scholarly journals Trypsin-induced increase in cyclic AMP concentration in rat thymocytes An effect independent of calcium and calmodulin

1986 ◽  
Vol 239 (3) ◽  
pp. 603-607 ◽  
Author(s):  
J Segal
Keyword(s):  
Cyclic Amp ◽  
Free Medium ◽  
Standard Medium ◽  
Intracellular Pool ◽  
Calmodulin Inhibitor ◽  
Related Increase ◽  
In Cells

Trypsin produces a dose-related increase in cellular cyclic AMP concentration in rat thymocytes [Shneyour, Patt & Trainin (1976) J. Immunol. 117, 2143-2149; Segal & Ingbar (1983) Clin. Res. 31, 277A]. In the present study, I examined whether this effect of trypsin requires Ca2+ and whether it is modified by calmodulin. In fresh thymocytes suspended in standard medium (containing 1 mM-Ca2+), trypsin produced a concentration-dependent increase in cytoplasmic free Ca2+ concentration, which was evident at a concentration of 50 micrograms of trypsin/ml and reached maximal values at about 1 mg/ml. This effect of trypsin was very prompt in onset, almost immediate, and reached maximal values within 2-3 min. But in cells suspended in essentially Ca2+-free medium (6 nM free Ca2+), trypsin had no effect on cytoplasmic free Ca2+ concentration, which indicates that trypsin acted by increasing Ca2+ uptake rather than Ca2+ release from an intracellular pool. However, the increase in thymocyte cyclic AMP concentration produced by trypsin was independent of extracellular Ca2+ and was not influenced by calmodulin, because it was the same in the presence or absence of Ca2+ and was not changed by the calmodulin inhibitor trifluoperazine. I therefore suggest that in rat thymocytes the trypsin-induced increase in cyclic AMP concentration does not require Ca2+ and is not influenced by calmodulin.

scholarly journals Heat treatment induces an increase in intracellular cyclic AMP content in human epidermoid A-431 cells

1991 ◽  
Vol 276 (3) ◽  
pp. 683-689 ◽  
Author(s):  
J G Kiang ◽  
Y Y Wu ◽  
M C Lin
Keyword(s):  
Heat Treatment ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
G Proteins ◽  
Phosphodiesterase Inhibitors ◽  
Metabolic Inhibitors ◽  
Thermally Induced ◽  
Heat Treated ◽  
Atp Breakdown ◽  
In Cells

The basal level of intracellular cyclic AMP (cAMPi) in A-431 cells incubated at 37 degrees C in Na(+)-containing Hanks solution is 2086 +/- 139 fmol/10(6) cells. When cells are exposed to 45 degrees C for 10 min, cAMPi increases by 40 +/- 4%, and then returns to basal levels within 30 min. Incubating cells in Ca(2+)-free or Mg(2+)-free Hanks solution has no effect on the heat-induced increase in cAMPi, but the increase is inhibited by acid-loading cells to intracellular pH 7.0 or 6.8. In unheated cells, cAMPi increases by 16 +/- 8%, 53 +/- 7%, or 39 +/- 8%, when incubated with isobutyl-1-methylxanthine (1 mM), Ro 20-1724 (0.5 mM), or theophylline (1 mM) respectively. However, heat treatment further elevates cAMPi in cells treated with phosphodiesterase inhibitors, indicating that heat treatment and phosphodiesterase inhibitors elevate cAMPi by a different pathway(s). Heat treatment increases adenylate cyclase activity 2.5-fold. When forskolin (150 microM), an adenylate cyclase stimulator, is applied to cells, the basal cAMPi increases 28 +/- 6-fold compared with controls. Subsequent heating of these cells lowers cAMPi levels to 7.0 +/- 0.5 times that in control cells. This decrease is prevented by pretreatment with pertussis toxin (30 ng/ml, 24 h), suggesting that G-proteins are involved in the process of heat-induced cAMPi increase. 2-Deoxy-D-glucose (10 mM), NaN3 (10 mM) and 2,4-dinitrophenol (1 mM) also increase cAMPi in A-431 cells. However, application of these metabolic inhibitors to cells before heat treatment does not result in cAMPi levels greater than that observed in cells with heat alone. Similar observations are obtained in heat-treated cells previously exposed to adenosine, but not to AMP or ADP. These data are the first to suggest that thermally induced increase in cAMPi is due to a combination of activation of adenylate cyclase and G-proteins, and an increase in adenosine owing to ATP breakdown caused by hyperthermia.

scholarly journals Calcium metabolism in rat hepatocytes

1978 ◽  
Vol 170 (3) ◽  
pp. 615-625 ◽  
Author(s):  
S Foden ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Ionophore A23187 ◽  
Total Calcium ◽  
Free Medium ◽  
Adrenergic Agonists ◽  
Calcium Efflux ◽  
Similar Time ◽  
Carbonyl Cyanide

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.

scholarly journals Agonist-induced Ca2+ influx in human neutrophils is secondary to the emptying of intracellular calcium stores

1991 ◽  
Vol 277 (1) ◽  
pp. 73-79 ◽  
Author(s):  
M Montero ◽  
J Alvarez ◽  
J Garcia-Sancho
Keyword(s):  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Time Course ◽  
Human Neutrophils ◽  
Calcium Stores ◽  
Free Medium ◽  
Prolonged Incubation ◽  
Intracellular Calcium Stores ◽  
Cytochrome P 450 ◽  
In Cells

Emptying of the intracellular calcium stores of human neutrophils, by prolonged incubation in Ca(2+)-free medium, by treatment with low concentrations of the Ca2+ inophore ionomycin, or by activation with cell agonists, increased the plasma-membrane permeability to Ca2+ and Mn2+. The chemotactic peptide formylmethionyl-leucyl-phenylalanine and the natural agonists platelet-activating factor and leukotriene B4 released different amounts of calcium from the stores and induced Ca2+ (Mn2+) uptake, the rate of which correlated inversely with the amount of calcium left in the stores. The increased Mn2+ uptake induced by these agonists was persistent in cells incubated in Ca(2+)-free medium, but returned to basal levels in cells incubated in Ca(2+)-containing medium, with the same time course as the refilling of the calcium stores. The calcium-stores-regulated Mn2+ influx, including that induced by agonists, was prevented by cytochrome P-450 inhibitors. We propose that agonist-induced Ca2+ (Mn2+) influx in human neutrophils is secondary to the emptying of the intracellular stores which, in turn, activates plasma-membrane Ca2+ channels by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197].

Multiple calcium mobilization pathways in single avian salt gland cells

1990 ◽  
Vol 258 (2) ◽  
pp. C289-C298 ◽  
Author(s):  
E. L. Stuenkel ◽  
S. A. Ernst
Keyword(s):  
Salt Gland ◽  
Transient Increase ◽  
Secretory Cells ◽  
Free Medium ◽  
Subsequent Removal ◽  
Intracellular Pool ◽  
Gland Cells ◽  
Agonist Stimulation ◽  
Induced Changes ◽  
Sustained Increase

Agonist-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in individual secretory cells from the avian salt gland were detailed using dual-wavelength microspectrofluorimetry of the Ca2(+)-sensitive fluorescent probe fura-2. Resting [Ca2+]i averaged 42 +/- 5 nM. Stimulation with the cholinergic agonist carbachol (1 microM) resulted in a rapid increase in [Ca2+]i to 308 +/- 26 nM, which was sustained at a nearly constant elevated level (328 +/- 31 nM) throughout agonist application. In the absence of extracellular Ca2+ or in the presence of an inorganic blocker of Ca2+ entry (Ni2+, 1 mM), only a transient increase in [Ca2+]i occurred on agonist stimulation, whereas subsequent readmission of Ca2+ or washout of Ni2+ reinitiated a sustained increase in [Ca2+]i. The initial transient response results from Ca2+ release from intracellular stores, whereas the sustained phase represents entry of extracellular Ca2+ into the cytoplasm. Repetitive stimulations in Ca2(+)-free medium alternating with Ca2(+)-containing medium were performed to examine the mechanisms involved in refilling of the agonist-sensitive intracellular pool. After depletion of the intracellular pool by stimulation in Ca2(+)-free medium, removal of the agonist and readmission of Ca2+ resulted in a rapid transient increase in [Ca2+]i that could be blocked by Ni2+, La3+, or elevated K+. Subsequent removal of extracellular Ca2+ and restimulation nonetheless showed that complete refilling of the intracellular pool had occurred in each case. These results suggest that two separate Ca2(+)-entry mechanisms, one sensitive to Ni2+, La3+, and elevated K+ and responsible for the agonist-induced increase in [Ca2+]i and one insensitive to the blockers and involved in refilling of the intracellular pool, may exist in salt gland cells. Spontaneous oscillations of [Ca2+]i that are independent of extracellular Ca2+ have also been observed in 10% of the cells. The abolition of the oscillations by depletion of the agonist-sensitive pool suggests this pool as the Ca2+ source for the oscillations.

[Na]i modulates isoproterenol's effect on Ca permeability in cultured heart cells

1987 ◽  
Vol 253 (2) ◽  
pp. C253-C262 ◽  
Author(s):  
D. Kim ◽  
T. W. Smith
Keyword(s):  
Resting Membrane Potential ◽  
Heart Cells ◽  
Ca Channel ◽  
Control Medium ◽  
Free Medium ◽  
Cardiac Muscle Cells ◽  
45Ca Uptake ◽  
Ca Uptake ◽  
Chick Heart ◽  
In Cells

Isoproterenol (ISO) augments the slow inward Ca current in cardiac muscle cells. We examined the role of intracellular Na (Nai) on ISO-mediated alterations in Ca uptake in cultured chick heart cells. In 140 mM Na medium, 1 microM ISO did not measurably alter 45Ca uptake. When cells were first preincubated in Na-free medium for 5 min and then incubated in control medium with 45Ca, ISO increased 45Ca uptake by 30%. Nifedipine (10 microM), verapamil (1 microM), or dl-propranolol (1 microM) abolished the effect of ISO on 45Ca uptake. CGP 28392 (1 microM), a Ca channel agonist, increased Ca influx in a manner that was augmented by decreased Nai, similar to the ISO response. Neither ISO nor CGP 28392 altered 45Ca uptake when cells preincubated in Na-free medium were further incubated in Na-free medium containing 45Ca. Exposure of cells to Na-free medium or 25 mM K+ medium caused depolarization of the resting membrane potential to approximately -40 mV. In the absence of ISO, the 45Ca uptake in cells preincubated in Na-free or 25 mM extracellular K (Ko) medium was significantly greater than in cells preincubated in control medium. This appeared to be due partly to increased 45Ca uptake via nifedipine-sensitive pathways. These findings support the hypothesis that reduction in Nai concentration ([Na]i) enhances the ISO-induced augmentation of Ca uptake via nifedipine-sensitive pathways (presumably via slow Ca channels), probably by a direct effect on the channels.

Potential mechanism of insulin resistance in ageing: impaired insulin-stimulated glucose transport due to a depletion of the intracellular pool of glucose transporters in Fischer rat adipocytes

1990 ◽  
Vol 126 (1) ◽  
pp. 99-107 ◽  
Author(s):  
S. Matthaei ◽  
H. Benecke ◽  
H. H. Klein ◽  
A. Hamann ◽  
G. Kreymann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Insulin Binding ◽  
Low Density ◽  
Intracellular Pool ◽  
Subcellular Membrane ◽  
Impaired Insulin ◽  
In Cells

ABSTRACT To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (30MG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 30MG (12±4 vs 13±3 fmol/5 × 104 cells, means ± s.e.m.); in contrast, in FR-24 rats insulin-stimulated 30MG was markedly decreased by 43% when compared with that in FR-6 rats (158±14 vs 90±8 fmol/5 × 104 cells; P < 0·01). Insulin binding to adipocytes from FR-6 rats was 2·40±0·38% compared with 2·28±0·47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48·6±7·2 vs 29·8±5·5 pmol/mg membrane protein; P < 0·01). In consequence, in FR-24 there were fewer glucose transporters available for insulin-induced translocation to the plasma membrane (insulin-treated plasma membrane: 23·9±4·2 (FR-6) vs 14·4±3·1 (FR-24) pmol/mg membrane protein; P < 0·01). These results were confirmed by immunoblotting. In conclusion, (1) maximal insulin-stimulated 30MG was decreased by 43% in cells from FR-24 rats compared with those from FR-6 rats, while basal 30MG was similar in both groups, (2) neither insulin binding nor IRKA were significantly altered in cells from FR-24 rats, and (3) impaired insulin-stimulated 30MG was associated with reduced numbers of glucose transporters in the plasma membrane as a consequence of a depletion of the intracellular pool of glucose transporters in cells from FR-24 rats. Journal of Endocrinology (1990) 126, 99–107

Dibutyryl cyclic AMP inhibits transport dependent QO2 in cells isolated from the rabbit medullary ascending limb

1987 ◽  
Vol 409 (1-2) ◽  
pp. 74-80 ◽  
Author(s):  
Patricio Silva ◽  
Barbara Koenig ◽  
Stephanie Lear ◽  
Jill Eveloff ◽  
Rolf Kinne
Keyword(s):  
Cyclic Amp ◽  
Dibutyryl Cyclic Amp ◽  
In Cells
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