Effects of high extracellular calcium concentrations on phosphoinositide turnover and inositol phosphate metabolism in dispersed bovine parathyroid cells
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Abstract We previously showed that high extracellular calcium (Ca2+) concentrations raise the levels of inositol phosphates in bovine parathyroid cells, presumably via the G protein-coupled, “receptor-like” mechanism through which Ca2+ is thought to regulate these cells. To date, however, there are limited data showing Ca2+-evoked hydrolysis of phosphoinositides with attendant increases in the levels of the biologically active 1,4,5 isomer of inositol trisphosphate (IP3) that would be predicted to arise from such a receptor-mediated process. In the present studies we used HPLC and TLC, respectively, to quantify the high Ca2+-induced changes in various inositol phosphates, including the isomers of IP3, and phosphoinositides in bovine parathyroid cells prelabeled with [3H]inositol. In the absence of lithium, high Ca2+ dose dependently elevated the levels of inositol-1,4,5-trisphosphate [I(1,4,5)P3], with a maximal, 4- to 5-fold increase within 5 s; the levels of inositol 1,3,4-trisphosphate [I(1,3,4)P3] first rose significantly at 5–10 s and remained 5- to 10-fold elevated for at least 30 minutes. These changes were accompanied by reciprocal 29–36% decreases in PIP2 (within 5–10 s, the earliest time points examined), PIP (within 60 s), and PI (within 60 s). These results document that, as in other cells responding to more classic “Ca2+-mobilizing” hormones, the high Ca2+-evoked increases in inositol phosphates in bovine parathyroid cells arise from the hydrolysis of phosphoinositides, leading to the rapid accumulation of the active isomer of IP3. The latter presumably underlies the concomitant spike in the cytosolic calcium concentration (Cai) in parathyroid cells.Keywords:
Inositol trisphosphate
Inositol phosphate
Inositol trisphosphate receptor
Parathyroid chief cell
Second messenger system
In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.
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The metabolism of inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate was studied in a cytosolic fraction prepared from the bovine adrenal cortex. The activity of the partially purified inositol 1,4,5-trisphosphate 3-kinase was dependent on Ca2+/calmodulin, Mg2+, and pH, and was inhibited by 2,3-bisphosphoglycerate. The enzyme exhibited Michaelis-Menten behavior toward its two substrates, inositol 1,4,5-trisphosphate and ATP, with Km values of 0.42 μmol/L and 0.4 mmol/L, respectively. The presence of other inositolphosphate metabolizing enzymes in the cytosolic fraction was indicated by the appearance of additional inositol polyphosphates during prolonged incubation with inositol 1,4,5-trisphosphate. These included inositol 1,3,4-trisphosphate, inositol 1,3,4,6-tetrakisphosphate, and inositol pentakisphosphate. These findings are consistent with the rapid phosphorylation of inositol 1,4,5-trisphosphate to the 1,3,4,5- tetrakisphosphate by the calcium/calmodulin-dependent 3-kinase, and its subsequent conversion to inositol 1,3,4-trisphosphate and thence to inositol 1,3,4,6-tetrakisphosphate in angiotensin-stimulated bovine glomerulosa cells. The formation of inositol pentakisphosphate during prolonged incubations suggests that inositol 1,3,4,6-tetrakisphosphate is slowly phosphorylated and serves as a source of inositol pentakisphosphate in the adrenal. The metabolic conversion of inositol 1,4,5-trisphosphate to several higher inositol polyphosphates provides potential new messengers for intracellular regulation in agonist-stimulated target cells. Am J Hypertens 1989;2:387–394
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Rat hepatocytes respond to α-adrenergic stimulation by intracellular production of myo-inositol 1,4,5-trisphosphate (IP3) which stimulates the periodic release and reuptake of intracellular store (IS) Ca2+. The generation of these Ca2+ oscillations was investigated by simultaneously monitoring Ca2+ changes in the cytosol and IS by combined fluorescence microscopy and whole-cell patch clamp. Intracellular IP3 perfusion (1-50 μM in the pipette) produced three types of Ca2+ response: understimulation, oscillations and overstimulation, i.e. with Ca2+ levels not returning to baseline. In a total of 57 experiments, only three displayed oscillations during continuous IP3 infusion, in a narrow range of IP3 concentration centred around 5-8 μM in the pipette. In oscillating cells, cytosolic Ca2+ spikes were synchronized with transient Ca2+ depletions of the IS, consistent with a direct exchange of Ca2+ between the two compartments. Application of 8-Br-cAMP to cells infused with IP3 increased the probability of eliciting Ca2+ oscillations by a factor of 4-5 for IP3 concentrations in the range 1-10 μM, whereas IP3 concentrations above 10 μM always resulted in overstimulation. IP3 photorelease experiments and measurements of IS Ca2+ content indicated that 8-Br-cAMP enhanced the affinity of the IP3 receptor and increased the pool of releasable Ca2+. We propose that cAMP has a permissive role in the generation of IP3-induced Ca2+ oscillations by extending the window of IP3 concentrations able to elicit oscillations.
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Receptors that mobilize calcium stimulate the hydrolysis of phosphoinositides. Addition of 5-HT to the fly salivary gland causes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). The resulting increases in inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (I P2) that begin to accumulate with no apparent delay is fast enough to account for the calcium-dependent component of the physiological response. In contrast, the levels of inositol 1-phosphate (I P) and free inositol begin to rise with lag periods of 20 and 60 seconds respectively. This analysis of how fast these inositol phosphates accumulate following stimulation is consistent with the idea that the primary action of hormones is to hydrolyse PIP2 to diacylglycerol and IP3. The idea that IP3 may function as a second messenger to mobilize intracellular calcium has been supported by observations on pancreatic and liver cells with 'leaky' plasma membranes. Addition of IP3 in the micromolar range induces a release of calcium from an internal membrane pool that is most likely located in the endoplasmic reticulum. The effect of IP3 appears to be specific in that there was no release in response to IP2, IP or inositol 1,2-cyclic phosphate. It is proposed that the IP3 might be the long sought-after second messenger that functions to mobilize intracellular calcium.
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Abstract We previously showed that high extracellular calcium (Ca2+) concentrations raise the levels of inositol phosphates in bovine parathyroid cells, presumably via the G protein-coupled, “receptor-like” mechanism through which Ca2+ is thought to regulate these cells. To date, however, there are limited data showing Ca2+-evoked hydrolysis of phosphoinositides with attendant increases in the levels of the biologically active 1,4,5 isomer of inositol trisphosphate (IP3) that would be predicted to arise from such a receptor-mediated process. In the present studies we used HPLC and TLC, respectively, to quantify the high Ca2+-induced changes in various inositol phosphates, including the isomers of IP3, and phosphoinositides in bovine parathyroid cells prelabeled with [3H]inositol. In the absence of lithium, high Ca2+ dose dependently elevated the levels of inositol-1,4,5-trisphosphate [I(1,4,5)P3], with a maximal, 4- to 5-fold increase within 5 s; the levels of inositol 1,3,4-trisphosphate [I(1,3,4)P3] first rose significantly at 5–10 s and remained 5- to 10-fold elevated for at least 30 minutes. These changes were accompanied by reciprocal 29–36% decreases in PIP2 (within 5–10 s, the earliest time points examined), PIP (within 60 s), and PI (within 60 s). These results document that, as in other cells responding to more classic “Ca2+-mobilizing” hormones, the high Ca2+-evoked increases in inositol phosphates in bovine parathyroid cells arise from the hydrolysis of phosphoinositides, leading to the rapid accumulation of the active isomer of IP3. The latter presumably underlies the concomitant spike in the cytosolic calcium concentration (Cai) in parathyroid cells.
Inositol trisphosphate
Inositol phosphate
Inositol trisphosphate receptor
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