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Biomedical subjects

E Gylfe

Publications and source records attributed to E Gylfe.

At least 91 records · Page 5Linked to original sources
PubMed1989

Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells.

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.

Antibodies, Monoclonal↗
PubMed1989

Plasma membrane associated ATP as a regulator of the secretory activity of the pancreatic beta-cell.

beta-Cell-rich pancreatic islets from ob/ob-mice were used for evaluating how ATP associated with the plasma membrane participates in the regulation of insulin release. Increase of Ca2+ initiates insulin release from permeabilized beta-cells only in the presence of Mg-ATP. When bound to the inner part of the plasma membrane ATP depolarizes the beta-cells by closing a glucose-regulated K+-channel. It is possible that ATP in a plasma membrane compartment modulates insulin release also by stimulating ion pumps and exchange processes. ATP can regulate the secretory activity by binding also to the exterior of the beta-cells. The addition of ATP resulted in stimulation of insulin release related to polyphosphoinositide breakdown. It is suggested that the granule fusion with the plasma membrane is followed by release of sufficient amounts of ATP and ADP for activating a P2-purinoceptor. This receptor may consequently be part of a system for amplifying the secretory response to glucose and other agents facilitating the entry of Ca2+.

Adenosine Triphosphate↗
PubMed1989

Glucose effects on cytoplasmic Ca2+ of individual pancreatic beta-cells recorded by two procedures for dual-wavelength fluorometry.

Two different dual-wavelength microfluorometric systems were used to measure the cytoplasmatic Ca2+ concentration (Cai2+) of individual pancreatic beta-cells loaded with the indicator fura-2. Fluorescence was excited continuously at 340 nm with frequent manual checks at 360 nm. Alternatively, the 340/380 nm fluorescence excitation ratio was recorded based on automated samples at the two wavelengths every 10 msec. Irrespective of the experimental technique a rise of the glucose concentration from 3 to 20 mM resulted in an initial lowering of Cai2+ followed by a rise with a peak and a subsequent sustained intermediary level. The increase of Cai2+ depended on the presence of extracellular Ca2+. In some cells the pattern differed and the increase of the glucose concentration induced large oscillations of Cai2+. Glucose-induced increase of Cai2+ was associated with small rapid fluctuations, which were detectable only with the fast time-sharing approach. The characteristic Cai2+ response to glucose was maintained by some beta-cells after culture for more than 3 weeks. The results indicate that the glucose sensitivity of individual beta-cells varies, and that many electrically coupled cells within an islet collectively determine the characteristic pattern of rhythmic depolarization.

Animals↗
PubMed1989

Phorbol ester desensitization of clonal insulin-releasing cell response to carbachol involves depletion of an intracellular calcium pool.

The mechanism by which 12-o-tetradecanoylphorbol-13-acetate (TPA) desensitizes carbachol mobilization of glucose-incorporated calcium (Ca2+) was studied in clonal insulin-releasing cells (RINm5F) using colour indicators and dual wavelength spectrophotometry. The net uptake of Ca2+ stimulated by 20 mM glucose reached saturation after 19 +/- 2 min when it corresponded to 1.21 +/- 0.09 mmol calcium kg-1 protein. Carbachol then induced a release of 0.21 +/- 0.03 mmol calcium kg-1 protein. Half of the remaining Ca2+ was liberated by antimycin A and the rest with the Ca2+ ionophore A-23187. When 0.1 microM TPA was added initially, the cells lost 0.29 +/- 0.08 mmol calcium kg-1 protein within 10 min. The subsequent addition of glucose resulted in a sluggish uptake of only 0.58 +/- 0.09 mmol calcium kg-1 protein reaching equilibrium after 35 +/- 3 min. Carbachol now failed to induce any Ca2+ release. The actions of TPA were essentially unchanged by previous exposure to glucose, removal of Na+ from the medium and even when some of the glucose-incorporated Ca2+ had been liberated with carbachol. The results indicate that TPA desensitization of carbachol-induced mobilization of Ca2+ in the RINm5F cells is due to the disappearance of Ca2+ from the sensitive pool, an effect which may depend on stimulated extrusion of Ca2+ from the cells by the (Ca2+-Mg2+)-ATPase.

Animals↗
PubMed1989

Ca2+ oscillations in pancreatic beta-cells exposed to leucine and arginine.

Dual-wavelength microfluorometry with the fura-2 indicator was employed for continuous recordings of cytoplasmic Ca2+ (Ca2+i) in individual pancreatic beta-cells isolated from ob/ob-mice. When added to a medium containing 3 mmol l-1 glucose, both 10 mmol l-1 leucine and 20 mmol l-1 arginine induced rises in Ca2+i with periodic fluctuations. In the case of leucine, this increase was preceded by initial lowering followed by high-amplitude oscillations with a periodicity of 2-6 min. In a glucose-free medium arginine had no effect, and leucine was unable to induce more than a single peak of Ca2+i increase. When present at a concentration of 1 mmol l-1, leucine sometimes induced a couple of high-amplitude oscillations at 3 mmol l-1 glucose but lowered Ca2+i permanently in a glucose-free medium. It is likely that the high-amplitude oscillations of Ca2+i are related to the electrical activity of the beta-cells. Provided that some glucose was present, leucine initiated a similar type of Ca2+i response as obtained during glucose-induced insulin release. The observed leucine effect is therefore compatible with a role of glycolysis in generating high-amplitude Ca2+ oscillations and pulsatile insulin release.

Animals↗
PubMed1989

The actions of growth hormone and prolactin on rat hepatocytes are not mediated by changes in cytoplasmic Ca2+.

In connection with measurement of the binding of growth hormone and prolactin to rat hepatocytes we investigated whether such binding is associated with changes in the cytoplasmic Ca2+ concentration. Whereas hepatocytes from male animals were found to have essentially only somatogenic receptors, lactogenic receptors were dominant in females. All hepatocyte preparations responded to epinephrine and vasopressin with transient peaks of cytoplasmic Ca2+. However, no effects on cytoplasmic Ca2+ were obtained when cells from female or male animals were exposed to growth hormone or prolactin. We therefore conclude that signal transduction of the growth hormone and prolactin responses in the rat liver does not involve an early messenger function for Ca2+.

Animals↗
PubMed1989

Leucine induces initial lowering of cytoplasmic Ca2+ in pancreatic beta-cells without concomitant inhibition of insulin release.

The early effects of glucose and leucine on cytoplasmic Ca2+ and insulin release were compared in suspensions of cells prepared by dispersal of the beta-cell-rich pancreatic islets of ob/ob-mice. Adequate temporal resolution was achieved by continuously recording the 340/380 nm fluorescence excitation ratio from cells loaded with the Ca2+ indicator fura-2 and measuring insulin in the perifusate from cells mixed with polyacrylamide beads. Raising the glucose concentration from 3 to 20 mM resulted in concomitant reductions of cytoplasmic Ca2+ and insulin release during the first minute. Whereas 10 mM leucine was as efficient as glucose in inducing temporary lowering of cytoplasmic Ca2+, this amino acid did not depress insulin release. It is concluded that the initial decrease of cytoplasmic Ca2+ is a phenomenon coupled to stimulation of the metabolism. The leucine-induced lowering of Ca2+ may essentially reflect changes in cytoplasmic pools other than in a peripheral one regulating insulin release.

Animals↗
PubMed1989

Microfluorometric measurements of cytoplasmic calcium in chief and oxyphil parathyroid cells of adenomatous and hyperplastic glands and of normal-sized glands associated with adenomas.

The effects of extracellular calcium on the cytoplasmic Ca2+ concentration (Ca2+i) were studied by dual-wavelength microfluorometry in individual human parathyroid cells obtained from adenomatous glands and normal-sized glands associated with adenomas in hypercalcemic hyperparathyroidism (HPT), as well as from enlarged glands of patients with uremia with HPT. In comparison with the normal parathyroid tissue, chief cells of the adenomatous and hyperplastic glands showed significantly lower Ca2+, and also right-shifted responses of Ca2+i to increases in the extracellular calcium concentration within the 0.5 to 3.0 mmol/L range. This pathophysiologic disturbance apparently was independent of the cell size. Oxyphil cells of nodules from the hyperplastic glands had lower Ca2+i and responded less to increments in extracellular Ca2+ than the chief cells from the surrounding parts of the same glands. Also the chief cells from the normal-sized glands associated with single adenomas exhibited a disturbance of the regulation of Ca2+i, which was less pronounced than that in the cells of the adenomas. These findings support the presence of relative calcium insensitivity of Ca2+i in chief and oxyphil parathyroid cells from adenomatous and hyperplastic glands. This derangement may also be found in all parathyroid glands of individuals with adenomatous HPT.

Adenoma↗
PubMed1988

Nutrient secretagogues induce bimodal early changes in cytoplasmic calcium of insulin-releasing ob/ob mouse beta-cells.

The cytoplasmic calcium concentration (Ca2+i) was measured in suspensions of fura-2 loaded mouse pancreatic beta-cells by continuously recording the 340/380 nm fluorescence excitation ratio. When the glucose concentration was raised from 3 to 20 mM, there was an initial lowering of Ca2+i followed by a sustained increase. Whereas the reduction in Ca2+i was related to the extracellular glucose concentration in a hyperbolic manner, the increasing component exhibited a sigmoidal dose-response relationship. Both effects became maximal at 15-20 mM of the sugar. Qualitatively similar bimodal Ca2+i responses were obtained with 30 mM mannose, 2 mM alpha-ketoisocaproic acid, 10 mM leucine, and 10 mM metabolism-stimulating leucine analogue beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. Fructose (30 mM) had virtually no effect on Ca2+i in the presence of extracellular Ca2+, and 10 mM arginine induced only a rise. The results indicate that nutrient secretagogues stimulate both the entry of Ca2+ into the beta-cells and its elimination from the cytoplasm by processes like organelle sequestration and outward transport. Consequently, the Ca2+i level determining insulin secretion results from the balance between two opposing actions.

Amino Acids↗
PubMed1988

Phorbol-ester-induced stable changes in the regulation of DNA synthesis and intracellular pH are accompanied by altered expression of protein kinase C in the monoblastoid cell line U-937.

12-O-tetradecanoylphorbol-13-acetate (TPA)-induced changes in cytoplasmic pH, cytoplasmic Ca2+-concentration, rate of DNA synthesis, and concentration and activity of protein kinase C (PKC) were studied in human monoblastoid cell lines. The cell line U-937 GTB was compared to the subline U-937 RES (adapted to growth in the presence of 10(-9) M TPA) and another subline U-937 RESREV (U-937 RES grown in TPA-free medium) established in order to analyze the stability of the TPA-induced differences. TPA induced half maximal inhibition of DNA synthesis in the wild-type U-937 GTB cell line at 10(-9) M, whereas 10 times higher concentrations of phorbol ester were needed for a corresponding inhibition of the U-937 RES and U-937 RESREV lines. Furthermore, the U-937 RES cells exhibited a decreased sensitivity to TPA, and the U-937 RESREV cells did not respond at all to this agent with regard to cytoplasmic alkalinization by an intracellular mechanism independent of Na+/H+ exchange. A Na+-dependent system for extrusion of protons, which was activated by the Ca2+ ionophore ionomycin, was also severely depressed as a result of TPA-adaptation. The concentration of PKC, measured by immunoblotting, was reduced by 34 and 24% in U-937 RES and U-937 RESREV cells, respectively, as compared to the wild-type U-937 GTB line. The corresponding reductions in PKC activity were 32 and 54% when histone III-S was used as substrate. The data suggest that adaptation to growth in the presence of TPA results in stable modifications of several parameters, which are assumed to be involved in the regulation of proliferation and differentiation. Furthermore, the data from the U-937 RESREV cells question a causal relationship between cytoplasmic alkalinization and control of proliferation.

Cell Line↗
PubMed1988

Kinetic evidence for cytoplasmic calcium as an inhibitory messenger in parathyroid hormone release.

A sudden change of extracellular Ca2+ from 0.5 to 3.0 mM resulted in a transient rise of the cytoplasmic Ca2+ concentration (Ca2+i) followed by a sustained increase in parathyroid cells loaded with the Ca2+-indicator fura-2. The initial transient could be eliminated by increasing the Ca2+ buffering capacity of the cytoplasm. Under such conditions the rise of Ca2+i exhibited kinetics reminiscent of those for 45Ca uptake and cell depolarization. Addition of 0.5 mM Mn2+ mimicked the effect of raising the extracellular Ca2+ concentration, since there was an initial Ca2+i transient followed by a slower entry of Mn2+ into the cells. This reaction pattern was different from that of pancreatic alpha 2-cells in which there was no substantial influx of Mn2+ before depolarization with arginine. When measuring the kinetics of parathyroid hormone (PTH) release it was apparent that Ca2+ inhibition of secretion followed Ca2+i and thus became substantially delayed after eliminating the initial transient. The results support the concept of a depolarizing Ca2+ permeability in the parathyroid cell membrane which can be activated by external Ca2+, and indicate that Ca2+i is an inhibitory messenger of importance for the physiological regulation of PTH release.

Animals↗
PubMed1988

Glucose-induced early changes in cytoplasmic calcium of pancreatic beta-cells studied with time-sharing dual-wavelength fluorometry.

The cytoplasmic calcium concentration (Ca2+i) was measured in suspensions of fura-2-loaded mouse pancreatic beta-cells by recording the 340:380 nm fluorescence excitation ratio. Exposure to 20 mM glucose resulted in an initial reduction and later increase of Ca2+i irrespective of preincubation in medium containing 0.5 or 1.28 mM Ca2+ and 0 or 3 mM glucose. When elevating the Ca2+ concentration to 5 or 10 mM only 5 min before raising glucose to 20 mM, the sugar-induced reduction of Ca2+i became more pronounced like the subsequent increase. However, when the Ca2+ concentration was increased from 1.28 to 10 mM 2 min after stimulation with glucose, there was a sudden pronounced Ca2+i transient, which was followed by a decrease and a slower secondary rise. After preincubation in 20 mM glucose the glucose-induced initial reduction of Ca2+i was only seen in a Ca2+-deficient medium. Reintroduction of the sugar in the presence of extracellular Ca2+ resulted in an immediate rise of Ca2+i, the rapidity of which depended on the transmembrane Ca2+ gradient. The results emphasize the role of a saturable beta-cell pool of Ca2+ in glucose-induced reduction of Ca2+i and indicate that the first phase of insulin release depends on an influx of extracellular Ca2+.

Animals↗
PubMed1988

Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic beta-cell.

The cytoplasmic calcium concentration (Ca2+i) was measured in individual mouse pancreatic beta-cells loaded with fura-2 by recording the 340/380 nm fluorescence excitation ratio. An increase of the glucose concentration from 3 to 20 mM, caused initial lowering of Ca2+i followed by a rise with a peak preceding constant elevation at an intermediary level. However, at 11 mM glucose there were large Ca2+i oscillations with a frequency of 1 cycle per 2-6 min. The results indicate that both first and second phase secretion depend on elevated Ca2+i, and that many electrically coupled cells collectively determine the pace of rhythmic depolarization.

Animals↗
PubMed1988

Modulation of the Ca2+-sensing function of parathyroid cells in vitro and in hyperparathyroidism.

When raising the extracellular Ca2+ concentration stepwise from 0.5 to 3.0 mM, bovine parathyroid cells reacted with initial transient and sustained elevations of the cytoplasmic Ca2+ concentration (Ca2+i), as well as more than 50% inhibition of parathyroid hormone (PTH) release. Human parathyroid adenoma cells and bovine cells cultured for 1 day or exposed to a low concentration of a monoclonal antiparathyroid antibody exhibited right-shifted dependencies of PTH release and Ca2+i on extracellular Ca2+ and reduced Ca2+i transients. The protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) further right-shifted the dose response relationship for Ca2+ regulated Ca2+i of the adenoma cells, whereas the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) tended to normalize it, without affecting Ca2+i of normal bovine cells. In cells from an oxyphil adenoma and a parathyroid carcinoma as well as in bovine cells cultured 4 days or exposed to a high concentration of the antiparathyroid antibody, there were no Ca2+i transients, very small increases in steady-state Ca2+i and nonsuppressible PTH release. The results suggest that reduced availability of a putative Ca2+-receptor and increased protein kinase C activity may be important factors in the decreased Ca2+ sensitivity of abnormal parathyroid cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Dual effect of glucose on cytoplasmic Ca2+ in single pancreatic beta-cells.

The cytoplasmic Ca2+ concentration (Ca2+i) was measured in single pancreatic beta-cells from ob/ob-mice using the fluorescent indicator fura-2. Raising the glucose concentration from 3 to 20 mM resulted in 25% initial lowering of Ca2+i, followed by 250% rise above the basal level of 49 +/- 3 nM. Tolbutamide (100 microM) was as effective as glucose in increasing Ca2+i, although its action was more rapid and not preceded by any reduction. The results support the concept that stimulated removal of Ca2+ from the cytoplasm is an essential part of the physiological glucose effect on the pancreatic beta-cells.

Animals↗
PubMed1988

Cytoplasmic pH is differently regulated in the monoblastic U-937 and erythroleukemic K-562 cell lines.

Regulation of cytoplasmic pH (pHi) of the human monoblastic U-937 and erythroleukemic K-562 cell lines was investigated. The apparent resting pHi, as assessed by the fluorescent pH probe quenel, were 6.61 and 6.75 for the U-937 and K-562 cells, respectively. When extracellular Na+ was substituted by equimolar choline+, pHi decreased by about 0.2 units. The protein kinase C activating beta-form of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10(-10) and 10(-7) M) induced a dose-dependent alkalinization in both cell types of 0.03-0.12 units, whereas the alpha-form was inactive. The response was detectable after about 2 min and reached steady-state 10-15 min later. In the K-562 cells the alkalinization was mediated by Na+/H+ exchange as it was accompanied by stimulation of H+ extrusion and abolished by Na+ removal. The TPA response in the U-937 cells, however, was unaffected by Na+ removal, not accompanied by H+-efflux, and thus unrelated to Na+/H+ exchange. Since electron microscopy indicated development of multivesicular bodies with an acidic interior, the alkalinization can probably be accounted for by an intracellular mechanism. Ionomycin (10(-5) M) induced a rapid increase in the cytoplasmic Ca2+ concentration of both cell types and this response was accompanied by acidification followed by a Na+-dependent recovery. In the U-937, but not in the K-562, cells this recovery was followed by a net alkalinization. It is concluded that both cell types possess a Na+/H+ exchange of importance for pHi but that this mechanism is regulated differently in the U-937 and K-562 cells.

Calcium↗