Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intracellular events that regulate parathyroid hormone (PTH) release are not well understood. Cyclic AMP (cAMP) and cAMP-dependent protein kinases play a role in the regulation of release due to several agonists, but these factors do not fully explain PTH release that is mediated by extracellular ionized calcium (Ca++). A calcium-phospholipid-dependent (non-cAMP-dependent) protein kinase can be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether this protein kinase regulates PTH release, we examined the effect of TPA on PTH release from human parathyroid tissue. Cell suspensions of abnormal parathyroid tissue removed at surgery were prepared by enzymatic dispersion and incubated for several hours with and without 10(-7) mol/L TPA at low and high calcium levels. In ten preparations in the absence of TPA, increasing Ca++ from 0.25 to 2.5 mmol/L reduced PTH release to an average of 39% of maximal release (range, 11% to 67%). The effect on TPA on Ca++-regulated PTH release appeared biphasic. At low (0.25 mmol/L) Ca++ level, TPA suppressed PTH release to an average of 78% of maximal release without TPA (95% confidence interval, 67% to 88%) (p less than 0.01 compared to cells incubated without TPA). At high (2.25 mmol/L) Ca++ level, TPA augmented PTH release from an average of 39% of maximal release without TPA to 62% of maximal release without TPA (95% confidence level, 48% to 78%), an average augmentation of 22% (95% confidence level, 9% to 36%) (p less than 0.01 compared with cells incubated without TPA). TPA appeared to make PTH release independent of Ca++. Both inhibitory and stimulatory effects were dose dependent. Incubations with TPA demonstrated no toxicity as judged by trypan blue dye exclusion, linearity of PTH release, and cellular incorporation of tritiated leucine. TPA had no effect on the radioimmunoassay for PTH. We conclude that a calcium/phospholipid-dependent, non-cAMP-dependent protein kinase may play a role in mediating Ca++-regulated PTH release from abnormal human parathyroid cells. Its site of action and integration with other regulatory pathways remain to be determined.
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PMID:The effect of phorbol ester on in vitro release of parathyroid hormone from abnormal human parathyroid cells. 368 56

Hormonal activation of cAMP-dependent protein kinase has been studied in cultured cells derived from a rat osteogenic sarcoma and in osteoblast-rich cells grown from newborn rat calvaria. Both cell strains contain adenylate cyclase activities which respond to parathyroid hormone (PTH) and a variety of prostanoids. PTH, prostaglandin E2 (PGE2), and prostacyclin (PGI2) were all capable of activating cAMP-dependent protein kinase(s) in suspensions of the two cell types. Activation was very rapid in all cases, being detectable at 10 sec and maximal between 30-60 sec. Using saturating concentrations of hormones, the protein kinase activity ratio remained elevated (between 0.6-0.9) for up to 35 min after the start of PGE2 stimulation, but declined toward basal activity ratio 5-10 min after stimulation with PTH or PGI2. Each of the hormones caused a dose-dependent increase in activation of cAMP-dependent protein kinase in both cell types. Half-maximal activation of the enzyme occurred at 2 X 10(-9) M bovine PTH for calvarial cells, at 10(-8) M bPTH for osteogenic sarcoma cells, and at 2-4 X 10(-8) M PGE2 and 1-3 X 10(-7) M PGI2 for both cell types. Maximal activation of protein kinase occurred before maximal cAMP accumulated, implying that only a fraction of cAMP is biologically significant. These two cell strains provide a useful means of analyzing postreceptor events in the hormonal regulation of bone cells.
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PMID:Activation of adenosine 3',5'-monophosphate-dependent protein kinase in normal and malignant bone cells by parathyroid hormone, prostaglandin E2, and prostacyclin. 625 86

Mouse renal cortical slices were incubated with parathyroid hormone (30 U/ml) for 2 min. Brush border membrane vesicles isolated from the treated slices had a decreased Na+ gradient-dependent uptake of phosphate. Concomitantly, the hormone elicited the activation of adenylate cyclase, the increase in tissue level of cAMP, and the enhancement of cAMP-dependent protein kinase.
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PMID:In vitro effects of parathyroid hormone on kidney cortical slices: cAMP responses and concomitant inhibition of the Na+ gradient-dependent uptake of phosphate by brush border membrane vesicles isolated from the renal slices. 627 24

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.
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PMID:Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport. 627 74

Parathyroid hormone, prostaglandin E2, and prostacyclin activate cAMP-dependent protein kinase in osteoblast-rich normal rat calvarial cells and in clonal rat osteogenic sarcoma cells of osteoblastic phenotype. The present study was undertaken to determine the activation of the enzyme in relation to cellular cAMP concentrations at increasing doses of the three hormones and also to test that the activity ratio measurement of the enzyme (ratio of the activity in the absence of cAMP to the activity in the presence of excess cAMP) was a true reflection of intracellular activation of the enzyme. With each hormone, using either normal or malignant osteoblasts, activation of the enzyme took place at hormone concentrations lower than those required to produce detectable changes in cAMP concentrations in the incubations. Stimulation of activity was abolished by addition of the heat-stable inhibitor of cAMP-dependent protein kinase, indicating that activation was of cAMP-dependent protein kinase alone. To demonstrate that protein kinase activation occurred intracellularly and not during sample preparation, charcoal was added at the time of cell disruption to absorb free cAMP. Under these conditions, no change was observed in the concentration of bovine parathyroid hormone required to cause activation of cAMP-dependent protein kinase. Finally, addition of purified cAMP-dependent protein kinase type I or type II to treated cells at the time of lysis did not result in significant activation of added isoenzyme, except at hormone concentrations sufficient to increase the total cAMP concentration of incubations. It is concluded that activity ratio measurement reflects the intracellular state of activation of cAMP-dependent protein kinase in the osteoblast-like cells treated by hormones and, furthermore, that only a fraction of the maximally generated cAMP is necessary for full enzyme activation.
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PMID:Activity ratio measurements reflect intracellular activation of adenosine 3',5'-monophosphate-dependent protein kinase in osteoblasts. 628 67

The regulation of endogenous protein phosphorylation by parathyroid hormone (PTH) was investigated using confluent monolayer cultures of chick kidney cells. Homogenates and subcellular fractions of PTH (bovine 1-34)-treated cells were subjected to an endogenous protein phosphorylation assay using ((gamma- 32P]ATP in the presence or absence of 2.0 microM cAMP or 0.5 mM Ca2+ with 25 micrograms/ml of phosphatidylserine and reactions terminated with sodium dodecyl sulfate. In other experiments, cultures were incubated in a phosphate-free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline containing 50 muCi/ml of [32P]PO4 and incubations were terminated with sodium dodecyl sulfate. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cyclic AMP stimulated 32P incorporation into proteins having molecular weights of 17,000, 22,000, 35,000, 42,000, 54,000, 75,000, 80,000, 120,000, and 143,000. Calcium-phosphatidylserine stimulated the phosphorylation of proteins of 20,000, 52,000, 58,000, 60,000, and 143,000. The protein phosphorylation patterns in cultured kidney cells and freshly isolated kidney tissue were quite similar. Treatment of cultured cells with 5-50 ng/ml of PTH resulted in stimulated phosphorylation of the 35,000 and 42,000 dalton proteins as assessed by endogenous phosphorylation in homogenates. In intact cells incubated with [32P]PO4, PTH stimulated most noticeably the phosphorylation of the 35,000-dalton protein. Based on studies with cultured and fresh kidney cells, the majority of the substrate proteins for cAMP and calcium-dependent protein kinases were located in the cytoplasm with the exception of the 42,000-dalton protein which was located in the brush-border-plasma membrane fraction. The cytoplasmic cAMP-dependent protein kinase activity was responsible for the majority of PTH-stimulated protein phosphorylation.
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PMID:Protein phosphorylation in chick kidney. Response to parathyroid hormone, cyclic AMP, calcium, and phosphatidylserine. 629 12

Obstructive uropathy is associated with changes in several renal functions. As some of these are mediated by cAMP, we examined whether changes in cAMP metabolism could contribute to the pathophysiology of unilateral ureteral ligation in rats. We found that basal adenylate cyclase in the cortex of the obstructed kidney doubled after 3-7 days, but the response to NaF and parathyroid hormone was decreased. Similarly, in vivo infusion of parathyroid hormone resulted in lower cAMP levels in the cortex of the obstructed kidney. Parathyroidectomy or pretreatment with beta-adrenergic blocker had no effect. Prostaglandin inhibition by indomethacin decreased, but did not abolish, the differences. On the other hand, basal adenylate cyclase in glomeruli from contralateral kidneys was higher. Pretreatment with indomethacin reduced the basal activity in contralateral glomeruli, and led to increased degrees of stimulation with parathyroid hormone and prostaglandins. Furthermore, we demonstrated that isolated glomeruli contain cAMP-dependent protein kinase that is found predominantly in the 30,000 g supernatant fraction and that by DEAE-cellulose chromatography has the characteristics of a type II kinase. In glomeruli from rats with unilateral ureteral ligation, the distribution and degree of activation of the cAMP-dependent protein kinase are affected differently on the ligated and contralateral sides, probably reflecting the change in glomerular cAMP generation. These results indicate that changes in cAMP metabolism may contribute to the altered cortical tubular function in unilateral ureteral ligation and may be partially related to enhanced prostaglandin synthesis in the obstructed kidney. On the other hand, the higher adenylate cyclase activity and the change in cAMP-dependent protein kinase activity in glomeruli from the contralateral kidney may in part reflect increased PGE2 production by these glomeruli. The results support the concept that similar to their in vitro action, locally generated prostaglandins may in vivo affect cortical tubular function by influencing the tubular cAMP system while those generated in the glomerulus alter glomerular cAMP metabolism.
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PMID:Changes in glomerular and cortical tubular cAMP metabolism in kidneys from rats with unilateral ureteral obstruction. 631 Jul 10

To ascertain whether cAMP-dependent phosphorylation could be demonstrated in brush border membrane vesicles (BBMV) isolated from kidneys of mice with X-linked hypophosphatemic rickets (HYP/Y) and normal littermates (+/Y) and, if so, to determine whether the absence of dephosphorylation might underlie differences in Na+-dependent 32Pi transport in BBMV, we measured 1) 32Pi transport, 2) cAMP-dependent phosphorylation, and 3) dephosphorylation in BBMV from +/Y and HYP/Y mice. Na+ gradient-dependent 32Pi transport was decreased in BBMV from HYP/Y mice as reflected in a decreased apparent Vmax. cAMP-dependent phosphorylation of a 62,000 Mr protein was demonstrated in sodium dodecyl sulfate polyacrylamide gels of BBMV from +/Y and HYP/Y mice and was associated with decreased Na+-dependent 32Pi transport. Dephosphorylation of the 62,000 Mr band was demonstrable in both types of membranes. Thus, both cAMP-dependent protein kinase and phosphoprotein phosphatase activities were demonstrable in BBMV isolated from +/Y and HYP/Y mice. These results are consistent with the renal tubular defect in the HYP/Y mouse reflecting an intrinsic abnormality of Pi transport in the brush border membrane independent from mediation of the phosphaturic effect of parathyroid hormone.
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PMID:Pi transport, phosphorylation, and dephosphorylation in renal membranes from HYP/Y mice. 666 Feb 93

Both metabolic acidosis and phosphorus (Pi) deprivation have been shown to alter not only renal Pi metabolism independent of parathyroid hormone (PTH), but also the phosphaturic response to PTH. In the present studies, we examined the interaction between metabolic acidosis and Pi deprivation on renal handling of Pi in an animal model in which metabolic acidosis was superimposed on 3 days of dietary Pi deprivation. The effect of metabolic acidosis was evaluated after acute (for 3 h) and chronic (for 3 days) administration of HCl. In Pi-deprived and thyroparathyroidectomized rats, chronic acidosis increased both plasma Pi and the basal fractional excretion of Pi (FEPi). Furthermore, chronic acidosis partially restored the phosphaturic response to PTH, which was totally absent in nonacidotic Pi-deprived rats. These effects metabolic acidosis were not demonstrable in acutely acidotic animals. Determination of PTH-dependent cAMP generation and cAMP-dependent protein kinase activation revealed that neither of these parameters was altered by chronic metabolic acidosis in vivo. These results show that in Pi-deprived rats chronic metabolic acidosis induced by HCl administration further modifies the renal handling of Pi associated with Pi deprivation. Further, the renal interaction between acidosis and Pi deprivation is at a step (or steps) after cAMP generation and protein kinase activation.
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PMID:Effect of metabolic acidosis on renal response to parathyroid hormone in phosphorus-deprived rats. 678 36

Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.
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PMID:Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells. 693 60


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