Margret Oddsdottir

Spasmolytic polypeptide-expressing metaplasia (SPEM) associated with gastric cancer in Iceland

Recent studies have described a spasmolytic polypeptide-expressing metaplastic cell lineage (SPEM) in the gastric fundic mucosa associated with both chronic H. pylori infection and gastric adenocarcinoma. We investigated the association of SPEM both with early gastric adenocarcinoma and in biopsies taken from patients prior to diagnosis of cancer. Two cohorts were examined. First, gastric resections from 29 patients with early gastric cancer were examined. Second, biopsies taken from 18 patients prior to the diagnosis of gastric cancer were compared with their respective resection specimens as well as with control biopsies from a cohort of 19 patients diagnosed with gastritis without subsequent development of cancer. The presence of SPEM and intestinal metaplasia (IM) adjacent to and distant from the cancer was compared and spasmolytic polypeptide (SP) immunostaining within dysplastic/cancerous cells was identified. SPEM was present adjacent to cancer in all early cancer cases where the tumor was located in the body or at the body/antrum junction, and was present in the body mucosa distant from the cancer in 76% of cases. Intestinal metaplasia was found adjacent to the tumor in 76% of cases and in body sections in 52% of resections. SP immunostaining was noted within cancer cells in 62% of tumors, and within dysplastic cells in 76% of resections where dysplasia was present. SPEM was present in 82% of the biopsies obtained prior to the diagnosis of cancer, compared with only 37% in the gastritis cohort. IM was present in only 57% of biopsies. In conclusion, SPEM is strongly associated with early gastric cancers and is observed in gastric biopsies prior to the development of cancer. In addition, early gastric cancers demonstrated a high incidence of SP expression. These results suggest that SPEM merits consideration as an important pre-neoplastic gastric lesion.

A specific histamine-stimulated phosphoprotein in isolated parietal cells

Histamine-stimulated phosphorylation was studied in isolated rabbit parietal cells. Secretion of acid, as assessed by aminopyrine uptake, was linear at 15 min of stimulation with histamine. By utilizing two dimensional gels, a specific 30,000-Da protein (pp30) was identified whose phosphorylation was prominently stimulated by histamine after 15 min of incubation. The pp30 protein displayed an isoelectric point of 6.0. Furthermore, cAMP-dependent pp30 phosphorylation could also be demonstrated in vitro in a preparation of parietal cell cytosol. The results suggest that pp30 may represent an important histamine-stimulated cAMP-dependent phosphoprotein involved in the initiation or maintenance of parietal cell secretion.

A micromethod for the assay of cellular secretory physiology: Application to rabbit parietal cells

A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of [14C]-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of [14C]aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in [14C]aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes.

Identification and characterization of a cytosolic 30 kDa histamine stimulated phosphoprotein in parietal cell cytosol

Histamine stimulated acid secretion is mediated by an increase in intracellular cAMP. Cytosolic protein phosphorylation stimulated by histamine was investigated in isolated rabbit parietal cells. Histamine stimulated the phosphorylation of a 30 kDa phosphoprotein with an isoelectric point of 5.6. Cimetidine completely inhibited histamine-stimulated pp30 phosphorylation. However, omeprazole had no effect on the phosphorylation of pp30. Forskolin and 8-bromo-cAMP also stimulated the phosphorylation of pp30. The results suggest that pp30 is a histamine-stimulated, cAMP-dependently phosphorylated protein substrate in parietal cell cytosol.

A novel protein kinase activity in rabbit gastric gland cytosol.

A novel protein kinase activity was characterized from the cytosolic fraction of isolated rabbit gastric glands. The kinase phosphorylated a major 33,000 Da endogenous protein (pp33) and was stimulated by Zn2+ and Mn2+ with Kact of 1.0 and 7.5 mM, respectively. Mg2+ and Ca2+ failed to stimulate any pp33 kinase activity. The kinase utilized both ATP and GTP as phosphate donors with a Km of 10 microM for both. The pp33 protein displayed an isoelectric point of 7.5 to 7.8 and was phosphorylated predominantly on threonine residues. The kinase activity is clearly differentiable from all reported kinase activities and appeared to be enriched in rabbit gastric fundic mucosa. The results indicate that gastric fundic mucosa contains a novel protein kinase activity.

Somatostatin inhibition of intrinsic factor secretion from isolated guinea pig gastric glands.

The aim of our study was to examine the direct effect of somatostatin on histamine- and pentagastrin-stimulated intrinsic factor (IF) release in collagenase-dispersed guinea pig gastric glands. The effect of somatostatin (10(-11) M to 10(-6) M) on half-maximal doses of histamine (10(-6) M), pentagastrin (10(-6) M), and both histamine and pentagastrin together was tested. All tested concentrations of histamine significantly stimulated IF release. Pentagastrin (10(-10) M to 10(-6) M) inconsistently stimulated IF release. The quantity of IF release stimulated by histamine and pentagastrin together was approximately the additive sum of that produced by either agent alone. Somatostatin (10(-6) M) inhibited histamine-stimulated (10(-6) M) IF release by 69.9 +/- 7.2% and the combination of histamine (10(-6) M) and pentagastrin (10(-6) M) by 64.2 +/- 9.1%. This is the first in vitro demonstration that somatostatin inhibits IF release.

A calmodulin dependent protein kinase in parietal cells

An enriched population of isolated rabbit gastric parietal cells, from the fundic mucosa of New Zealand White rabbit, contained an active cytosolic calmodulin-dependent protein kinase activity with a prominent 100 kDa substrate (pp100). The latter focused as a doublet with isoelectric point of 6.8-7.0. The pp100 protein was phosphorylated only on threonine residues on a single tryptic peptide. Trifluoperazine inhibited the pp100 kinase activity with a KI of 10-15 microM. Addition of exogenous calmodulin was able to restore activity to uninhibited levels. A protein band with a molecular weight and phosphopeptide map identical to pp100, phosphorylated by calcium-dependent kinase, was also observed in rabbit pancreatic cytosol. The data suggest that a type III calmodulin-dependent kinase is present in parietal cell cytosol.