augmented, changes not accompanied by altered content of PLCbeta1 or PLCbeta3 protein. In contrast to chronic morphine, acute morphine treatment of opioid na?ve longitudinal muscle myenteric plexus tissue attenuates PLCbeta3 phosphorylation, an effect also manifested by endogenous opioids that is reflected by the ability of acute naloxone to substantially augment PLCbeta3 phosphorylation. This indicates that PLCbeta phosphorylation is dynamically regulated. PLCbeta1 and PLCbeta3 activities are negatively modulated by phosphorylation. Thus, their concomitant reciprocal phosphorylation would alter the relative contribution of these isoforms to PLC/Ca2+ signaling, a significant shift in light of their differential regulatory characteristics. Reciprocal modulation of the phosphorylation (activity) of two isoforms within the same subclass of signaling enzyme, proteins that have a high degree of structural similarity and subserve the same biological function, represents an adaptation modality to chronic morphine that has heretofore not been recognized.     

Roles of PLC-γ2 and PKCα in TPA-induced apoptosis of gastric cancer cells

AIM: To investigate the roles of PLCγ2 and PKCα in TPA-induced apoptosis of gastric cancer cells. METHODS: Human gastric cancer cell line MGCS0-3 was used. Protein expression levels of PLCγ2 and PKCα were detected by Western blot. Protein localization of PLCγ2 and PKCα was shown by immunofluoscence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among I 000 cells randomly. RESULTS: Treatment of gastric cancer cells MGCS0-3 with TPA not only up-regulated expression of PLC-γ2 protein, but also induced PLC-γ2 translocation from the cytoplasm to the nucleus. However, this process was not directly associated with apoptosis induction. Further investigation showed that PKCa translocation from the cytoplasm to the nucleus was correlated with initiation of apoptosis. To explore the inevitable linkage between PLC-γ2 and PKCα during apoptosis induction, PLC inhibitor U73122 was used to block PLC-γ2 translocation,in which neither stimulating PKCα translocation nor inducing apoptosis occurred in MGC80-3 cells. However, when U73122-treated cells were exposed to TPA, not only PLC-γ2, but also PKCα was redistributed. On the other hand, when cells were treated with PKC inhibitor alone, PLC-γ2 protein was still located in the cytoplasm. However, redistribution of PLC-γ2 protein occurred in the presence of TPA, no matter whether PKC inhibitor existed or not. CONCLUSION: PLC-γ2 translocation is critical in transmittingTPA signal to its downstream molecule PKCα. As an effector, PKCα directly promotes apoptosis of MGC80-3 cells. Therefore, protein translocation of PLCγ2 and PKCα is critical event in the process of apoptosis induction.