Summary. Glucose-stimulated 45calcium uptake and total calcium content of rat pancreatic islets has been studied, using a new fluorometric micro-method to estimate total calcium. Extracellular calcium was separated from incubated tissue by a rapid micro-filtration procedure. Islets incubated up to 60 min with calcium chloride 2.5mmol/1 and glucose 2.5mmol/1 maintained the same calcium content (670 + 7.5 pmol/gg DNA). When the glucose concentration was raised to 15mmol/1 no change in the total calcium content could be detected. On incubation with glucose 2.5 mmol/1 in the absence of calcium, the calcium content decreased to 488 + 27 pmol/gg DNA. On incubation with 4Scalcium chloride 2.5 mmol/1 for 5 or 30min at 2.5 mmol/1 glucose, islets exchanged 21 + 2 and 28 _ 1% of their total calcium content and, at 15 mmol/1 glucose, 30 + 3 and 45 ___ 2%, respectively. Thus, islet calcium has a high turn-over rate. Glucose stimulation results in an increase of the calcium uptake without enhancing the total calcium content and hence must increase the calcium-exchangeable pool.

Effects of fasting on glucose-induced changes of various calcium parameters were investigated. The total calcium content of islets of fed rats and obese mice, determined by fluorometric microanalysis, amounted to 575 +/- 25 and 600 +/- 75 pmol/microgram DNA, respectively. Fasting for 24 or 72 h had no effect on the calcium content of rat islets, but increased that of mice by 30%. Glucose stimulation did not alter their total calcium content. The amount of 45Ca (as percentage of total islet calcium content) taken up over 30 min by fed mouse islets at 2.5, 10, and 15 mM glucose was equivalent to 32%, 41%, and 44% and by fed rat islets to 25%, 31%, and 34%, respectively. The net uptake of fed rat islets over 60 min at 2.5 and 15 mM glucose amounted to 36% and 61%. Fasting for 24 h inhibited insulin secretion over 30 min but not 45Ca uptake, whereas both parameters were inhibited after fasting for 72 h. Insulin secretion of mouse islets, was inhibited after 24 and 72 h of fasting, but 45Ca uptake was not significantly affected. Since glucose stimulates 45Ca uptake without changing the total islet calcium content, it must represent 45Ca- 40Ca exchange. Stimulation of insulin secretion of rat islets is associated with an increased calcium influx rate and increased exchangeable calcium pool(s) size. Calcium pool(s) size seems not to be altered by 24 h of fasting; however, 72 h of fasting tends to decrease the pool(s) size. The inhibition of insulin secretion induced by 24 h of fasting is not due to an altered calcium uptake, but it may be due to a modification of intracellular handling of calcium.

Secretory granules of pancreatic B-cells contain high concentrations of zinc and calcium. The effect of gradual degranulation (induced by tolbutamide over a period of 3 days) and the subsequent regranulation (over a period of 4 days) on the histochemically detectable zinc (Zn) and calcium (Ca) content of B-cells was investigated. Zn was stained by dithizone, Ca by glyoxal-bis-(2-hydroxyanil), (GBHA), and B-granules by aldehyde fuchsin (AF). The staining intensities were determined cytophotometrically. A decrease of the granulation by 50% causes a comparable decrease of the Zn content. Almost complete degranulations, however, hardly further diminished the Zn content. Regranulation restores the Zn content parallel to the granulation. The presence of 40% of the initial Zn content in degranulated B-cells suggests the existence of a non-granular Zn fraction. The Zn content of B-cells may be partly involved in the storage of insulin as a Zn-insulin complex in the secretory vesicles. A-cells, however, contain even more (+ 30%) Zn than B-cells. Degranulation of B-cells is accompanied by a moderate decrease of the zinc content of the A-cells. The function of Zn in A-cells is completely unknown. Degranulation of B-cells causes the GBHA-Ca content to decrease to a very low level parallel to the AF-positive granulation. During regranulation the GBHA-Ca content restores parallel to the granulation and reaches after complete regranulation a slightly higher level than in untreated control rats. Almost complete disappearance of CBHA-Ca in the B-cells is accompanied by a decrease of the total islet calcium content of 33%. The results indicate that GBHA stains a Ca fraction which is mainly localized to the secretory granules. The stainability of granular Ca by GBHA is probably based on: a) the high Ca concentration in the granules, b) the presence of ionized Ca in the granules, due to the low intragranular pH, and c) on the properties of GBHA, which stains, under conditions used, only ionized (possibly also readily ionizable) Ca.

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Rat pancreatic islets contain an ionized or readily ionizable calcium fraction that can be determined by the metallochromic indicator glyoxal-bis-(2- hydroxyanil ) ( GBHA ). This calcium fraction is mainly localized in the secretory granules. The relationship between the effects of glucose on 45Ca uptake and on this ionized calcium fraction was investigated. In addition, the effects of glucose on total islet calcium content were also studied. Stimulation of isolated islets for 30 min with 15 mM glucose in the presence of 2.5 mM CaCl2 increased the 45Ca uptake but decreased the GBHA -Ca content, while the total calcium content was not affected. Deletion of CaCl2 caused, at 2.5 mM glucose, an abrupt decrease of GBHA -Ca, which did not occur at 15 mM glucose. Total islet calcium content decreased slowly at 2.5 mM glucose, but this was not significantly affected by glucose stimulation. Islet GBHA -Ca can be reduced by 70% and total islet calcium by 30% by means of washing with calcium-free buffer. Reintroduction of calcium at 2.5 mM glucose partly restored, but glucose 15 mM completely restored, the GBHA -Ca level within 5 min. The total calcium content was restored within 15 min independent of the glucose concentration. The increase of the islet calcium content equalled the 45Ca uptake at 2.5 mM glucose. The 45Ca uptake at 15 mM glucose was higher than the increase of the islet calcium content. The results indicate that the intragranular Ca2+ pool, as measured by GBHA , is rapidly and dramatically altered by glucose stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)