| 1. Isolation of Gastric Parietal Cells from RatPronase-EDTA method was used to isolate gastric mucosal cells from everted gastric sacs of rat, Percoll linear density gradient to enrich parietal cells. The purity of parietal cells was over 65%, the viability over 92% excluded by trypan blue test. Purified gastric parietal cells from rat were obtained first time in China, which will be useful experimented models for parietal cells-related studies.2.Establishment of Radioligand Binding Assay of Gash-in Receptors in Rat Gastric Parietal CellsUsing 125I-labeled [Leu15]-gastrin-17-I, radioligand binding assay of gastrin receptor was established in rat gastric parietal cell preparations after an incubation period of 30min at 37癈(pH7.4) with a cell concentration of 1.8 x 105cells/L per assay tube. In the range of the study, a Scatchard plot of the binding was made by Ligand programwith an equilibrium Kd of approximately 1.249 x 10'10M and a maximum binding capacity of 4.4604x 10~12M. The amount of gastrin binding was strongly associated with the cell concentration of rat gastric parietal cell preparations ranged from 0.25 x 109 to 2 x 109cells/L. The variant coefficent of the binding assay was 7.04% within the same sample. The competitive inhibiting analyses showed that 125I-gastrin was bound to gastric parietal cell preparations with a high specificity. These results suggested that the radioligand binding assay of gastrin receptor in rat gastric parietal cell preparations meets the criteria for establishing receptor binding assay.S.Measurement of Gastrin Receptor Binding Capacity of Parietal Cells of Spleen-Deficency Model in Rat and the Regulating Effects of Herbal HuangqiThe results showed that the gastrin receptor binding capacity was markedly decreased in isolated gastric parietal cell of spleen-deficency rat induced by administration of herbal medicine Dahuang (vs. the normal rat, P<0.01). After the treatmeat with Hangqi, which functions are replenishing Qi and invigorating spleen, the gastrin receptor binding capacity was increased significantly (vs. the model rat, P<0.01). The present study confirmed our previous results using rat gastric mucosa as receptor preparation. We speculate that the spleen-deficiency syndrome might be a gastrin receptor related disease, and receptor-regulation might be a new method to treat this syndrome. 4. Measurement of Intracellular Calcium Level of Gastric Parietal Cells of Spleen-Deficiency Model in Rats and the Regulating Effects of HuangqiGastric parietal cells [Ca2+]i of spleen-deficiency model in rat was measured using fura-2/ AM fluorescent indicator. (1) The resting [Ca2+]i of the model and its elevating percentage under gastrin stimulation were significantly lower than those of normal rats (P<0.01); (2) The resting [Ca2+]i of treating group treated with Huangqi was increased in a degree, but had no statistical significance compared with that of the model. Stimulating by gastrin, the percentage of increased [Ca2+]i of the treating group was significantly higher than that of the model (P<0.01), and recovered to the normal level (P>0.05). These results indicated that gastric parietal cells might be in a lower metabolic condition when the body was suffered with spleen-deficiency syndrome. We speculates that calcium pool volume of parietal cell might be decreased, or gastrin receptor sites and/or their function were decreased which could not mediate extracellular signals. The Chinese herbal Huangqi, which function is replenishing Qi and invigorating spleen, can elevate intracellular calcium pool volume or upregulate gastrin receptor sites of gastric parietal cells, these might be the cellular mechanism of Huangqi's function.
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