Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Secretin stimulates ductal secretion by activation of cAMP --> PKA --> CFTR --> Cl(-)/HCO(3)(-) exchanger in cholangiocytes. We evaluated the expression of alpha(2A)-, alpha(2B)-, and alpha(2C)-adrenergic receptors in cholangiocytes and the effects of the selective alpha(2)-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in purified cholangiocytes. alpha(2)-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. alpha(2)-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases.
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PMID:The alpha2-adrenergic receptor agonist UK 14,304 inhibits secretin-stimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats. 1763 18

Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches, that three MAPKs exhibit unique interrelationships with the Na(+)/H(+) exchanger, NHE1, after osmotic cell shrinkage: Extracellular Signal Regulated Kinase (ERK1/2) is inhibited in an NHE1-dependent, pH(i)-independent manner, c-Jun N-terminal kinase (JNK1/2) is stimulated, in part through NHE1-mediated intracellular alkalinization, and p38 MAPK is activated in an NHE1-independent manner, and contributes to NHE1 activation and ERK inhibition. Shrinkage-induced ERK1/2 inhibition was attenuated in Ehrlich Lettre Ascites cells by NHE1 inhibitors (EIPA, cariporide) or removal of extracellular Na(+), and mimicked by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pH(i)-independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO(3)(-). Basal JNK activity was augmented at alkaline pH(i). Shrinkage-activation of p38 MAPK was NHE1-independent, and p38 MAPK inhibition (SB203580) attenuated NHE1 activation and ERK1/2 inhibition. Long-term shrinkage elicited caspase-3 activation and a loss of cell viability, which was augmented by ERK1/2 or JNK1/2 inhibition, and attenuated by p38 MAPK inhibition.
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PMID:The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells. 1798 56

The present study evaluated the activity of Cl(-)/HCO(3)(-) exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO(3)(-) was identical in WKY and SHR PTE cells, but V(max) values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly (P<0.05) increased the Cl(-)/HCO(3)(-) exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl(-)/HCO(3)(-) exchanger. The Cl(-)/HCO(3)(-) exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.
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PMID:Short-term regulation of the Cl-/HCO3(-) exchanger in immortalized SHR proximal tubular epithelial cells. 1840 47

The present study evaluated the role of oxidative stress on alpha(2)-adrenoceptor-mediated events (Cl(-)/HCO (3) (-) exchanger activity and cAMP accumulation) in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) and its normotensive control (Wistar Kyoto rat; WKY). The exposure of cells to alpha(2)-adrenoceptor agonist UK 14,304 reduced Cl(-)/HCO (3) (-) exchanger activity with EC(50) of 2.0 microM in SHR PTE cells, whereas in WKY PTE cells no effects were observed. These effects were abolished by yohimbine, an alpha(2)-adrenoceptor antagonist, but insensitive to prazosin. Both forskolin and dibutyryl cAMP stimulated Cl(-)/HCO (3) (-) exchanger activity in WKY and SHR PTE cells, which was prevented by the PKA inhibitor H-89. Forskolin increased cAMP levels in both WKY and SHR PTE cells to a similar extent, but UK 14,304 significantly reduced the forskolin-induced increase in cAMP levels in only SHR PTE cells. Immunoblotting showed that expression of alpha(2B)-adrenoceptors was 12-times greater in SHR PTE cells than in WKY PTE cells. SHR PTE cells have increased levels of H(2)O(2) and overexpress type 2 NADPH oxidase (NOX2) and p22(phox) compared with WKY cells. In SHR PTE cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H(2)O(2) and abolished the inhibitory effects of UK 14,304 on Cl(-)/HCO (3) (-) exchanger activity and cAMP accumulation. It is concluded that differences between WKY and SHR PTE cells on their sensitivity to alpha(2)-adrenoceptor agonists correlate with the expression of alpha(2B)-adrenoceptors. The increased generation of H(2)O(2) amplifies the response downstream to alpha(2)-adrenoceptor activation in SHR PTE cells.
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PMID:Oxidative stress plays a permissive role in alpha2-adrenoceptor-mediated events in immortalized SHR proximal tubular epithelial cells. 1849 Oct 35

H(2)O(2) is a highly reactive oxygen metabolite that has been implicated as an important mediator of inflammation-induced intestinal injury associated with ischaemia/reperfusion, radiation and inflammatory bowel disease. Previous studies have shown that H(2)O(2) inhibits NaCl absorption and activates Cl(-) secretion in the rat and rabbit colon. To date, however, almost no information is available with respect to its effect on the human intestinal apical anion exchanger Cl(-)/OH(-) (HCO(3)(-)). The present studies were, therefore, undertaken to examine the direct effects of H(2)O(2) on OH(-) gradient-driven DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate)-sensitive (36)Cl(-) uptake utilizing a post-confluent transformed human intestinal epithelial cell line, Caco-2. Our results demonstrate that H(2)O(2) (1 mM for 60 min) significantly inhibited (approx. 60%; P<0.05) Cl(-)/OH(-) exchange activity in Caco-2 cells. H(2)O(2)-mediated inhibition of Cl(-)/OH(-) exchange activity involved the Src kinase Fyn and PI3K (phosphoinositide 3-kinase)-dependent pathways. H(2)O(2) also induced phosphorylation of Fyn and p85 (the regulatory subunit of PI3K) in Caco-2 cells. Moreover, an increased association of Fyn and p85 was observed in response to H(2)O(2), resulting in the activation of the downstream target PLCgamma1 (phospholipase Cgamma1). Elevated intracellular Ca(2+) levels and PKCalpha (protein kinase Calpha) functioned as downstream effectors of H(2)O(2)-induced PLCgamma1 activation. Our results, for the first time, provide evidence for H(2)O(2)-induced Src kinase Fyn/PI3K complex association. This complex association resulted in the subsequent activation of PLCgamma1 and Ca(2+)-dependent PKCalpha, resulting in the inhibition of Cl(-)/OH(-) exchange activity. These findings suggest that H(2)O(2)-induced inhibition of the Cl(-)/OH(-) exchange process may play an important role in the pathophysiology of diarrhoea associated with inflammatory disorders, where the amount of reactive oxygen species is markedly elevated.
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PMID:Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl-/OH- exchange activity in human intestinal epithelial cells. 1856 62

It is well known that Glucagon (Gl) is released after a high protein diet and participates in water excretion by the kidney, principally after a protein meal. To study this effect in in vitro perfused inner medullary collecting ducts (IMCD), the osmotic water permeability (Pf; mum/s) at 37 degrees C and pH 7.4 in normal rat IMCDs (n = 36) perfused with Ringer/HCO(3) was determined. Gl (10(-7) M) in absence of Vasopressin (AVP) enhanced the Pf from 4.38 +/- 1.40 to 11.16 +/- 1.44 microm/s (P < 0.01). Adding 10(-8), 10(-7), and 10(-6) M Gl, the Pf responded in a dose-dependent manner. The protein kinase A inhibitor H8 blocked the Gl effect. The specific Gl inhibitor, des-His(1)-[Glu(9)] glucagon (10(-7) M), blocked the Gl-stimulated Pf but not the AVP-stimulated Pf. There occurred a partial additional effect between Gl and AVP. The cAMP level was enhanced from the control 1.24 +/- 0.39 to 59.70 +/- 15.18 fm/mg prot after Gl 10(-7) M in an IMCD cell suspension. The immunoblotting studies indicated an increase in AQP2 protein abundance of 27% (cont 100.0 +/- 3.9 vs. Gl 127.53; P = 0.0035) in membrane fractions extracted from IMCD tubule suspension, incubated with 10(-6) M Gl. Our data showed that 1) Gl increased water absorption in a dose-dependent manner; 2) the anti-Gl blocked the action of Gl but not the action of AVP; 3) Gl stimulated the cAMP generation; 4) Gl increased the AQP2 water channel protein expression, leading us to conclude that Gl controls water absorption by utilizing a Gl receptor, rather than a AVP receptor, increasing the AQP2 protein expression.
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PMID:Aquaporin 2 expression increased by glucagon in normal rat inner medullary collecting ducts. 1882 41

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.
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PMID:Chloride Is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation. 1895 26

Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (Calpha) of PKA confers sensitivity to the pyrazolo[3,4-d]pyrimidine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the CalphaM120A mutant protein is expressed and the wild-type Calpha is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express CalphaM120A mutant protein. For CalphaM120A sperm, 10 microM of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (<90 s) evoked by the HCO(3)(-) anion. A continuous (90 min) inhibition with 10 microM of 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in CalphaM120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional CalphaM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.
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PMID:Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation. 1914 27

Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.
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PMID:AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells. 1921 18

Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signaling pathways involved. We show here that protein kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP), which activates mitochondrial PKA, does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO(2)-HCO(3)(-)-sAC-cAMP-PKA (mito-sAC) signaling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability.
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PMID:Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation. 1925 71


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