Gastrin-regulated expression of p53 in transformed enterochromaffin-like cells in the African rodent mastomys.

The tumor suppressor p53 functions at the G1/S-phase checkpoint of the cell cycle to direct cells that have accumulated somatic mutations toward apoptosis and away from mitosis. The p53 gene is commonly mutated in human cancers, but the molecular mechanisms regulating this event are not clear. The African rodent mastomys exhibits a genetic predisposition to develop gastric carcinoids derived from enterochromaffin-like (ECL) cells. The ECL cell transformation can be accelerated by acid inhibition-induced hypergastrinemia. This study evaluates the alteration of p53 during the rapid ECL cell transformation. Hypergastrinemia was generated by the irreversible histamine-2 receptor antagonist loxtidine for 8 weeks (hyperplasia) and 16 weeks (neoplasia). p53 expression was evaluated in fundic mucosa from different stages of transformation by Western blot analysis and immunohistochemistry using monoclonal antibodies against wild-type p53. RT-PCR and molecular sequence analysis of p53 were undertaken with mRNA isolated from purified ECL cells. Overproduction of the wild type of p53 was evident in ECL cells during hypergastrinemia, and the molecular characteristics of p53 were determined in naive and transformed ECL cells. p53 was mutated at the C-terminus in ECLoma induced by hypergastrinemia. Therefore, p53 is altered from overproduction to mutation during the development of hypergastrinemia-induced ECLoma and it may therefore play a role in the cell transformation.

Gastrin receptor expression and function during rapid transformation of the enterochromaffin-like cells in an African rodent.

The enterochromaffin-like cell (ECL) cells of the stomach are principally regulated by gastrin via a gastrin/CCK(B) receptor (G[R]) which modulates both histamine secretion and cell proliferation. In the African rodent (mastomys) hypergastrinemia generated by the histamine-2 receptor antagonist (loxtidine) results in ECL cell hyperplasia and neoplasia at 8 and 16 weeks respectively. The expression, structure and function of the G(R) during transformation is however unknown. We utilized a pure (approximately 90%) preparation of ECL cells to evaluate alterations in the G(R) utilizing immunocytochemistry, Western blot analysis, reverse transcription polymerase chain reaction (RT-PCR), 5-bromo-2-deoxyuridine uptake and phosphorylation site analysis. Although the expression of ECL cell G(R) was upregulated at both mRNA (PT-PCR) and protein (Western analysis) level, its affinity to gastrin was decreased in the hyperplastic phase and lost during transformation. The coding sequence of the G(R) of mastomys tumor ECL cells was identical to that of normal ECL cells, parietal cells and the brain. However, the mRNA sequence of the third introcytoplasmic loop of the G(R) was significantly different to other species. In addition, the G(R) exhibited phosphorylation site on serine residue(s). We have thus noted a direct correlation between hypergastrinemia and G(R) alteration and function during ECL cell transformation. It is possible that the unique mastomys gastrin receptor mediated ECL cell transformation involves the novel phosphorylation sites and a divergence in the introcytoplasmic domain.

Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum).

Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.

Pathophysiology of the fundic enterochromaffin-like (ECL) cell and gastric carcinoid tumours.

The genesis of human gastric carcinoma is ill understood but is invariably related to achlorhydria. Gastrin secretion is negatively regulated by luminal acid and hypergastrinaemia is thus associated with low acid states which may be natural (atrophic gastritis) or owing to acid inhibitory therapy. Apart from its acid secretory activity, gastrin is trophic to the mucosa, via stimulation of the fundic enterochromaffin-like (ECL) cells to secrete histamine. In conditions of elevated gastrin levels, ECL cell hyperplasia and even neoplasia have been noted. The relationship between low acid, hypergastrinaemia, ECL cell hyperplasia, and neoplasia may be of relevance since ECL cells secrete histamine and TGF alpha which are both recognised mitogens. We studied the rodent mastomys, which spontaneously develop gastric carcinoid tumours, which can be generated in 4 months under conditions of drug-induced acid inhibition and inhibited by octreotide administration. A pure (90-95%) cell preparation was used to evaluate ECL cell physiology and trophic regulation. A gastrin/CCKB receptor responsible for histamine secretion and DNA synthesis was identified, cloned and sequenced. Octreotide lowers plasma gastrin levels, decreases ECL cell neoplasia and, in vitro, inhibits ECL cell DNA synthesis. H1 receptor antagonists inhibited DNA synthesis in vitro and ECL neoplasia in vivo without altering gastrin levels. Hypergastrinaemia increased TGF alpha/EGF receptor and TGF alpha production and TGF alpha massively stimulated ECL cell DNA synthesis. Since ECL cells produce both histamine and TGF alpha and regulate parietal cells which produce TGF alpha, it is possible that achlorhydria-generated ECL cell dysfunction may play an initiative role in the pathobiology of gastric adenocarcinoma. The long-term clinical consequences of drug-induced sustained acid inhibition are worthy of further consideration.

The cardiac mutant gene c in axolotls: cellular, developmental, and molecular studies.

The cardiac mutant axolotl is an interesting model for studying heart development. The mutant gene results in a failure of heart cells to form organized myofibrils and as a consequence the heart fails to beat. Experiments have shown that mutant hearts can be "rescued" (i.e., turned into normally contracting organs) by the addition of RNA purified from conditioned media produced by normal embryonic anterior endoderm-mesoderm cultures. These corrected hearts form myofibrils of normal morphology. New advances in recombinant DNA technology applied to this system should provide significant insights into the regulatory mechanisms of myofibrillogenesis as well as the inductive processes related to the control of gene expression during embryonic heart development. In a broader biological sense, the use of gene c in axolotls is potentially capable of helping to solve major unanswered questions in modern biology related to the genetic regulation of differentiation in vertebrates.

Localization and distribution of gap junctions in normal and cardiomyopathic hamster heart.

Gap junctions in mammalian heart function to provide low-resistance channels between adjacent cells for passage of ions and small molecules. It is clear that the almost unrestricted passage of ions between cells, ionic coupling, is required for coordinate and synchronous contraction. This knowledge of gap junction function has made it important to study their properties in normal and abnormal tissues. In the present study, we analyzed gap junction distribution in normal and cardiomyopathic heart tissue utilizing immunofluorescent and electron microscopy techniques. Frozen, unfixed sections of age-matched normal and cardiomyopathic cardiac tissues were immunofluorescently stained using an antibody directed against a specific peptide sequence of the connexin-43 gap junction protein. These studies revealed a characteristic punctate staining pattern for the intercalated discs in normal tissues. Some of the intercalated discs in cardiomyopathic hearts appeared to stain normally; however, others stained diffusely. The pixel intensity distribution of the confocal images demonstrated a marked difference of up to 90% increase in the number of pixels in cardiomyopathic myocardium (CM), yet the pixel intensity of gap junctions had a decrease of approximately 60%. This suggests the possibility that connexin-43 is present in CM cells in significant quantity; however, it does not become localized on the membranes as in normal cells. Electron-microscopic findings corroborate these observations on CM cells by showing an irregular distribution of intercalated discs relatively smaller in size with abnormal orientation and distribution.

Molecular cloning, sequencing and expression of an isoform of cardiac alpha-tropomyosin from the Mexican axolotl (Ambystoma mexicanum).

The cDNA for alpha-tropomyosin (TM) was cloned by the polymerase chain reaction (PCR) from a lambda gt11 library constructed with mRNA from juvenile axolotl heart tissues. Subsequently, the nucleotide sequence of the cDNA was determined. This is the first reported cDNA for axolotl alpha-tropomyosin. Comparative analyses of the deduced amino acid sequence of this cDNA with Xenopus skeletal muscle alpha-tropomyosin sequences indicate that the axolotl heart cDNA has 93% and 96% homology in the regions of amino acids 39-80 (exon 2b) and 258-284 (exon 9a), respectively. However, there is about 86.55% homology at the nucleic acid level (coding region) and 97.5% homology at the amino acid level with that of Xenopus skeletal muscle alpha-tropomyosin cDNA. Northern blot analyses with polyA+ RNA from juvenile heart suggest the presence of at least two different transcripts for alpha-tropomyosin in axolotl heart. The results of 3'-RACE concur with those of northern blot analyses.

A simple method for the correction of biliary excretion curves distorted by the biliary dead space.

Biliary solute concentrations measured at the tip of the cannula suffer a delay with respect to bile flow due to the transit time through the biliary tree volume. This study proposes a simple method, which is valid under variable bile flow conditions, to correct the distortion introduced by the biliary tree volume on the kinetic curves of the biliary excretion rate. The biliary transit time (tt) was calculated as the time needed to excrete a bile volume equal to the biliary tree volume by means of the interpolation of biliary cumulative volume versus time curves. Such tt permits one to estimate the canalicular concentration at time t, interpolating the biliary concentration curves at time t-tt. The product between the estimated canalicular concentration and the bile flow allows the calculation of the corrected biliary excretion rate. This method was evaluated by a comparison between biliary excretion rate curves of [14C]taurocholate [( 14C]TC) injected as a bolus under basal and sodium dehydrocholate (DHC)-induced choleresis conditions. Since the canalicular excretion rate of [14C]TC is considered independent of bile flow, the significant differences observed in its excretion kinetics under both conditions were attributed to distortion due to the biliary tree volume. After the correction, both curves showed a significant overlapping. This result indicates that the method improves the time-course representation of canalicular events in biliary excretion kinetic studies.