PGE2 regulates cAMP production in cultured rabbit CCD cells: evidence for dual inhibitory mechanisms

1992 ◽  
Vol 263 (6) ◽  
pp. C1208-C1215 ◽  
Author(s):  
T. D. Noland ◽  
C. E. Carter ◽  
H. R. Jacobson ◽  
M. D. Breyer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Collecting Duct ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Camp Production ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Inhibitory Mechanisms ◽  
Protein Kinase C Inhibitor

In cultured cortical collecting duct (CCD) cells, exogenous prostaglandin E2 (PGE2) inhibited arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in a concentration-dependent manner. Although pertussis toxin (PT, 500 ng/ml) alone did not reverse the PGE2-dependent inhibition, PT and staurosporine, a protein kinase C inhibitor, together partially reversed the effect of exogenous PGE2. In contrast, PT completely reversed the inhibition of AVP-dependent cAMP production by sulprostone. These data suggest that exogenous PGE2 can inhibit AVP-stimulated cAMP production and that the inhibitory effects of PGE2 are mediated by staurosporine- and PT-sensitive component(s). Short-term (15-240 min) incubation with phorbol 12-myristate 13-acetate (PMA, 10(-7) M) inhibited PGE2-stimulated cAMP production. Long-term (20 h) incubation with PMA augmented PGE2-stimulated cAMP production. These data provide evidence for the maintenance of a PT-sensitive PGE2-dependent inhibitory pathway of cAMP production in cultured CCD cells. In addition, data are presented that support an inhibitory role for protein kinase C in the effects of PGE2 on the metabolism of cAMP in these cells.

Localization of IP in rabbit kidney and functional role of the PGI2/IP system in cortical collecting duct

2002 ◽  
Vol 283 (4) ◽  
pp. F689-F698 ◽  
Author(s):  
Rania Nasrallah ◽  
Rolf M. Nusing ◽  
Richard L. Hébert
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Functional Role ◽  
Collecting Duct ◽  
Inhibitory Effect ◽  
Rabbit Kidney ◽  
Cortical Collecting Duct ◽  
Kinase C ◽  
Protein Kinase C Inhibitor

To clarify the role of the PGI2/PGI2 receptor (IP) system in rabbit cortical collecting duct (RCCD), we characterized the expression of IP receptors in the rabbit kidney. We show by Northern and Western blotting that IP mRNA and protein was detectable in all three regions of the kidney. To determine how PGI2 signals, we compared the effects of different PGI2 analogs [iloprost (ILP), carba-prostacyclin (c-PGI2), and cicaprost (CCP)] in the isolated perfused RCCD. PGI2 analogs did not increase water flow ( L p). Although PGI2 analogs did not reduce an established L p response to 8-chlorophenylthio-cAMP, they equipotently inhibited AVP-stimulated L p by 45%. The inhibitory effect of ILP and c-PGI2 on AVP-stimulated L p is partially reversed by the protein kinase C inhibitor staurosporine and abolished by pertussis toxin; no effect was obtained with CCP. In fura 2-loaded RCCD, CCP did not alter cytosolic Ca2+concentration ([Ca2+]i), but, in the presence of CCP, individual infusion of ILP and PGE2 increased [Ca2+]i, suggesting that CCP did not cause desensitization to either ILP or PGE2. We concluded that ILP and c-PGI2 activate PKC and the liberation of [Ca2+]i but not CCP. This suggested an important role for phosphatidylinositol hydrolysis in mediating ILP and c-PGI2 effects but not CCP in RCCD.

Prostaglandins mediate the defect in AVP-stimulated cAMP generation in immature collecting duct

1994 ◽  
Vol 267 (1) ◽  
pp. F44-F48 ◽  
Author(s):  
M. Bonilla-Felix ◽  
C. John-Phillip
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Camp Production ◽  
Direct Inhibition ◽  
Endogenous Prostaglandin ◽  
Kinase C ◽  
Camp Generation ◽  
Camp Levels

The immature kidney is characterized by resistance to arginine vasopressin (AVP). In the immature cortical collecting duct (iCCD), AVP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation is decreased, but the mechanisms involved are not known. We examined cAMP production in isolated CCD from immature and mature rabbits. Cellular cAMP levels were measured by radioimmunoassay under basal conditions and after stimulation with hormone. Basal cAMP production in the iCCD was not different from that in the mature CCD (mCCD). In contrast, AVP- and forskolin-stimulated cAMP generation were severely decreased in the iCCD. Inhibition of endogenous prostaglandin production by indomethacin increased AVP-stimulated cAMP generation in the iCCD to levels that were not different from the mCCD. Inhibition of protein kinase C (PKC) by staurosporine and inhibition of Gi by pertussis toxin elicited a mature cAMP response in the iCCD. These data suggest that the defect in AVP-stimulated cAMP production in the iCCD is mediated by prostaglandins via 1) activation of Gi and 2) direct inhibition of the adenylyl cyclase catalytic subunit. In addition, PKC appears to play a significant role.

DIACYLGLYCEROL AND PHORBOL ESTER REDUCE AEQUORIN-INDICATED [Ca++] ELEVATIONS INDUCED BY THROMBIN OR ADP

1987 ◽  
Author(s):  
J A Ware ◽  
M Smith ◽  
E W Salzman
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Time Dependent ◽  
Dependent Manner ◽  
Kinase C ◽  
Protein Kinase C Activators ◽  
Protein Kinase C Inhibitor ◽  
Ca Homeostasis

Platelet aggregation and secretion induced by phorbol ester (PMA) or diacylglycerol (DAG) are preceded by an increase in [Ca++] that is detected byaequorin, but not by quin2, fura-2, or indo-1, suggesting that these indicatorsreflect different aspects of Ca++ homeostasis, possibly different functional Ca++ pools. Addition of two conventional agonists in subthreold concentrations synergistically enhances the [Ca++] rise and aggregation.However, if PMA or DAG is the first agonist the subsequent quin2-indicated [Ca++] rise after thrombin is reduced.Whether aequorin-indicated [Ca++] is similarly affected is unknown. We studied gel-filtered platelets loaded with aequorin or a fluorophore and added PMA, DAG, thrombin or ADP, alone or in combination. Either PMA or DAG alone caused a concentration-dependent increase in [Ca++] detectable with aequorin but not with the fluorophores; simultaneous addition of thrombin or ADP with DAG or PMA produced a larger [Ca++] rise than either alone. However, addition of DAG or PMA as a first agonist reduced subsequent aequorin-indicated [Ca++] rises following thrombin or ADP in a concentration and time-dependent manner. Inhibition of ADP or thrombin-induced [Ca++] rise was not always accompanied by inhibition of aggregation or secretion. Combination of subthreshold concentrations of ADP and thrombin produced an enhanced [Ca++] rise and aggregation. However, this synergistic effect was inhibited by preincubation with DAG or PMA. Neither this effect nor DAG-induced [Ca++] rise was inhibited by the protein kinase C inhibitor H-7. In genera^ preincubation of platelets with an agonist enhances Ca rise and aggregation in response to a second agonist; in contrasl protein kinase C activators, which themselves elevate [Ca++] as shown by aequorin, inhibit aequorin-indicated Ca rises after ADP or thrombin, and limit synergism between these two agonists.

Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+

1993 ◽  
Vol 264 (5) ◽  
pp. F845-F853
Author(s):  
M. M. Friedlaender ◽  
D. Jain ◽  
Z. Ahmed ◽  
D. Hart ◽  
R. L. Barnett ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Intracellular Signaling ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Eta Receptor ◽  
Ca2 Channels ◽  
Stimulation Of

Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.

Carvedilol-induced elevation in cytosolic free Ca2+ level and apoptosis in SIRC corneal epithelial cells

2009 ◽  
Vol 29 (6) ◽  
pp. 477-487
Author(s):  
Pochuen Shieh ◽  
Chih-Hung Lee ◽  
Ng Ling Yi ◽  
Chung-Ren Jan
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Phospholipase C ◽  
Dependent Manner ◽  
Corneal Epithelial Cells ◽  
Concentration Dependent Manner ◽  
Corneal Epithelial ◽  
Kinase C

The effect of the cardiovascular drug carvedilol on cytosolic free Ca2+ concentrations ([Ca 2+]i) and viability was examined in Statens Seruminstitut rabbit cornea (SIRC) corneal epithelial cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1), respectively. Carvedilol at concentrations between 1 and 30 μM increased [Ca 2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Carvedilol induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was inhibited by suppression of protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor), carvedilol-induced [Ca2+]i rise was reduced; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca 2+]i rise. Addition of the phospholipase C inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U73122; 2 μM) did not change carvedilol-induced [Ca2+]i rise. At concentrations between 5 and 70 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 μM carvedilol was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5—70 μM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca 2+ influx via protein kinase C-regulated Ca2+ channels. Carvedilol-caused cytotoxicity was mediated by Ca2+-independent apoptosis in a concentration-dependent manner.

scholarly journals G-protein βγ subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-β1

1997 ◽  
Vol 326 (3) ◽  
pp. 701-707 ◽  
Author(s):  
Irene LITOSCH
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
G Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Negative Feedback Regulation ◽  
Kinase C ◽  
Stimulation Of

Protein kinase C (PKC) isoforms phosphorylated phospholipase C-β1 (PLC-β1) in vitro as follows: PKCα ≫ PKCϵ; not PKCζ. PLC-β3 was not phosphorylated by PKCα. G-protein βγ subunits inhibited the PKCα phosphorylation of PLC-β1 in a concentration-dependent manner. Half-maximal inhibition occurred with 500 nM βγ. G-protein βγ subunits also antagonized the PKCα-mediated inhibition of PLC-β1 enzymic activity. PKCα, in turn, inhibited the stimulation of PLC-β1 activity by βγ. There was little effect of PKCα on the stimulation of PLC-β1 by αq/11–guanosine 5′[γ-thio]triphosphate (GTP[S]). These findings demonstrate that G protein βγ subunits antagonize PKCα regulation of PLC-β1. Thus βγ subunits might have a role in modulating the negative feedback regulation of this signalling system by PKC.

The role of protein kinase C in LHRH-induced LH and FSH release and LHRH self-priming in rat anterior pituitary glands in vitro

1988 ◽  
Vol 116 (2) ◽  
pp. 231-239 ◽  
Author(s):  
M. S. Johnson ◽  
R. Mitchell ◽  
G. Fink
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Pituitary Glands

ABSTRACT We have investigated the role of protein kinase C (PKC) in LHRH-induced LH and FSH secretion and LHRH priming. Hemipituitary glands from pro-oestrous rats were incubated with agents known to affect PKC and with or without LHRH, during which time the secretion of gonadotrophins was measured. Phorbol esters and phospholipase C, activators of PKC, released LH and FSH in a concentration-dependent manner and potentiated the LHRH-induced secretion of gonadotrophins in parallel with their ability to release these hormones alone. Inhibitors of PKC had either no effect on LH release (1-(5-isoquinolinesulphonyl)-2-methylpiperazine hydrochloride) or they augmented LHRH-induced gonadotrophin release (polymyxin B and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate). Neither the activators nor the inhibitors of PKC, when present with LHRH, caused any change in LHRH priming, even though the activators alone produced a release of gonadotrophins that showed a temporal pattern similar to that produced by LHRH priming. The profiles of effects on LH and FSH secretion were always qualitatively similar. These results show that PKC may be involved in general regulation of gonadotrophin release but that it is not important in acute responses to LHRH nor in LHRH self-priming. J. Endocr. (1988) 116, 231–239

scholarly journals Modulation of Calmodulin and Protein Kinase C Activities by Pencillium Mycotoxins

1999 ◽  
Vol 18 (2) ◽  
pp. 91-96 ◽  
Author(s):  
I. Pala ◽  
A. Srinivasan ◽  
P. J. S. Vig ◽  
D. Desaiah
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Mammalian Brain ◽  
Dependent Manner ◽  
Active Conformation ◽  
Cellular Functions ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Ic50 Values

Calmodulin (CaM), a calcium-binding protein, is found in high concentrations in mammalian brain where it plays a pivotal role in a large number of cellular functions. Protein kinase C (PKC), a multifunctional cytosolic enzyme, in the presence of both Ca2+ and phospholipids, transduce extracellular signals into intracellu-lar events. Both CaM and PKC are partially involved in maintaining Ca2+ homeostasis in the cell. Any fluctuations in the intracel-lular Ca2+ can modulate cellular functions and may contribute to neuronal dysfunction. Hence, the present investigation was initiated to study the effects of some selected penicillium (naturally occurring tremorgenic) mycotoxins like secalonic acid, citreoviridin, and verruculogen on CaM activity, active conformation of CaM and PKC activity. Stimulation of CaM-deflcient bovine brain 3′-5′ phosphodieste rase (PDE) indicated CaM activity. The modification of CaM active conformation was studied by the binding of fluorescent probe N-phenyl-1-napthylamine (NPN) to CaM. Alterations in the fluorescence of dansyl-CaM was used to study the effect of these compounds on complex formation between CaM and PDE. Rat brain cytosolic PKC was studied using 32P-ATP as a measure of altered protein phosphorylation. The concentrations of mycotoxins used were in the range of 10 to 50 μM. All three mycotoxins inhibited CaM-stimulated PDE activity in a concentration-dependent manner. Citreoviridin and secalonic acid inhibited NPN fluorescence and Ca2+-dependent complex formation of dansyl-CaM and PDE. The IC50 values for NPN fluorescence of citreoviridin and secalonic acid were 13 μM and 19 μM respectively. However, verruculogen showed little effect on NPN fluorescence and the Ca2+-dependent complex formation of dansyl-CaM and PDE. These mycotoxins also inhibited PKC activity in a concentration-dependent manner with IC50 values of 19.8, 25.7, and 38.4 μM for secalonic acid, citreoviridin, and verruculogen, respectively. The results of our study suggest that these mycotoxins at very low concentrations are interacting with CaM and PKC. Such an effect could lead to impairment of neurotransmission and result in neurotoxicity.

Bradykinin-stimulated cPLA2phosphorylation is protein kinase C dependent in rabbit CCD cells

1997 ◽  
Vol 273 (6) ◽  
pp. F907-F915 ◽  
Author(s):  
Mark A. Lal ◽  
Chris R. J. Kennedy ◽  
Pierre R. Proulx ◽  
Richard L. Hébert
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Collecting Duct ◽  
Specific Inhibitor ◽  
Calcium Ionophore ◽  
Serine Phosphorylation ◽  
Established Cell Line ◽  
Similar Degree ◽  
Cortical Collecting Duct ◽  
Kinase C

We have used an established cell line of rabbit cortical collecting duct (RCCD) epithelial cells representing a mixed population of principal and intercalated cell types to determine which phospholipase A2(PLA2) enzyme therein is responsible for bradykinin (BK)-stimulated arachidonic acid (AA) release and how its activation is regulated. BK-stimulated AA release was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2(cPLA2). Examination of PLA2 activity in vitro demonstrated that BK stimulation resulted in a greater than twofold increase in PLA2 activity and that this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2. To determine a possible role for protein kinase C (PKC) in the BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA2 activity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA release and cPLA2 activity by 51 and 58%, respectively. cPLA2 activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] displayed a similar degree of activation and was associated with an increase in serine phosphorylation identical to that caused by BK. The phosphorylation-induced activation of this enzyme was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated cPLA2 activity. In addition, we have also found that PMA stimulation did not cause a synergistic potentiation of BK-stimulated AA release as did calcium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken together, indicate that cPLA2 is the enzyme responsible for BK-mediated AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA2 to BK.

scholarly journals The diacylglycerols dioctanoylglycerol and oleoylacetylglycerol enhance prostaglandin synthesis by inhibition of the lysophosphatide acyltransferase

1987 ◽  
Vol 247 (3) ◽  
pp. 773-777 ◽  
Author(s):  
M Goppelt-Strübe ◽  
H J Pfannkuche ◽  
D Gemsa ◽  
K Resch
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Direct Interaction ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Key Enzyme ◽  
Free Arachidonic Acid

Prostanoids are synthesized by resident macrophages upon stimulation with diacylglycerols. Oleoylacetylglycerol and dioctanoylglycerol induced prostaglandin E and thromboxane synthesis in a time- and concentration-dependent manner. Both diacylglycerols inhibited the lysophosphatide acyltransferase, which is the key enzyme in the reacylation of arachidonic acid. By this mechanism the pool of free arachidonic acid available for prostanoid synthesis is increased. Both diacylglycerols were able to inhibit the membrane-bound lysophosphatide acyltransferase by a direct interaction independent of protein kinase C. Thus lysophosphatide acyltransferase could be shown to be a new target of these diacylglycerols, known as activators of protein kinase C.

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