Role of calcium and phosphoinositides in the actions of certain hormones and neurotransmitters.
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Animals↗
Biomedical subjects
J H Exton
Publications and source records attributed to J H Exton.
The relationships between receptor binding capacity for norepinephrine, angiotensin II, and vasopressin and release of inositol trisphosphate, Ca2+ mobilization, and phosphorylase activation in rat liver.
Concentration-response relationships for norepinephrine-, angiotensin II-, and vasopressin-stimulated changes in cell Ca2+ content, phosphorylase activation, and cytosolic free Ca2+ and myo-inositol-P3 levels were examined in isolated hepatocytes. The specific binding of radioligands to alpha 1-adrenergic, vasopressin, and angiotensin II receptors was also examined in rat liver plasma membranes. Disparities occurred between the concentration-response curves for myo-inositol-P3 formation and the Ca2+ and phosphorylase responses, with the greatest difference being observed with vasopressin and the smallest with norepinephrine. It was also observed that all three agonists produced the same maximum changes in phosphorylase, cell Ca2+, and cytosolic Ca2+, but the maximum capacity of each agonist to generate myo-inositol-P3 varied greatly and was correlated with the maximum receptor binding capacity. The data indicated that a very small and submaximal elevation of myo-inositol-P3 was sufficient to maximally elevate cytosolic Ca2+ and activate phosphorylase. In addition, the relationship between the accumulation of myo-inositol-P3 and the elevation of cytosolic Ca2+ was similar, irrespective of whether the agonist was norepinephrine, angiotensin II, or vasopressin. It is proposed that the large differences between the concentration-response curves for myo-inositol-P3 formation and Ca2+ and phosphorylase changes observed with vasopressin and angiotensin II are due to the higher density of their receptors on liver cell plasma membranes compared with alpha 1-adrenergic receptors.
Angiotensin II↗
Multiple phosphorylation of rat-liver glycogen synthase by protein kinases.
The phosphorylation sites in liver synthase were studied using gel filtration and high performance liquid chromatography of 32P-labeled tryptic peptides. Phosphorylase b kinase, calmodulin-dependent glycogen synthase kinase and glycogen synthase kinase 4 from liver phosphorylated the same low Mr tryptic peptide. cAMP-dependent protein kinase mainly phosphorylated the low Mr tryptic peptide, but also incorporated phosphate into two other peptides. Glycogen synthase kinase 5 phosphorylated a single tryptic peptide, whereas glycogen synthase kinase 3 phosphorylated several tryptic peptides. Calcium-phospholipid-dependent protein kinase phosphorylated two tryptic peptides, the major one of which had the same chromatographic properties as the low Mr peptide described above. These findings confirm that liver glycogen synthase undergoes multi-site phosphorylation and suggest that the topography of the sites is generally similar to that in muscle glycogen synthase.
Animals↗
The Mg2+ requirements of nonactivated and activated rat liver phosphorylase kinase. Inhibition of the activated form by free Mg2+.
Incubation of rat liver phosphorylase kinase in the presence of MgATP results in a time-dependent increase in activity, i.e., activation. Determination of the magnitude of activation depends, in large part, on the relative concentrations of Mg2+ and ATP used in the phosphorylase kinase activity assay, such that as the Mg2+ to ATP ratio increases less activation is detectable. Prior to activation, maximal activity of nonactivated phosphorylase kinase requires a 2-3-fold molar excess of Mg2+ (i.e., free Mg2+) over ATP. MgATP-dependent activation of the enzyme results in an alteration in the free Mg2+ requirement such that the activity of the activated enzyme is sharply inhibited by the free cation. Inhibition by free Mg2+ of the activated enzyme is rapidly reversed by removal of free Mg2+ but is not affected by addition of Ca2+. Both nonactivated and activated forms of the enzyme appear to be inhibited by free ATP4-. The results show that the use of high concentrations of free Mg2+ in the phosphorylase kinase activity assay can blunt or completely obscure changes in enzyme activity following activation of the enzyme.
Adenosine Triphosphate↗
Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases.
A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.
Animals↗
Vasopressin-, angiotensin II-, and alpha 1-adrenergic-induced inhibition of Ca2+ transport by rat liver plasma membrane vesicles.
A rapid method for isolating highly purified rat liver plasma membrane vesicles using isotonic medium and Percoll self-forming gradient centrifugation is described. The vesicles were characterized by enzyme markers and electron microscopy. The method also yielded a fraction rich in nuclei. The vesicles transported Ca2+ in an ATP-dependent manner and this was enhanced by oxalate. The Vmax for Ca2+ uptake was 0.65 +/- 0.08 nmol/mg X min, which was approximately 18-fold higher than for other liver plasma membrane preparations, and the Km for Ca2+ was 5.2 +/- 0.4 nM. Calcium uptake was inhibited by 40-50% in vesicles isolated from rat livers perfused for 3 min with 10(-7)M vasopressin. The half-maximally effective concentration of vasopressin was 5 X 10(-10)M which correlates with that for raising cytosolic Ca2+ and phosphorylase a. Inhibition was not significant in vesicles from livers perfused with vasopressin for only 1 min, indicating that inhibition of the Ca2+ pump may not be involved in the rise in cytosolic Ca2+ observed at 1-2 s with this hormone. Epinephrine (10(-5)M) and angiotensin II (10(-7)M) inhibited Ca2+ uptake by 31 +/- 10 and 26 +/- 5%, respectively, at 3 min. Glucagon (10(-7)M) had no effect. It is proposed that the inhibitory action of the Ca2+-dependent hormones on the plasma membrane Ca2+ pump plays an important role in the actions of these hormones by prolonging the elevation in cytosolic Ca2+.
Angiotensin II↗
Potentiation of alpha 1-adrenergic responses in rat liver by a cAMP-dependent mechanism.
Treatment of isolated hepatocytes with the alpha 1-adrenergic agonist norepinephrine induced a dose-dependent increase in free cytosolic Ca2+, as judged by fluorescence increases, in cells loaded with the Ca2+ indicator (2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8 -bis [carboxymethyl]aminoquinoline (quin-2). Pretreatment with either glucagon or dibutyryl cAMP increased the rate and magnitude of the quin-2 fluorescence response in hepatocytes treated with submaximal doses of norepinephrine and increased the cell sensitivity such that a physiological concentration of norepinephrine (7.5 nM) was able to provoke a quin-2 fluorescence response. Similar enhancement of norepinephrine-induced phosphorylase activation and pyridine nucleotide reduction in isolated hepatocytes and Ca2+ efflux from the perfused liver was also observed in the presence of glucagon. These potentiated responses correlated with a cAMP-dependent increase (mediated by glucagon, dibutyryl cAMP, or forskolin) in the binding of [3H]norepinephrine or [3H]epinephrine to sites present on isolated hepatocytes bearing the characteristics of alpha 1-adrenergic receptors. The data suggest that a cAMP-dependent mechanism is involved in the regulation of alpha 1-agonist binding to liver cells and, thereby, in the control of hepatic carbohydrate metabolism in response to catecholamines.
Animals↗
Characterisation of the alpha 1-adrenergic control of hepatic cAMP in male rats.
alpha 1-Adrenergic agonists characteristically elicit a mobilization of intracellular Ca2+ in rat liver. These agents also induced accumulation of cAMP in mature male rats (greater than 300 g body weight) and in Ca2+-depleted hepatocytes from 200 g rats although not in Ca2+-depleted cells from juvenile (less than 100 g) male rats. Comparison of these two responses revealed a similar agonist potency order in both cases, although cAMP accumulation was approximately 5-fold less sensitive to agonists. A variety of alpha-antagonists, including prazosin, phenoxybenzamine and dihydroergocryptine were equipotent as inhibitors of each response, although the alpha 1-adrenergic cAMP response was more sensitive to inhibition by WB-4101 and phentolamine. These data are discussed and a model proposed whereby in mature male rats, the same alpha 1-adrenergic receptor population becomes simultaneously coupled to two separate signal transduction mechanisms, namely Ca2+ mobilization and cAMP generation.
Adrenergic alpha-Agonists↗
Studies on the mechanism of inhibition of hepatic cAMP accumulation by vasopressin.
Vasopressin elicited a dose-dependent inhibition of glucagon-induced cAMP accumulation in isolated hepatocytes. This response was not diminished by incubation of cells with the calmodulin antagonists trifluoperazine or chlorpromazine and was only slightly reduced in Ca2+-depleted hepatocytes. Half-maximal inhibition of cAMP accumulation occurred at 8 X 10(-11) M vasopressin, a dose which does not increase cytosolic Ca2+ in hepatocytes. Direct activation of adenylate cyclase by forskolin was significantly inhibited by vasopressin in Ca2+-depleted cells. It is concluded that inhibition of hormone-induced cAMP accumulation by vasopressin in liver is not dependent on cellular Ca2+ mobilisation but may involve direct inhibition of adenylate cyclase.
Adenylyl Cyclases↗
Time course of alpha1-adrenergic and vasopressin actions on phosphorylase activation, calcium efflux, pyridine nucleotide reduction, and respiration in hepatocytes.
Calcium efflux from rat liver perfused with nonrecirculating medium was observed at 1.4 s following 10(-6) M (-)epinephrine infusion, when the perfusate Ca2+ was 60 microM. Net calcium efflux was also seen in livers perfused with 1.3 microM Ca2+ at approximately 8 s. In isolated rat hepatocytes, phosphorylase, a cytosolic enzyme, was activated significantly at 3 s and maximally at approximately 15 s by phenylephrine (10(-5) M), epinephrine (10(-6) M), and vasopressin (10(-8) M). Hexose phosphates were elevated at between 3 and 6 s with vasopressin. Phenylephrine and vasopressin stimulated hepatocyte respiration relatively slowly. The effects took 10 s to become evident, were dependent on the presence of Ca2+, and were probably the result of increased total cellular reduced pyridine nucleotide observed at 5 s. The slowness of the increase in respiration indicates that it cannot be the cause of the Ca2+ mobilization, but is more likely to be a consequence of it. From these studies, it is proposed that, following binding of catecholamines to alpha 1-adrenergic receptors, Ca2+ is first mobilized from the plasma membrane resulting in an elevation of the free Ca2+ ion concentration in the cytosol (Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H. (1983) J. Biol. Chem. 258, 8769-8773) which stimulates phosphorylase kinase and, hence, phosphorylase. These events begin to occur within the first 2 to 3 s. Following this, the concentration of reduced pyridine nucleotide(s) increases at 5 s resulting in the stimulation of respiration seen at 10 s. These events occur more slowly than the mobilization of cell Ca2+ and activation of phosphorylase, and may be secondary to the rise in cytosolic Ca2+. The time at which mitochondrial Ca2+ decreases is not known, but it accounts for most of the Ca2+ mobilized.
Animals↗
Changes in free cytosolic Ca2+ in hepatocytes following alpha 1-adrenergic stimulation. Studies on Quin-2-loaded hepatocytes.
The Ca2+ selective fluorescent indicator, Quin-2, was employed to monitor continuously the concentration of free cytosolic Ca2+ [ Ca2+ ]i in isolated rat hepatocytes. Epinephrine (10(-6) M) and phenylephrine (10(-5) M), acting via alpha 1-adrenergic receptors, increases [ Ca2+ ]i from a basal concentration of approximately 0.2 microM to approximately 0.6 microM. This increase in [ Ca2+ ]i is evident as early as 1 to 1.5 s, the earliest time so far reported for any hepatic alpha 1-adrenergic event. Vasopressin (10(-8) M), after a lag which is 2 to 3 s longer, increases [ Ca2+ ]i to the same extent and at the same rate as the alpha 1-adrenergic agonists. Glucagon (10(-8) M) also increases [ Ca2+ ]i but at a significantly slower rate and only after a lag of about 10 s. All of these agents also induce an increase in the fluorescence of control cells. This Quin-2 independent fluorescence, which is due to an increased reduction of pyridine nucleotides, must be corrected for before the maximum change in [ Ca2+ ]i can be calculated but is sufficiently slow so as not to contribute to the initial rate of increase in the Quin-2-dependent fluorescence.
Adrenergic alpha-Agonists↗
Effect of micromolar concentrations of manganese ions on calcium-ion cycling in rat liver mitochondria.
The effects of micromolar concentrations of Mn2+ on the rat liver mitochondrial Ca2+ cycle were investigated. It was found that the addition of Mn2+ to mitochondria which were cycling 45Ca2+ led to a rapid dose dependent decrease in the concentration of extramitochondrial 45Ca2+ of about 1 nmol/mg of protein. The effect was complete within 30 s, was half maximal with 10 microM Mn2+ and was observed in the presence of 3 mM Mg2+ and 1 mM ATP. It occurred over a broad range of incubation temperatures, pH and mitochondrial Ca2+ loads. It was not observed when either Mg2+ or phosphate was absent from the incubation medium, or in the presence of Ruthenium Red. These findings indicate that micromolar concentrations of Mn2+ stimulate the uptake of Ca2+ by rat liver mitochondria, and provide evidence for an interaction between Mg2+ and Mn2+ in the control of mitochondrial Ca2+ cycling.
Animals↗
Regulation of rat liver glycogen synthase. Roles of Ca2+, phosphorylase kinase, and phosphorylase a.
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Adenosine Triphosphate↗
Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats.
Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.
Aging↗
Modulation of the alpha 1-adrenergic control of hepatocyte calcium redistribution by increases in cyclic AMP.
The Ca2+ content of hepatocytes from juvenile male rats (80-110 g) or adult female rats (135-155 g) displayed a biphasic dose-response curve to epinephrine. Low concentrations (less than or equal to 10(-7) M) caused efflux of Ca2+ from the cells, while higher concentrations (10(-6) M and 10(-5) M) induced net Ca2+ uptake which correlated with a large beta 2-adrenergic-mediated increase in cAMP (Morgan, N. G., Blackmore, P. F., and Exton, J. H. (1983) J. Biol. Chem. 258, 5103-5109). Calcium accumulation could be induced in cells from older male rats (180-230 g) by combining a Ca2+-mobilizing hormone with either exogenous cAMP or glucagon (10(-8) M). Readdition of Ca2+ in the presence of glucagon to cells treated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also resulted in enhanced Ca2+ accumulation compared with controls. Addition of vasopressin plus glucagon to the medium perfusing male rat livers also led to cell Ca2+ accumulation, as evidenced by uptake of Ca2+ from the perfusate. Incubation of hepatocytes with antimycin A, oligomycin, and carbonyl cyanide m-chlorophenylhydrazone prevented net Ca2+ accumulation suggesting that mitochondria play a role in the uptake response. This was confirmed by isolation of mitochondria from cells incubated under conditions which promote Ca2+ accumulation. Within 5 min of incubation, the Ca2+ content of these mitochondria was increased 2-fold relative to controls, an effect which was inhibited by oligomycin. These studies demonstrate that a rise in hepatic cAMP can reverse hormonally induced Ca2+ mobilization and point to a major role for the mitochondria in this effect.
Angiotensin II↗
Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in hepatocytes by vasopressin.
Hepatocyte phosphatidylinositol 4,5-bisphosphate (4,5-P2), phosphatidylinositol 4-phosphate (4-P), and phosphatidylinositol were labeled with 3H when rats were injected intraperitoneally with 200 microCi of [2-3H] myo-inositol 18 h previously. Phosphatidylinositol 4,5-P2 and phosphatidylinositol 4-P accounted for 0.84 +/- 0.06 and 7.48 +/- 0.36%, respectively, of the total [3H] myo-inositol containing phospholipids. The breakdown of phosphatidylinositol 4,5-P2 was stimulated transiently (maximum effect seen at 15 s) and in a Ca2+-dependent manner by 10(-8) M vasopressin. Phosphatidylinositol 4-P breakdown was enhanced to a smaller, but longer, extent by vasopressin, whereas no changes in phosphatidylinositol were detected up to 120 s. Subcellular fractionation studies also showed no preferential breakdown of phosphatidylinositol in plasma membranes at 5-20 min. Only doses of vasopressin (10(-8) and 10(-7) M) in excess of those producing maximum effects on phosphorylase activation and Ca2+ efflux (10(-9) M) were effective at stimulating phosphatidylinositol 4,5-P2 breakdown. It is concluded that phosphatidylinositol 4,5-P2 breakdown induced by vasopressin in rat hepatocytes is not responsible for the mobilization of Ca2+ which leads to the activation of phosphorylase. On the contrary, it is Ca2+-dependent and appears to require the occupation of more receptors than are required for Ca2+ mobilization and phosphorylase activation.
Animals↗
Angiotensin II inhibits hepatic cAMP accumulation induced by glucagon and epinephrine and their metabolic effects.
Incubation of isolated hepatocytes containing normal Ca2+ levels with angiotensin II, vasopressin or A23187 caused significant inhibition of the cAMP response to glucagon. Angiotensin II also inhibited cAMP accumulation induced by either glucagon or epinephrine in Ca2+-depleted hepatocytes. When submaximal doses of hormone were employed such that cell cAMP was elevated only 3-4-fold (approximately 2 pmol cAMP/mg wet wt cells) inhibition by angiotensin II was correlated with a decrease in phosphorylase activation. The data demonstrate that inhibition of hepatic cAMP accumulation results in reduced metabolic responses to glucagon and epinephrine and do not support the contention that the hepatic actions of glucagon are independent of cAMP.
Angiotensin II↗
Stimulation of hepatic glycogenolysis by alpha 1- and beta 2-adrenergic agonists. Evidence against short term agonist-induced desensitization of the responses.
Addition of alpha 1-adrenergic agonists or vasopressin to the medium perfusing rat livers was associated with a rapid efflux of Ca2+, which was rapidly reaccumulated on removal of the stimulus. The magnitudes of the respective Ca2+ efflux and influx responses were similar, suggesting that Ca2+ was being mobilized from, and then reaccumulated into, the same pool(s). Addition of combinations of alpha 1-agonists, vasopressin and angiotensin II to isolated hepatocytes revealed that the Ca2+ efflux response induced by each individual hormone could only be augmented by addition of a second hormone when submaximal doses of each were employed. This suggests that an identical pool(s) of hepatocyte Ca2+ is mobilized in response to each of these agents. No desensitization of the alpha 1-adrenergic glycogenolytic response was observed in the perfused liver, upon repeated or more prolonged (25 min) exposure to agonist, providing cell Ca2+ reaccumulation occurred before addition of each successive stimulus. Selective short term stimulation of the beta 2-adrenergic glycogenolytic response in immature male rats (less than 150 g, body weight) was not associated with desensitization to subsequent stimulation. These data demonstrate that the glycogenolytic response of liver cells does not become desensitized by successive short term stimulation with alpha 1- or beta 2-adrenergic agonists.
Angiotensin II↗