PGE2 inhibits AVP-induced water flow in cortical collecting ducts by protein kinase C activation

1990 ◽  
Vol 259 (2) ◽  
pp. F318-F325 ◽  
Author(s):  
R. L. Hebert ◽  
H. R. Jacobson ◽  
M. D. Breyer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Water Flow ◽  
Collecting Duct ◽  
Inhibitory Effect ◽  
Cortical Collecting Duct ◽  
Independent Evidence ◽  
Kinase C ◽  
Pi Hydrolysis

It is well known that prostaglandin E2 (PGE2) both inhibits arginine vasopressin (AVP)-stimulated water permeability (hydraulic conductivity, Lp) in the cortical collecting duct (CCD) or, if administered alone, modestly increases Lp in the CCD. These bifunctional effects on Lp correspond to PGE2's capacity to inhibit AVP-stimulated adenylate cyclase (AC) activity, or to singularly stimulate AC activity in the collecting duct. The present studies suggest that the inhibitory effect of PGE2 on Lp may also be mediated by phosphatidylinositol (PI) hydrolysis. Using in vitro microperfused rabbit CCDs, we show that PGE2 releases Ca from intracellular stores. We also demonstrate that the inhibitory effect of PGE2 on AVP-stimulated Lp in the CCD is significantly reversed by the protein kinase C (PKC) inhibitor, staurosporine (SSP). Although PGE2 does not reduce an established water flow response to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPTcAMP), when the sequence of addition is reversed and PGE2 is added first, marked inhibition of 8-CPTcAMP-induced Lp is observed. This provides independent evidence that PGE2 can act through a mechanism separate from modulating AC activity. PGE2 inhibition of 8-CPTcAMP-induced Lp is reversed by SSP pretreatment. Finally, SSP pretreatment also markedly potentiates the capacity of PGE2 itself to increase Lp. We conclude that PGE2 releases Ca from intracellular stores and, by activating PKC, inhibits AVP-induced osmotic water flow. This suggests an important role for PI hydrolysis in mediating PGE2's effects on the CCD.

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.

scholarly journals Annexin 5 as a potential regulator of annexin 1 phosphorylation by protein kinase C. In vitro inhibition compared with quantitative data on annexin distribution in human endothelial cells

1993 ◽  
Vol 292 (3) ◽  
pp. 759-765 ◽  
Author(s):  
P Raynal ◽  
F Hullin ◽  
J M F Ragab-Thomas ◽  
J Fauvel ◽  
H Chap
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Inhibitory Effect ◽  
Human Endothelial Cells ◽  
Annexin 1 ◽  
Kinase C ◽  
Vitro Effect

In vitro phosphorylation of annexin 1 by purified rat brain protein kinase C (PKC) has been studied in the presence of annexin 5, which is not a substrate for PKC. Annexin 5 promoted a dose-dependent inhibition of annexin 1 phosphorylation, which could be overcome by increasing the concentration of phosphatidylserine (PtdSer). In addition, a close relationship was found between the amount of PtdSer uncovered by annexin 5 and the residual phosphorylation of annexin 1. These data fit with the ‘surface depletion model’ explaining the antiphospholipase activity of annexins. In order to check the possibility that the in vitro effect of annexin 5 could be of some physiological relevance, annexins 1, 2, and 5, as well as the light chain of calpactin 1 (p11), have been quantified in human endothelial cells by measuring the radioactivity bound to the proteins after Western blotting with specific antibodies and 125I-labelled secondary antibody. Our data indicate that annexins 1 and 5, PKC and PtdSer are present in human endothelial cells in relative amounts very similar to those used in vitro under conditions permitting the detection of the inhibitory effect of annexin 5. Since annexin 1 remained refractory to PKC-dependent phosphorylation in intact cells, we suggest that annexin 5 might exert its inhibitory effect towards PKC in vivo, provided that its binding to phospholipids can occur at physiological (micromolar) concentrations of Ca2+. This was previously shown to occur in vitro using phosphatidylethanolamine/phosphatidic acid vesicles [Blackwood and Ernst (1990) Biochem. J. 266, 195-200]. Using identical assay conditions, which also allowed expression of PKC activity, annexin 5 again inhibited annexin 1 phosphorylation without interfering with PKC autophosphorylation. These data suggest that annexins 1 and 5 might interact with each other on the lipid surface, resulting in a specific inhibition of annexin 1 phosphorylation by PKC. Whether a similar mechanism also occurs in vivo remains to be determined.

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 Inhibitory effect of shikonin on protein kinase C in vitro and in vivo.

1991 ◽  
Vol 55 ◽  
pp. 224
Author(s):  
Keiko Kurihara ◽  
Kazuhiko Qishi ◽  
Hiromi Takano-Ohmuro
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Inhibitory Effect ◽  
Kinase C

scholarly journals Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function

1992 ◽  
Vol 286 (1) ◽  
pp. 197-203 ◽  
Author(s):  
S J Winder ◽  
M D Pato ◽  
M P Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Smooth Muscle Contraction ◽  
Inhibitory Effect ◽  
Chicken Gizzard ◽  
Kinase C

Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.

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.

Muscarinic receptor activation inhibits AVP-induced water flow in rabbit cortical collecting ducts

1991 ◽  
Vol 260 (6) ◽  
pp. F929-F936 ◽  
Author(s):  
H. M. Snyder ◽  
D. M. Fredin ◽  
M. D. Breyer
Keyword(s):  
Muscarinic Receptor ◽  
Water Flow ◽  
Calcium Concentration ◽  
Collecting Duct ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Cortical Collecting Duct ◽  
Kinase C ◽  
Collecting Ducts ◽  
Subsequent Addition

We examined the effect of carbachol, an acetylcholine analogue, on hydraulic conductivity (Lp) response to 10 microU/ml arginine vasopressin (AVP) in rabbit cortical collecting duct (CCD). In CCDs in which water flow had been established with AVP, subsequent addition of carbachol caused Lp (X10(-7) cm.atm-1.s-1) to fall from 251 +/- 32 to 146 +/- 19. Carbachol washout resulted in recovery of Lp to 217 +/- 38. In CCDs in which water flow had been established using 10(-4) M 8-chlorophenylthioadenosine 3',5'-cyclic monophosphate (8-CPT-cAMP), addition of carbachol had no effect. These posttreatment studies suggest that carbachol's effects on modulating established water flow occur at a "pre-cAMP" step. With carbachol added first, AVP-induced Lp was reduced from 233 +/- 24 (controls) to 105 +/- 19 (carbachol-pretreated). Pretreatment with 10(-6) M atropine, a muscarinic receptor antagonist, totally reversed the inhibitory effect of carbachol, consistent with a receptor-mediated effect of carbachol. Carbachol pretreatment also inhibited 8-CPT-cAMP-induced Lp, indicating that carbachol's effects also occur at a "post-cAMP" step. Pretreatment with 10(-7) M staurosporine, a protein kinase C (PKC) inhibitor, reversed inhibitory effect of carbachol on AVP-induced Lp (193 +/- 26), suggesting that carbachol's effects are mediated by PKC. Intracellular calcium concentration [( Ca2+]i) was measured in fura-2-loaded CCDs. Carbachol also increased [Ca2+]i from 229 +/- 120 to 389 +/- 160 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Involvement of calcium in modulation of neutrophil function by phorbol esters that activate protein kinase C isotypes and related enzymes

1993 ◽  
Vol 289 (3) ◽  
pp. 919-926 ◽  
Author(s):  
J E Merritt ◽  
K E Moores ◽  
A T Evans ◽  
P Sharma ◽  
F J Evans ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Biological Activities ◽  
Inhibitory Effect ◽  
Human Neutrophils ◽  
Published Data ◽  
Bivalent Cation ◽  
Kinase C

In this study, the effects of a series of phorbol esters with different spectra of biological activities and different patterns of activation of the isoenzymes of protein kinase C (PKC) have been studied in human neutrophils. The aim was to gain more information on which isoenzymes of PKC are involved in neutrophil activation, specifically inhibition of fMet-Leu-Phe (fMLP)-stimulated bivalent cation influx and stimulation of O2-. release (either alone or potentiation of the response to fMLP). Prior addition of both phorbol 12-myristate 13-acetate (PMA) and sapintoxin A (SAPA) inhibited fMLP-stimulated Mn2+ influx. Higher concentrations of resiniferatoxin (RX) were also inhibitory, inhibition being more apparent at longer preincubation times. However, 12-deoxyphorbol 13-O-phenylacetate (DOPPA) showed only a slight inhibitory effect and required a prolonged preincubation. PMA, SAPA and RX, but not DOPPA, stimulated O2-. release by themselves. Lower concentrations of PMA, SAPA and RX, which were ineffective alone, considerably potentiated O2-. release stimulated by fMLP, whereas DOPPA had little or no effect. These results rule out a major role for PKC-delta (not activated by SAPA) and PKC-beta 1 (activated by DOPPA), but suggest the involvement of RX kinase in addition to PKC in the inhibition of fMLP-stimulated Mn2+ influx and potentiation of fMLP-stimulated O2-. release. However, when the cytosolic free Ca2+ concentration ([Ca2+]i) was elevated with the Ca2+ ionophore ionomycin, DOPPA was able to stimulate O2-. release, which probably reflects the known Ca2+ requirement for activation of PKC-beta 1 by DOPPA in vitro. The effects of the other phorbols were also enhanced when [Ca2+]i was elevated; all of the phorbols synergize, to variable extents, with Ca2+ to activate PKC in vitro. Enhancement of RX-stimulated O2- release by elevation of [Ca2+]i was unexpected, since RX kinase has been reported to be inhibited by high concentrations of Ca2+ in vitro. Finally, use of fura-2 and SK&F 96365 to manipulate the fMLP-stimulated rise in [Ca2+]i showed that when fMLP was able to evoke its normal rise in [Ca2+]i (to a peak of 700-900 nM), O2-. release was potentiated by PMA, SAPA and RX. However, when fMLP was only able to evoke a small increase in [Ca2+]i (to a peak of 400 nM), potentiation by PMA was unaffected but potentiation by SAPA and RX was considerably reduced. This observation agrees with published data demonstrating that activation of PKC in vitro by SAPA is more Ca(2+)-dependent than activation by PMA.(ABSTRACT TRUNCATED AT 400 WORDS)

ANF and bradykinin synergistically inhibit transport in M-1 cortical collecting duct cell line

1992 ◽  
Vol 263 (1) ◽  
pp. F1-F6 ◽  
Author(s):  
B. A. Stoos ◽  
O. A. Carretero ◽  
J. L. Garvin
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Duct Cell ◽  
Cortical Collecting Duct ◽  
Water Excretion ◽  
Kinase C

Previous data suggest that atrial natriuretic factor (ANF) and bradykinin (BK) interact to increase Na+ and water excretion. We propose that this interaction is due to a synergistic action that inhibits Na+ absorption in the distal nephron. We examined the effects of BK and ANF on transport by monolayers of a cortical collecting duct cell line, M-1. BK (10(-8) M) had no effect on short-circuit current (Isc). Similarly, ANF (10(-8) M) did not inhibit Isc. In contrast, Isc decreased by 18% (from 57 +/- 8 to 46 +/- 6 microA/cm2) when BK and ANF were added simultaneously at this concentration (P less than 0.05). Because guanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase C are implicated in the second messenger cascades of ANF and BK, we investigated their potential roles in mediating this interaction. Dibutyryl-cGMP (10(-4) M) inhibited Isc from 33 +/- 4 to 22 +/- 3 microA/cm2 (P less than 0.05) in the presence of BK but not in its absence. Staurosporine and calphostin C, inhibitors of protein kinase C, completely blocked the decrease in Isccaused by simultaneous addition of ANF and BK. cAMP levels in M-1 cells were not affected by either ANF alone or BK alone; however, when cultures were treated with both hormones, cAMP decreased from 856 +/- 56 to 332 +/- 26 fmol/10(6) cells (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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