Protein kinase A, cytosolic calcium, and phosphate uptake in human proximal renal cells

1989 ◽  
Vol 257 (4) ◽  
pp. F631-F638
Author(s):  
J. P. Middleton ◽  
C. B. Dunham ◽  
J. J. Onorato ◽  
D. A. Sens ◽  
V. W. Dennis
Keyword(s):  
Protein Kinase ◽  
Phosphate Uptake ◽  
Human Kidney ◽  
Cytosolic Calcium ◽  
Intracellular Camp ◽  
Renal Cells ◽  
Camp Pathway ◽  
Half Maximal Effective Concentration ◽  
Dependent Protein ◽  
Short Term Culture

Phosphate uptake by proximal renal cells derived from the human kidney occurs by a saturable process that is approximately 85% dependent on the presence of sodium. Kinetic analysis is consistent with two distinct transport events with Km of 0.08 and 0.63 mM and Vmax of 3.4 and 11.0 nmol.mg-1.3 min-1, respectively. Parathyroid hormone (PTH), isoproterenol, and prostaglandin E2 (PGE2) increased cellular adenosine 3',5'-cyclic monophosphate (cAMP). PTH-stimulated cAMP prevented binding of the photolabel 8-azido[32P]cAMP with a half-maximal effective concentration (EC50) of 1 nM PTH, 30-fold lower than the EC50 for intracellular cAMP accumulation. These data are qualitatively similar to those observed in OK cells. PTH did not inhibit phosphate uptake in human cells, although it activated cAMP-dependent protein kinase and increased cytosolic calcium. Thus phosphate uptake in human proximal renal cells maintained in short-term culture is unresponsive to PTH in spite of increased cytosolic calcium and activation of the cAMP pathway.

Effect of calcium on transport characteristics of cultured proximal renal cells

1985 ◽  
Vol 249 (3) ◽  
pp. F346-F355
Author(s):  
L. M. Sakhrani ◽  
N. Tessitore ◽  
S. G. Massry
Keyword(s):  
Phosphate Uptake ◽  
Cytosolic Calcium ◽  
Ruthenium Red ◽  
Extracellular Calcium ◽  
Transport Processes ◽  
Renal Cells ◽  
Sodium Uptake ◽  
Calcium Loading ◽  
Sodium Dependent ◽  
Acute Changes

We examined the effects of acute changes in extracellular and intracellular calcium on transport processes in primary culture of proximal rabbit renal cells. A change in extracellular calcium from 0 to 3 mM inhibited amiloride-sensitive sodium uptake by 30%, and this effect was maximal at 1 mM calcium. Other polyvalent cations (Mn2+, Mg2+, La3+, and Ba2+) produced quantitatively similar inhibition of amiloride-sensitive sodium uptake compared with calcium. An increase in cytosolic calcium produced by calcium loading (20 mM) or by A23187 (20 microM) resulted in an inhibition of 25-40% of amiloride-sensitive sodium uptake. Moreover, quinidine (10(-4)M) and ruthenium red (3 microM), agents presumed to increase cytosolic calcium, inhibited amiloride-sensitive sodium uptake by 20-60%. Both these agents also inhibited sodium-dependent phosphate uptake by 20% but had no effect on ouabain-sensitive 86Rb+ uptake or on sodium-dependent alpha-methylglucoside uptake. Our data indicate that increases in extracellular calcium inhibit amiloride-sensitive sodium uptake and increases in cytosolic calcium inhibit sodium-dependent phosphate and amiloride-sensitive sodium uptakes. The effect of extracellular calcium may be due to charge screening and/or binding to the negatively charged plasma membrane or due to alterations in membrane fluidity.

scholarly journals Interaction of PKC and NOS in signal transduction of microvascular hyperpermeability

1997 ◽  
Vol 273 (5) ◽  
pp. H2442-H2451 ◽  
Author(s):  
Qiaobing Huang ◽  
Yuan Yuan
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
No Production ◽  
Apparent Permeability ◽  
Apparent Permeability Coefficient ◽  
Dependent Protein ◽  
Transient Elevation ◽  
Oxide Synthase

Our previous studies have shown that inflammatory mediators increase microvascular permeability through a phospholipase C-nitric oxide synthase (NOS)-guanylate cyclase cascade. The aim of this study is to delineate in more detail the signaling pathway leading to microvascular hyperpermeability. Endothelial cytosolic calcium and the apparent permeability coefficient of albumin ( P a) were measured in isolated and perfused coronary venules. Histamine stimulated a rapid increase in cytosolic calcium followed by a transient elevation in P a. The NOS inhibitor N G-monomethyl-l-arginine (l-NMMA) and the guanosine 3′,5′-cyclic monophosphate-dependent protein kinase G (PKG) inhibitor KT-5823 abolished the hyperpermeability but did not affect the calcium response to histamine. Similarly, the calcium ionophore ionomycin produced a calcium spike preceding venular hyperpermeability. Blockage of the NOS-PKG cascade inhibited the increase in P a, whereas the endothelial calcium was still elevated on administration of ionomycin. Furthermore, the relationship between protein kinase C (PKC) and the calcium-NOS-PKG pathway in modulation of venular permeability was investigated. Stimulation of PKC with phorbol 12-myristate 13-acetate (PMA) dramatically increased basal P a without significantly changing the cytosolic calcium level. The selective PKC inhibitor bisindolylmaleimide abolished the effect of PMA but did not alter the effect of histamines on P a. In contrast, both l-NMMA and KT-5823 were able to greatly attenuate the increase in P a caused by PMA. These results suggest that 1) elevation of endothelial cytosolic calcium is an early signaling event preceding nitric oxide (NO) synthesis in the transduction of endothelial hyperpermeability, and 2) activation of PKC may alter the endothelial barrier function partially through the modulation of NO production.

Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells

1997 ◽  
Vol 272 (5) ◽  
pp. L865-L871 ◽  
Author(s):  
B. Tolloczko ◽  
Y. L. Jia ◽  
J. G. Martin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Airway Smooth Muscle Cells ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
High Concentrations

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

scholarly journals Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.

1993 ◽  
Vol 4 (10) ◽  
pp. 993-1002 ◽  
Author(s):  
A T Harootunian ◽  
S R Adams ◽  
W Wen ◽  
J L Meinkoth ◽  
S S Taylor ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Camp Dependent Protein Kinase ◽  
Block Movement ◽  
C Subunit ◽  
Dependent Protein

The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.

Phosphorylation of myristoylated alanine-rich C kinase substrate is involved in the cAMP-dependent amylase release in parotid acinar cells

2009 ◽  
Vol 296 (6) ◽  
pp. G1382-G1390 ◽  
Author(s):  
Keitaro Satoh ◽  
Miwako Matsuki-Fukushima ◽  
Bing Qi ◽  
Ming-Yu Guo ◽  
Takanori Narita ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Acinar Cells ◽  
Cellular Migration ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Amylase Release ◽  
Kinase Substrate ◽  
Parotid Acinar Cells ◽  
Dependent Protein ◽  
Rat Parotid

Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major cellular substrate for protein kinase C (PKC). MARCKS has been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as phagocytosis, endocytosis, and exocytosis. The involvement of MARCKS phosphorylation in secretory function has been reported in Ca2+-mediated exocytosis. In rat parotid acinar cells, the activation of β-adrenergic receptors provokes exocytotic amylase release via accumulation of intracellular cAMP levels. Here, we studied the involvement of MARCKS phosphorylation in the cAMP-dependent amylase release in rat parotid acinar cells. MARCKS protein was detected in rat parotid acinar cells by Western blotting. The β-adrenergic agonist isoproterenol (IPR) induced MARCKS phosphorylation in a time-dependent manner. Translocation of a part of phosphorylated MARCKS from the membrane to the cytosol and enhancement of MARCKS phosphorylation at the apical membrane site induced by IPR were observed by immunohistochemistry. H89, a cAMP-dependent protein kinase (PKA) inhibitor, inhibited the IPR-induced MARCKS phosphorylation. The PKCδ inhibitor rottlerin inhibited the IPR-induced MARCKS phosphorylation and amylase release. IPR activated PKCδ, and the effects of IPR were inhibited by the PKA inhibitors. A MARCKS-related peptide partially inhibited the IPR-induced amylase release. These findings suggest that MARCKS phosphorylation via the activation of PKCδ, which is downstream of PKA activation, is involved in the cAMP-dependent amylase release in parotid acinar cells.

A neurochemical mechanism for hypoxia-induced anapyrexia

2002 ◽  
Vol 283 (6) ◽  
pp. R1412-R1422 ◽  
Author(s):  
Alexandre A. Steiner ◽  
Maria J. A. Rocha ◽  
Luiz G. S. Branco
Keyword(s):  
Protein Kinase ◽  
Body Temperature ◽  
Preoptic Region ◽  
Dependent Protein Kinase ◽  
Hypoxia Exposure ◽  
Cgmp Dependent Protein Kinase ◽  
Camp Pathway ◽  
Male Wistar Rats ◽  
Dependent Protein ◽  
Camp And Cgmp

Hypoxia evokes a regulated decrease in body temperature, a response that has been termed anapyrexia, but the mechanisms involved are poorly understood. Therefore, the present study was undertaken to test the hypothesis that hypoxia-induced anapyrexia results from the activation of cAMP- and cGMP-dependent pathways in the preoptic region (PO). Adult male Wistar rats weighing 230–260 g were used. Body temperature was monitored by biotelemetry, and the levels of cAMP and cGMP were determined in the anteroventral third ventricular region (AV3V), where the PO is located. Using immunohistochemistry, we observed that the PO contains a high density of cAMP- and cGMP-containing cells. Interestingly, hypoxia exposure raised the levels of cAMP and cGMP in the AV3V. Intra-PO microinjection of Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, attenuated hypoxia-induced anapyrexia. Similarly, intra-PO microinjection of the mixed β-adrenoceptor/serotonin (5-HT1A) receptor antagonist propranolol also impaired the drop in body temperature in response to hypoxia. The reduction in body temperature evoked by intra-PO serotonin, but not epinephrine, was blocked by Rp-cAMPS, indicating the involvement of a preoptic serotonin-cAMP pathway in the development of anapyrexia. Moreover, microinjection of N G-monomethyl-l-arginine, an inhibitor of nitric oxide (NO) synthesis, or Rp-cGMPS, an inhibitor of cGMP-dependent protein kinase, into the PO also attenuated hypoxia-induced anapyrexia. In conclusion, the present study supports that hypoxia-induced anapyrexia results from the activation of the serotonin-cAMP and NO-cGMP pathways in the PO.

scholarly journals Regulation of jun-B expression by a cyclic AMP (cAMP)-dependent mechanism in human myeloid cells

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 83-88 ◽  
Author(s):  
R Datta ◽  
T Nakamura ◽  
ML Sherman ◽  
D Kufe
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Jun B

Abstract The present studies have examined the regulation of the jun-B early response gene by cyclic AMP (cAMP)-dependent signaling pathways. The 2.0-kb jun-B transcript was at low but detectable levels in uninduced human HL-60 myeloid leukemia cells. In contrast, treatment with 1 mmol/L8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) in the presence of isobutylmethylxanthine, an inhibitor of cAMP-dependent phosphodiesterase, was associated with increases in jun-B transcripts that were maximal by 1 hour and then decreased to near pretreatment levels by 6 hours. Similar findings were obtained with 8–(4- chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate (8-CPT-cAMP) and N6,2′–0-dibutyryladenosine 3′,5′-cyclic monophosphate (dBt-cAMP). jun-B transcripts were also increased with other agents that increase intracellular cAMP levels, such as prostaglandin E2 (PGE2) and forskolin. Moreover, inhibition of cAMP-dependent protein kinase by the isoquinolinesulfonamide H-8 blocked 8-Br-cAMP-induced increases in jun- B expression. The results of nuclear run-on assays demonstrate that treatment of HL-60 cells with PGE2, forskolin, 8-Br-cAMP, and dBt-cAMP is associated with increases in the rate of jun-B transcription. The present findings also demonstrate that the related jun-D gene is similarly regulated by a cAMP-dependent pathway. Taken together, these findings suggest that stimulation of cAMP-dependent protein kinase is involved in the induction of jun gene expression in myeloid leukemia cells.

scholarly journals AaPKAc Regulates Differentiation of Infection Structures Induced by Physicochemical Signals From Pear Fruit Cuticular Wax, Secondary Metabolism, and Pathogenicity of Alternaria alternata

2021 ◽  
Vol 12 ◽  
Author(s):  
Miao Zhang ◽  
Yongcai Li ◽  
Tiaolan Wang ◽  
Yang Bi ◽  
Rong Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mutant Strain ◽  
Alternaria Alternata ◽  
Intracellular Camp ◽  
Pear Fruit ◽  
Appressorium Formation ◽  
Dependent Protein Kinase ◽  
Camp Content ◽  
Infection Structures ◽  
Dependent Protein

Alternaria alternata, the casual agent of black rot of pear fruit, can sense and respond to the physicochemical cues from the host surface and form infection structures during infection. To evaluate the role of cyclic AMP-dependent protein kinase (cAMP-PKA) signaling in surface sensing of A. alternata, we isolated and functionally characterized the cyclic adenosine monophosphate-dependent protein kinase A catalytic subunit gene (AaPKAc). Gene expression results showed that AaPKAc was strongly expressed during the early stages of appressorium formation on hydrophobic surfaces. Knockout mutants ΔAaPKAc were generated by replacing the target genes via homologous recombination events. We found that intracellular cAMP content increased but PKA content decreased in ΔAaPKAc mutant strain. Appressorium formation and infection hyphae were reduced in the ΔAaPKAc mutant strain, and the ability of the ΔAaPKAc mutant strain to recognize and respond to high hydrophobicity surfaces and different surface waxes was lower than in the wild type (WT) strain. In comparison with the WT strain, the appressorium formation rate of the ΔAaPKAc mutant strain on high hydrophobicity and fruit wax extract surface was reduced by 31.6 and 49.3% 4 h after incubation, respectively. In addition, AaPKAc is required for the hypha growth, biomass, pathogenicity, and toxin production of A. alternata. However, AaPKAc negatively regulated conidia formation, melanin production, and osmotic stress resistance. Collectively, AaPKAc is required for pre-penetration, developmental, physiological, and pathological processes in A. alternata.

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