Electron probe X-ray microanalysis using freshly frozen hydrated and dried thin sections of dog submandibular gland was performed to determine the distribution of elements and water in the acinar cells of resting and stimulating states. The results obtained are as follows : (a) The secretory granules contained high concentrations of Ca and S while high concentrations of K and P were present in the cytoplasm and/or nucleus of acinar mucous cells of the gland in the resting state. (b) With pilocarpine stimulation, the concentration of Ca increased in the cytoplasm and decreased in the secretory granules, while there was an increase in the concentration of Na and Cl in both the cytoplasm and secretory granules of the cells. (c) The local dry-mass fractions of acinar cells, estimated by comparing the continuum radiation of X-ray spectrum from the frozen hydrated sections with that from the frozen dehydrated sections, were approximately 20 and 33 % in the cytoplasm and secretory granules of resting acinar cells, respectively, and each value was not significantly altered under conditions of stimulation having a tendency to decrease slightly. Therefore, the passive Na and Cl influx and the cytoplasmic Ca flowed in from extracellular spaces and released from secretory granules, an intracellular calcium store, by secretory stimulation probably triggers the passive or active Na and Cl extrusion and consequently the osmotic water flux from the basal part of acinar cells to the secretory granules and the lumen, as well as the serial exocytosis of the granules in the luminal side of the acinar cells.Key Words: electron probe X-ray microanalysis, dog salivary gland, pilocarpine stimulation, electrolyte transport, intracellular calcium store.Saliva is primarily secreted in the acini of the salivary glands and is followed by a secondary process of reabsorption in the duct system (YOUNG and MARTIN,
Changes in electrolytes of pig pancreatic acinar cells following application of gastrin-cholecystokinin (CCK) were investigated using the technique of X-ray microanalysis of hydrated and dehydrated sections of freshly frozen pancreas. After stimulation by CCK (10(-9) M), Na and Cl increased significantly in the cytoplasm [Na, from 10 mmol/kg wet wt. (48 mmol/kg dry wt.) to 19 mmol/kg (95 mmol/kg); Cl, from 22 mmol/kg (105 mmol/kg) to 49 mmol/kg (245 mmol/kg)] as well as in the luminal interspace [Na, from 53 mmol/kg (189 mmol/kg) to 65 mmol/kg (283 mmol/kg); Cl, from 65 mmol/kg (232 mmol/kg) to 102 mmol/kg (443 mmol/kg)]. In the secretory granules Cl increased significantly from 30 mmol/kg (86 mmol/kg) to 67 mmol/kg (203 mmol/kg). K decreased significantly from 120 mmol/kg (571 mmol/kg) to 81 mmol/kg (405 mmol/kg) in the cytoplasm, while both increased from 38 mmol/kg (109 mmol/kg) to 58 mmol/kg (176 mmol/kg) in the granules and from 46 mmol/kg (164 mmol/kg) to 48 mmol/kg (209 mmol/kg) in the luminal interspace. Ca increased significantly in the cytoplasm as well as in the luminal interspace, and decreased significantly in the secretory granules. CCK evoked Ca release from secretory granules in the secretory pole of acinar cells. The values were measured from dehydrated sections, and agreed well with those from hydrated sections. The effect of furosemide, an inhibitor of the Na+-K+-2Cl- co-transporter, on the ion transport of acinar cell was studied. When furosemide (10(-5) M) was added to the external solution, the cytoplasmic Cl and Ca concentrations decreased significantly, while there was a little decrease in Na and K concentrations under the secretory condition. These results indicate that Na+-K+-2Cl- co-transport, and Na+, Cl- and K+ exits into the lumen are involved in the mechanism of ion secretion in pig pancreatic acinar cells.
The effects of tension at the site of coaptation on recovery of sciatic nerve function after neurorrhaphy were studied by evaluating walking-track measurements, nerve conduction velocity measurements, histomorphometry, and electron probe X-ray microanalysis. Forty adult male Lewis rats underwent right sciatic nerve (SN) transection followed by one of four different nerve repair procedures (N = 10 rats per group). In Group 1, the gap was repaired by end-to-end epineural coaptation. In Group 2, a 5-mm segment of SN was resected, and the defect was repaired under high tension by epineural neurorrhaphy. In Group 3, a 5-mm segment of SN was resected, and the defect was repaired with a 5-mm interposition nerve graft. In Group 4, a 5-mm segment of SN was resected. Then, to lessen the tension that follows neurorrhaphy, an anchoring suture was added. Finally, end-to-end coaptation was performed. Walking-track analysis showed better functional recovery in Group 1 than in Group 2, and better recovery in Group 3 than in Group 2. Group 4 showed a tendency toward better recovery comparing with Group 2. Electron probe X-ray microanalysis revealed higher Na, Cl, and K peaks in axoplasm accompanied by increase in the endoneural fluid pressure (EFP) in Group 2 than those of Group 1. This higher level of Na, Cl and K may be due to impairment of axonal sodium and potassium transport mechanism in Group 2. Increase in EFP may affect nerve regeneration.
We have investigated intracellular Ca2+ mobilization in oscillations of cytoplasmic Ca2+ in response to glucagon-like peptide 1 (GLP-1) and glucose in clonal HIT insulinoma cells with a confocal laser-scanning microscope (CLSM). We also used electron probe X-ray microanalysis to determine the GLP-1- and glucose-induced changes in electrolyte levels in the cytoplasm and insulin granules of the cells. GLP-1 produced 10- to 35-s duration oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i), both with and without Ca2+ in the extracellular solution, suggesting that Ca2+ is mobilized from intracellular Ca2+ stores, namely secretory granules. Glucose caused 1- to 3-min duration oscillatory increases in [Ca2+]i when the extracellular solution contained Ca2+. When the cells were cultured without Ca2+ (no Ca2+ added, 1 mM EGTA), an oscillatory [Ca2+]i increase of amplitude and short duration (12-35 s) was produced by 11 mM glucose, and the oscillation was inhibited by ruthenium red. X-ray microanalysis showed that stimulation with glucose increased the total Ca concentration in the cytoplasm and decreased it in the insulin granules with and without Ca2+ in the extracellular solution. The application of glucose significantly decreased K, and increased Na and C1 in the cytoplasm when the extracellular solution contained Ca2+. Our result also suggests that the [Ca2+]i oscillation induced by glucose is involved in the release of Ca2+ from intracellular Ca2+ stores through the ryanodine receptor, which is blocked by ruthenium red, and/or through the inositol trisphosphate receptor that may be present in the membrane of insulin granules.
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