Receptor regulation of phosphoinositide and calcium metabolism.
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Animals↗
Biomedical subjects
J W Putney
Publications and source records attributed to J W Putney.
Size of the inositol 1,4,5-trisphosphate-sensitive calcium pool in guinea-pig hepatocytes.
Permeabilized hepatocytes accumulated 45Ca2+ into a non-mitochondrial pool when provided with ATP. 45Ca2+ efflux from this pool was revealed by removal of ATP with glucose and hexokinase or by inhibiting uptake with NaVO3. The effect of inositol 1,4,5-trisphosphate (IP3) on 45Ca2+ efflux from the pool was investigated. IP3 (5 microM) evoked a rapid increase in the rate of 45Ca2+ efflux. Kinetic analysis of the effect of IP3 indicated the existence of two distinct Ca2+ fractions within the pool; only one, accounting for about one-third of the ATP-dependent Ca2+ content of the pool, was responsive to IP3. The effect of IP3 on 45Ca2+ efflux from the non-mitochondrial pool does not require ATP, a finding that is inconsistent with a previous suggestion that this effect may be mediated by protein phosphorylation.
Adenosine Triphosphate↗
Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.
The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.
Angiotensin II↗
Properties of receptor-controlled inositol trisphosphate formation in parotid acinar cells.
Activation of muscarinic receptors in rat parotid cells results in breakdown of polyphosphoinositides liberating inositol phosphates, including inositol trisphosphate. Formation of inositol trisphosphate appears independent of agonist-induced Ca2+ mobilization, since neither formation nor degradation of inositol trisphosphate are appreciably altered in low-calcium media, and elevation of cytosolic Ca2+ with a calcium ionophore does not cause an increase in cellular inositol trisphosphate. Further, activation of substance P receptors and alpha 1-adrenoreceptors, but not beta-adrenoreceptors, increases inositol trisphosphate formation. The dose-response curve for methacholine activation of inositol trisphosphate formation more closely approximates the curve for receptor occupancy than for Ca2+-activated K+ release. These results are all consistent with the suggestion that inositol trisphosphate could function as a second messenger linking receptor occupation to cellular Ca2+ mobilization.
Animals↗
Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes.
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.
Adenosine Triphosphate↗
Inositol 1,4,5-trisphosphate may be a signal for f-Met-Leu-Phe-induced intracellular Ca mobilisation in human leucocytes (HL-60 cells).
Permeabilised, dimethyl sulphoxide-differentiated HL-60 human myelomonocytic leukemia cells accumulate 45Ca in an ATP-dependent manner. The 45Ca is taken up by a pool thought to be a component of the endoplasmic reticulum. Inositol trisphosphate induced a rapid release of Ca from this pool, suggesting that this molecule which is formed in these cells in response to f-Met-Leu-Phe may play a role in agonist-induced Ca metabolism.
Adenosine Triphosphate↗
Secretagogue-induced phosphoinositide metabolism in human leucocytes.
The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.
Calcium↗
Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate.
The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.
Animals↗
Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate.
Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.
Animals↗
Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.
The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.
Animals↗
Alpha-adrenergic stimulation of potassium efflux in guinea-pig hepatocytes may involve calcium influx and calcium release.
Either 86Rb or 42K appears to be a useful marker for monitoring the movement of cellular K in dispersed guinea-pig hepatocytes. Alpha-adrenergic stimulation of perifused hepatocytes causes a biphasic increase in 86Rb or 42K efflux from hepatocytes previously equilibrated with radio-isotope. The first phase is a large transient (about 5 min) increase which is followed by a slowly falling phase of release. Alpha-adrenergic stimulation of hepatocytes perifused with medium containing no added Ca plus 0.1 mM-EGTA evokes only the transient increase in 86Rb efflux. The addition of Ca to the medium in the continued presence of agonist restores the second phase of the response. Both phases of the response appear to be mediated by alpha 1-receptors. The magnitude of the second phase is dependent upon the concentration of Ca added to the perifusion medium. Other agonists that are believed to act by mobilizing Ca give similar results in this system. Angiotensin II, ATP and A23187 stimulate a transient increase in 86Rb efflux without extracellular Ca present, with the second phase of the response appearing upon the addition of Ca to the medium. These results suggest that the initial transient phase of 86Rb efflux, which is independent of extracellular Ca, is stimulated by Ca released from an intracellular pool. The second phase, which occurs only in the presence of extracellular Ca, is probably a result of Ca influx into the cell.
Adenosine Triphosphate↗
Phorbol ester-induced protein secretion in rat parotid gland. Relationship to the role of inositol lipid breakdown and protein kinase C activation in stimulus-secretion coupling.
When added to rat parotid gland slices incubated in vitro, 4 alpha-phorbol-dibutyrate (PDBu) induced a dose-dependent increase in protein secretion, but did not affect membrane permeability to K+ (as determined by 86Rb efflux). The response to PDBu was unaffected by the removal of extracellular Ca2+ and was not markedly potentiated by incubation with the phosphodiesterase inhibitor, methylisobutylxanthine. PDBu did not activate phospholipase C breakdown of inositol lipids as shown by a failure to increase formation of soluble inositol phosphates. When applied in combination with the Ca2+ ionophore, ionomycin, a secretory rate was obtained that was greater than the predicted sum of rates obtained when the two drugs were given alone. These results, when taken with the reported results of others, are consistent with an action of PDBu in activating protein kinase C and suggest that this enzyme plays an important role in the pathway linking receptor activation to protein secretion, but not K+ flux, in the parotid gland.
Animals↗
Calcium pools in saponin-permeabilized guinea pig hepatocytes.
The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.
Adenosine Triphosphate↗
Stimulation of glycogenolysis in hepatocytes by angiotensin II may involve both calcium release and calcium influx.
The stimulation of [3H]glucose release (a measure of glycogenolysis) from isolated guinea pig hepatocytes by Ca-mobilizing agonists can be resolved into two phases. The initial transient phase is independent of extracellular Ca, and is probably a result of Ca released from an intracellular pool. The second phase occurs only in the presence of extracellular Ca, which suggests that Ca-influx is also involved in the mechanism of Ca-mobilization by these agents in the guinea pig hepatocyte.
Angiotensin II↗
Effects of secretagogues on [32P]phosphatidylinositol 4,5-bisphosphate metabolism in the exocrine pancreas.
Experiments were carried out to assess the effects of secretagogues on the polyphosphoinositides phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] on preparations of exocrine pancreas in vitro. Carbachol and caerulein provoked a rapid (less than 1 min) breakdown of 15-20% of [32P]PtdIns(4,5)P2 in isolated pancreatic acini, but did not affect [32P]PtdIns4P. In contrast, the Ca2+ ionophore ionomycin had no immediate effect on the levels of either inositide but caused a parallel fall in both lipids after 5-10 min. A similar decrease in [32P]PtdIns(4,5)P2 due to carbachol was obtained with isolated acini and isolated cells, despite the fact that the secretory response of isolated cells was considerably less than that of isolated acini. Loss of [32P]PtdIns(4,5)P2 elicited by carbachol or caerulein was unaffected either by the addition of EGTA in excess of extracellular Ca2+ or when a protocol was employed that eliminated caerulein-induced intracellular Ca2+-release. These results suggest that agonist-induced PtdIns(4,5)P2 breakdown in the exocrine pancreas may be an early step in the stimulus-response coupling pathway and also suggest that this breakdown is not dependent on Ca2+-mobilization.
Animals↗
Activation by calcium of membrane channels for potassium in exocrine gland cells.
In the rat parotid salivary gland, fluid secretion is regulated by alterations in fluxes of monovalent ions. In vitro, stimulation of muscarinic, alpha-adrenergic or substance P receptors provokes a biphasic increase in membrane permeability to K+ which can be conveniently assayed as efflux of 86Rb. The increased 86Rb flux is thought to arise in response to a receptor mediated elevation in [Ca2+]i which activates Ca2+-activated K+-channels. The biphasic nature of the response is presumably due to a biphasic mode of Ca2+ mobilization by secretagogues; a transient response reflects release of a finite pool of Ca from an intracellular store while a more sustained phase results from Ca entry through receptor operated Ca channels or gates. Calcium also mediates an increased Na+ entry which in turn activates the Na+, K+-pump. The mechanism involved in the regulation of monovalent ion channels by Ca2+ is not understood.
Animals↗
Ionic mechanisms in secretagogue-induced morphological changes in rat parotid gland.
When 10(-5) M carbachol was added to parotid tissue slices incubated in buffer containing Ca++, watery vacuoles were formed in the cells. The percent volume density of vacuoles, as measured from 0.5-micron sections, increased from 0.64 +/- 0.15 SE (n = 7) to 3.09 +/- 0.99 (n = 5) in 10 min and, finally, to 7.27 +/- 1.88 (n = 4) in 30 min. In electron micrographs, most of the vacuoles appeared to arise from a location near the Golgi apparatus. Condensation of nuclear chromatin and a conformational change in mitochondria were also noted immediately after stimulation. The percent volume density values returned to basal levels with the addition of either 5 mM EGTA or 10(-6) M atropine after the addition of carbachol. Nuclei and mitochondria returned to normal configurations. In the presence of either 1 mM ouabain or high K+, or in the absence of added Ca++, carbachol failed to induce vacuole formation. However, low Na+ medium did not prevent the formation of vacuoles due to carbachol. Ultrastructural changes in nuclei and mitochondria were consistently associated with the appearance of vacuoles. Since both high K+ and ouabain blocked vacuole formation, it is unlikely that Na+ or K+ movements were important for the response. Rather, receptor-activated Ca++ influx, which is likely to be inhibited by depolarizing agents (such as high K+ or ouabain), is probably the more important factor in vacuole formation and other concomitant ultrastructural changes.
Animals↗
Effects of antimycin A on receptor-activated calcium mobilization and phosphoinositide metabolism in rat parotid gland.
The effects of the mitochondrial poison, antimycin A, on responses of parotid acinar cells to cholinergic stimuli were examined. Antimycin A (10 microM) partially inhibited the agonist-induced increase in 86Rb efflux. Specifically, the initial transient phase of the response, believed to arise from intracellular calcium release, was partially inhibited, while the sustained phase of the response, believed to result from calcium entering from the extracellular space, was completely blocked. The stimulation of 45Ca influx by a cholinergic agonist was also completely blocked. Antimycin A (10 microM) caused a rapid loss of [32P] polyphosphoinositides. Stimulation of [32P]phosphatidylinositol breakdown and [32P] phosphatidate synthesis by methacholine was blocked by antimycin A. Breakdown of [32P]phosphatidylinositol-4,5-bisphosphate in response to cholinergic stimulation was partially inhibited. These results indicated that the activation by cholinergic agonists of cellular calcium mobilization as well as effects on phosphoinositide metabolism are similarly inhibited by antimycin A. Furthermore, this presumably indicates a role for ATP in receptor-activated calcium mobilization and phosphoinositide turnover.
Animals↗