Effect Of Phorbol 12,13-dibutyrate Research Articles

PKC activation impairs endothelium‐dependent NO‐mediated dilation of coronary arterioles via enhanced superoxide production from xanthine oxidase

We examined the role of PKC‐mediated pathway in eliciting endothelial dysfunction using isolated and pressurized porcine coronary arterioles. Diameter changes were recorded using videomicroscopic techniques. Superoxide production was detected with dihydroethidium (DHE) staining. Incubation of coronary arterioles with the PKC activator phorbol 12, 13‐dibutyrate (PDBu) (1 nM, 60 minutes) attenuated arteriolar dilation to the endothelium‐dependent, NO‐mediated agonists adenosine and serotonin, but not the NO donor sodium nitroprusside. In the presence of superoxide scavenger TEMPOL (1 mM), xanthine oxidase inhibitor allopurinol (10 μM), c‐Jun N‐terminal kinase (JNK) inhibitor SP600125 (5 μM) or Rho‐kinase inhibitor Y‐27632 (0.1 μM), the adverse effects of PDBu on adenosine‐ and serotonin‐induced dilations were prevented. Nevertheless, the inhibitory effects of PDBu were insensitive to H2O2 scavenger PEG‐catalase (500 units/ml), NAD(P)H oxidase inhibitor apocynin (100 μM) or p38 kinase inhibitor SB203580 (0.1 μM). DHE staining revealed that PDBu generated TEMPOL‐sensitive superoxide production in the coronary arteriolar smooth muscle. In conclusion, activation of vascular PKC with PDBu inhibits endothelium‐dependent, NO‐mediated dilation in coronary arterioles by producing superoxide from xanthine oxidase, which appears to be linked with JNK and RhoA/Rho kinase activation.

Modulation of Kv3.4 channel N‐type inactivation by protein kinase C shapes the action potential in dorsal root ganglion neurons

Fast inactivation of heterologously expressed Kv3.4 channels is dramatically slowed upon phosphorylation of the channel's N-terminal (N-type) inactivation gate by protein kinase C (PKC). However, the presence and physiological importance of this exquisite modulation in excitable tissues were unknown. Here, we employed minimally invasive cell-attached patch-clamping, single-cell qPCR and specific siRNAs to unambiguously demonstrate that fast-inactivating Kv3.4 channels underlie a robust high voltage-activated A-type K(+) current (I(AHV)) in nociceptive dorsal root ganglion neurons from 7-day-old rats. We also show that PKC activation with phorbol 12,13-dibutyrate (PDBu) causes a 4-fold slowing of Kv3.4 channel inactivation and, consequently, accelerates the repolarization of the action potential (AP) by 22%, which shortens the AP duration by 14%. G-protein coupled receptor (GPCR) agonists eliminate I(AHV) fast inactivation in a membrane-delimited manner, suggesting a Kv3.4 channel signalling complex. Preincubation of the neurons with the PKC inhibitor bisindolylmaleimide II inhibits the effect of GPCR agonists and PDBu. Furthermore, activation of PKC via GPCR agonists recapitulates the effects of PDBu on the AP. Finally, transfection of the neurons with Kv3.4 siRNA prolongs the AP by 25% and abolishes the GPCR agonist-induced acceleration of the AP repolarization. These results show that Kv3.4 channels help shape the repolarization of the nociceptor AP, and that modulation of Kv3.4 channel N-type inactivation by PKC regulates AP repolarization and duration. We propose that the dramatic modulation of I(AHV) fast inactivation by PKC represents a novel mechanism of neural plasticity with potentially significant implications in the transition from acute to chronic pain.

Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells

Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). We determined the effects of PDBu (10(-7) M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. We used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10(-6) M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10(-6) M). We also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined.

1950 PROTEIN KINASE C INHIBITS SPONTANEOUS AND NON-VOIDING CONTRACTIONS IN DETRUSOR SMOOTH MUSCLE: ROLE AND INTERACTION WITH BK CHANNELS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Non-Neurogenic Voiding Dysfunction1 Apr 20111950 PROTEIN KINASE C INHIBITS SPONTANEOUS AND NON-VOIDING CONTRACTIONS IN DETRUSOR SMOOTH MUSCLE: ROLE AND INTERACTION WITH BK CHANNELS Joseph Hypolite, Qi Lei, Shaohua Chang, Anna Malykhina, Stephen Zderic, Alan Wein, Stephan Butler, and Samuel Chacko Joseph HypoliteJoseph Hypolite Glenolden, PA More articles by this author , Qi LeiQi Lei Glenolden, PA More articles by this author , Shaohua ChangShaohua Chang Glenolden, PA More articles by this author , Anna MalykhinaAnna Malykhina Glenolden, PA More articles by this author , Stephen ZdericStephen Zderic Philadelphia, PA More articles by this author , Alan WeinAlan Wein Philadelphia, PA More articles by this author , Stephan ButlerStephan Butler Philadelphia, PA More articles by this author , and Samuel ChackoSamuel Chacko Glenolden, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2154AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Partial bladder outlet obstruction (PBOO)-induced detrusor smooth muscle hypertrophy is associated with a downregulation of protein kinase C (PKC) and large conductance calcium-activated potassium channels (BK) channels, and an increase in spontaneous contractions in isolated muscle strips, and non-voiding contractions in patients with BPH-induced PBOO. The purpose of this study is to examine whether or not PKC is involved in the regulation of spontaneous and non-voiding contractions in DSM, and whether inhibition of PKC contributes to increased detrusor excitability. METHODS DSM strips (∼2mm × 10mm) were removed from 2-week PBOO rabbits and used for in vitro contractile studies. Sham-operated animals served as controls. The effect of inhibition of PKC on non-voiding contractions was studied using in vivo cystometry in rats, and whole cell patch clamping was used to study the effect of phorbol 12,13-dibutyrate (PDBU), a PKC activator, on the BK channel current. Iberiotoxin (IBX), a BK-channel inhibitor, was used to study its effect on spontaneous contractions and PDBU-induced inhibition of spontaneous contraction. RESULTS The results indicate that the PKC inhibitor, bisindolylmaleimide1, (BIS-1) and IBX increased the amplitude of spontaneous contractions in detrusor smooth muscle strips from normal rabbit bladders but that there was no significant change in the strips from PBOO. BIS-1 also increased the frequency of non-voiding contractions in normal rat bladder. Both BIS-1- and IBX-induced spontaneous contractions were inhibited by PDBU (10-50 nM). BIS-1-induced spontaneous contractions were significantly reduced by BK channel openers, NS1619 and isopimeric acid. Pre-treatment of isolated muscle strips with IBX significantly delayed the inhibitory effect of PDBU on spontaneous contractions. Whole cell patch clamp studies revealed that PDBU caused an increase in the BK channel current in detrusor myocytes isolated from both sham-operated and PBOO animals. CONCLUSIONS The data suggest that PKC may play an important role in maintaining the relatively quiescent, non-excitable state of the normal bladder by suppressing spontaneous and non-voiding contractions, and that down-regulation of PKC in PBOO may contribute to detrusor overactivity. The data also suggests that the mechanism of action may involve the ability of PKC to increase the BK-channel current during the filling phase of the micturition cycle. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e780 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joseph Hypolite Glenolden, PA More articles by this author Qi Lei Glenolden, PA More articles by this author Shaohua Chang Glenolden, PA More articles by this author Anna Malykhina Glenolden, PA More articles by this author Stephen Zderic Philadelphia, PA More articles by this author Alan Wein Philadelphia, PA More articles by this author Stephan Butler Philadelphia, PA More articles by this author Samuel Chacko Glenolden, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of protein kinase C in regulation of Na+- and K+-dependent ATPase activity and pump function in corneal endothelial cells

Na+- and K+-dependent ATPase (Na,K-ATPase) plays an important role in the pump function of the corneal endothelium. We investigated the possible role of protein kinase C (PKC) in regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to phorbol 12,13-dibutyrate (PDBu) to induce activation of PKC. ATPase activity of the cells was evaluated by using ammonium molybdate in spectrophotometric measurement of phosphate released from ATP, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with a Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. PDBu (10(-7) M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of PDBu were potentiated by the cyclooxygenase inhibitor indomethacin and the cytochrome P(450) inhibitor resorufin and were blocked by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results suggest that PKC bidirectionally regulates Na,K-ATPase activity in mouse corneal endothelial cells: it inhibits Na,K-ATPase activity in a cyclooxygenase- and cytochrome P(450)-dependent manner, whereas it stimulates such activity by activating protein phosphatases 1 or 2A.

Up-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-α in Adrenal Chromaffin Cells

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid ( 15 h) translocation of both conventional (c) protein kinase C-α (PKC-α) and novel PKC-e (but not atypical PKC-ζ) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-α. In the present study, chronic (≥12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4α-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the β-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor β-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor β-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-α up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Phospholipase A2 regulation of bovine endometrial (BEND) cell prostaglandin production

BackgroundProstaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production.MethodsCells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates.ResultsBEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity.ConclusionResults from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production.

Short-term regulation of the Cl−/HCO3− exchanger in immortalized SHR proximal tubular epithelial cells

The present study evaluated the activity of Cl−/HCO3− 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 HCO3− was identical in WKY and SHR PTE cells, but Vmax 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−/HCO3− 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−/HCO3− exchanger. The Cl−/HCO3− exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.

Implication of spinal protein kinase Cγ isoform in activation of the mouse brain by intrathecal injection of the protein kinase C activator phorbol 12,13-dibutyrate using functional magnetic resonance imaging analysis

The aim of the present study was to investigate whether direct activation of protein kinase C (PKC) in the spinal cord could change brain activation using a functional magnetic resonance imaging (fMRI) analysis in mice that lack the PKCgamma gene. The activation of spinal PKC by intrathecal (i.t.) injection with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused a time-dependent decrease in paw-withdrawal latency to the heat thermal stimulus. In contrast, i.t. injection of PDBu failed to cause thermal hyperalgesia in mice which lacked the PKCgamma gene. We found that the i.t. injection with PDBu caused a remarkable increase in the activity of several brain regions in wild-type mice compared with vehicle injection. In the somatosensory cortex and lateral and medial thalamus, i.t. injection of PDBu produced a dramatic and time-dependent increase in signal intensity at 1-6h after i.t. PDBu injection. In contrast, i.t. injection of PDBu produced a delayed but significant increase in signal intensity at 3-6h in the cingulate cortex, at 4-6h in the nucleus accumbens and at 3-6h in the ventral tegmental area. In addition, all effects of PDBu were abolished in mice that lacked the PKCgamma gene. These results suggest that the activation of spinal PKCgamma associated with the activation of ascending pain transmission may be an important factor in chronic pain-like hyperalgesia with changes in emotionality.

Effect of PDBu on [alpha]1‐adrenoceptor‐mediated contraction and changes of intracellular free Ca2+ concentration in uterine artery of pregnant sheep

PKC activation by phorbol 12, 13-dibutyrate (PDBu) shows inhibitory effects on phenylephrine (PE)-induced contractions in pregnant uterine arteries. We hypothesize that PDBu inhibits PE-induced increases of intracellular free Ca2+ concentration ([Ca2+]i). Uterine arteries were isolated from near term pregnant ewes. Vascular smooth muscle [Ca2+]i was measured simultaneously with muscle contraction in the same tissue loaded with Fura-2 by using a CAF-110 intracellular Ca2+ analyzer. PE-induced contraction and simultaneously measured [Ca2+]i were compared between before and after PDBu treatment. Compared with before PDBu treatment, both 0.1 and 1 μM PDBu produced a significant decrease in PE-induced contraction and completely blocked PE-induced increases of [Ca2+]i as indicated by no changes of Rf340/380. These results suggested that PKC activation in pregnant uterine artery plays an inhibitory role in α1-adrenoceptor-mediated contraction through blocking the availability of free Ca2+ to the contractile proteins. (Supported in part by NIH grants HL57787 and HD 31226)

μ-Calpain is functionally required for α-processing of Alzheimer’s β-amyloid precursor protein

Alzheimer’s β-amyloid precursor protein (APP) is normally processed by an unidentified α-secretase. A unique feature of this protease is its high sensitivity to phorbol esters, yet the mechanism involved is unclear. We have previously reported that phorbol 12,13-dibutyrate (PDBu) activates calpain, a Ca 2+-dependent protease, and PDBu-induced release of APPs (secreted APP) is sensitive to calpain inhibitors, suggesting that calpain is involved in APP α-processing. In the present study, we found that PDBu markedly promoted the expression of both μ- and m-calpains in cultured fibroblasts. Dose–response and time course studies revealed that μ-calpain was more sensitive to PDBu than m-calpain and the temporal course of the μ-calpain change coincides better with that of APPs release. Moreover, the stimulatory effect of PDBu on μ-calpain was selectively blocked by μ-calpain-specific siRNA (small interference RNA) and the blockage was accompanied by a concomitant decrease in APPs release. In contrast, m-calpain siRNA did not affect APPs release significantly. Measurement of amyloid β protein (Aβ) release in the μ-calpain siRNA-treated cells indicated that Aβ40 and Aβ42 levels inversely changed in relation to APPs, and the changes in Aβ42 were more prominent than in Aβ40. Together, these data suggest that calpain, particularly μ-calpain, is a potential candidate for α-secretase in the regulated APP α-processing, and that changes in this protease can affect the outcome of the overall APP processing.

Endothelin-1 Acts via Protein Kinase C to Block KATP Channels in Rabbit Coronary and Pulmonary Arterial Smooth Muscle Cells

We investigated the effects of the vasoconstrictor endothelin-1 (ET-1) on the whole-cell ATP-sensitive K+ (KATP) currents of smooth muscle cells that were isolated enzymatically from rabbit coronary artery (CASMCs) and pulmonary artery (PASMCs). The size of the KATP current did not differ significantly between CASMCs and PASMCs. ET-1 reduced the KATP current in a concentration-dependent manner, and this inhibition was greater in PASMCs than in CASMCs (half-inhibition values of 12.20 nM and 1.98 nM in CASMCs and PASMCs, respectively). However, the level of inhibition induced by other vasoconstrictors (angiotensin II, norepinephrine, and serotonin) were not significantly different between CASMCs and PASMCs. Pretreatment with the protein kinase C (PKC) inhibitors staurosporine (100 nM) and GF 109203X (1 microM) prevented ET-1-induced inhibition of the KATP current in both arterial smooth muscle cell preparations. The PKC activators phorbol-12,13-dibutyrate (PDBu) and 1-olelyl-2-acetyl-sn-glycerol (OAG) reduced the KATP current in dose-dependent manner. Although the numbers of ET receptors were not significantly different between the 2 arterial smooth muscle cell preparations, the effects of PDBu and OAG were greater on PASMCs. ET-1-induced inhibition of the KATP current was unaffected by the PKA inhibitor Rp-cAMPs (100 microM) and PKA inhibitory peptide (5 microM).

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C.

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C.

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

Protein kinase C may regulate the tonic component of intestinal smooth muscle contraction in response to substance P

(1) The study investigated a possible involvement of protein kinase C (PKC) in the substance P-induced contraction of the longitudinal muscle of the guinea-pig isolated ileum. (2) The predominant effect of the PKC activator, phorbol-12,13-dibutyrate (PDB), was to change the time course of the response to substance P. While the initial peak contraction was hardly influenced by PDB, the fading of the contraction was accelerated to an extent that any tonic contraction which normally followed the initial peak response was prevented. This inhibitory effect of PDB on the tonic contraction was immediate in onset and related to its concentration (20-200 nM); responses to half-maximally (2-7 nM) or maximally effective (0.74 microM) concentrations of substance P were affected in the same manner. Tetrodotoxin (0.6 microM) did not alter the effect of PDB. Phorbol-13-monoacetate (2 microM), a phorbol ester which does not stimulate PKC, failed to change the time course of the substance P-induced contraction. (3) The tonic component of half-maximal contractile responses to histamine (0.2-0.4 microM) was also depressed by PDB (0.2 microM) whereas the tonic component of maximal responses to histamine (9 microM) was enhanced. (4) PDB (0.2 microM) reduced desensitization to substance P as judged by the reduction of the peak response to substance P (2-7 nM) following a 10-min exposure to a high concentration of the peptide (0.74 microM). (5) The PKC inhibitor, polymyxin B (0.1-0.3 mM), reduced the peak contractile response to substance P, slowed the fading of the contraction, and antagonized the inhibitory effect of PDB on the tonic contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C regulates functional coupling of beta1-adrenergic receptors to Gi/o-mediated responses in cardiac myocytes.

The effect of protein kinase C (PKC) activation on beta1-adrenergic receptor (beta1-AR) regulation of the cardiac L-type Ca2+ current (ICa,L) was studied using the whole-cell patch clamp technique. Treatment of guinea pig ventricular myocytes with phorbol-12,13-dibutyrate (PDBu) caused a significant decrease in ICa,L sensitivity to stimulation by submaximal beta1-AR activation using isoproterenol (Iso). This decrease in sensitivity was also associated with the ability of higher concentrations of Iso to directly inhibit the stimulatory response. PDBu treatment produced similar effects on H2 histamine receptor-mediated ICa,L responses. In the presence of PDBu, higher concentrations of Iso inhibited the histamine stimulated ICa,L, and this effect was blocked by a selective beta1-AR antagonist. Higher concentrations of histamine also inhibited the Iso stimulated ICa,L, and this effect was blocked by a selective H2 receptor antagonist. The effects of PDBu were blocked by the PKC inhibitor bisindolylmaleimide I, and they were not mimicked by the inactive phorbol ester 4alpha-phorbol-12,13-didecanoate. The inhibitory effects of Iso and histamine were significantly reduced when Gi/o mediated responses were blocked with pertussis toxin. These results suggest that PKC promotes coupling of cardiac beta1-adrenergic and H2 histamine receptors to Gi/o mediated inhibitory responses.

Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4- d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.

Protein kinase C regulation of glycine and γ-aminobutyric acid release in brain stem auditory nuclei

Protein kinase C (PKC) can regulate transmitter release in several brain areas. We determined if PKC could regulate the electrically evoked release of radiolabeled glycine (Gly) and γ-aminobutyric acid (GABA) in dissected samples of several brain stem auditory nuclei, such as the major subdivisions of the cochlear nucleus (CN) and the main nuclei of the superior olivary complex (SOC). The PKC activators, phorbol 12,13-diacetate (PDA) or phorbol 12,13-dibutyrate (PDBu) (3 μM), elevated the release by 1.4- to 2.0-fold. The PKC inhibitor, Ro31-8220 (50 nM), did not alter the release in most of the tissues but blocked the stimulatory effects of PDA and PDBu. This suggested that PKC positively regulates glycinergic and GABAergic release in the sampled nuclei. In the dorsal CN (DCN), Ro31-8220 elevated the release of [ 14C]Gly by 23%, suggesting that PKC negatively regulates glycinergic release in a proportion of DCN synapses. We also determined if PKC could regulate release after unilateral cochlear ablation (UCA). In the anteroventral (AVCN) and posteroventral (PVCN) CN and in the lateral (LSO) and medial (MSO) superior olive, the stimulatory effects of PDBu declined after this lesion and Ro31-8220 failed to alter release. Since UCA failed to alter release in these tissues, the stability of the release correlated with the lack of regulatory capacity of PKC. In the DCN and the medial nucleus of the trapezoid body (MNTB), the stimulatory effects of PDBu persisted after UCA. We previously demonstrated a postablation decline of Gly release in the DCN and elevated GABA release in the MNTB. Treatment of these tissues with Ro31-8220 reversed these changes in release. These findings suggested that PKC regulation persisted in the DCN and MNTB after UCA. Moreover, endogenous regulatory mechanisms activated after UCA probably act through PKC to alter release in these tissues. Thus, limiting PKC activation or activity might ameliorate pathological symptoms that accompany hearing loss and that stem from these plasticities in the DCN and MNTB.

Regulation of mdm2 mRNA expression in human breast tumor-derived GI-101A cells

The MDM2 oncoprotein (p90) binds to p53 and inhibits its function. Here, the expression of mdm2 mRNA subsequent to phorbol 12,13-dibutyrate (PDB) or diethylstilbestrol (DES) treatment was analyzed in human breast tumor-derived GI-101A cell line. Expression of mdm2 mRNA was detected in rapidly growing GI-101A cells and that was similar to the expression seen in HL-60 cells. On the other hand PC12 (rat adrenal pheochromocytoma cells) did not show any mdm2 expression. GI-101A cells were treated with varying concentrations of DES or PDB, and mdm2 mRNA levels were determined by RT-PCR analysis. The RT-PCR results clearly showed that mdm2 mRNA expression was increased with increasing concentrations of PDB and DES treatments. To determine the specificity of the effects produced by DES and PDB the cells were treated with estrogen receptor antagonist tamoxifen and protein kinase C (PKC) specific inhibitor chelerythrine. Tamoxifen and chelerythrine co-treatments inhibited DES and PDB stimulated increases of mdm2 transcription respectively, in GI-101A cells. In an attempt to determine the upstream signaling pathway, the effects of PDB or DES on the mitogen activated protein kinase (MAPK) levels were determined by western blot analysis in the presence and absence of PD098059, a specific inhibitor of mitogen activated protein kinase kinase (MAPKK). The phospho-MAPK (p44/42) levels, an activated form of MAPK, increased in DES and PDB stimulated cells whereas PD098059 treatment inhibited this increase. Our data implicate MAPK as an upstream regulator of mdm2 expression and help to speculate on the intracellular regulation of mdm2 expression.

Inhibition of cAMP accumulation by kappa-receptor activation in isolated iris-ciliary bodies: role of phosphodiesterase and protein kinase C.

The present study was designed to examine the roles of protein kinase C (PKC) and phosphodiesterase (PDE) in modulating the action of kappa receptor stimulation on cAMP accumulation in isolated iris-ciliary bodies (ICBs) of New Zealand White rabbits. The kappa receptor agonist, (+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL), and the PKC activator, phorbol 12,13-dibutyrate (PDBu), both caused a concentration-dependent inhibition of forskolin-stimulated cAMP production. The inhibitory effect of BRL on cAMP levels was significantly reduced in the presence of the selective kappa receptor antagonist, norbinaltorphimine (10(-6) M), but the effect of PDBu was not, thus supporting the involvement of kappa-opioid receptors in the response to BRL. In the presence of 3-isobutyl-1-methylxanthine or rolipram (10(-5) M), the inhibitory effect of BRL or PDBu (10(-6) M) on cyclic AMP accumulation was abolished. In the presence of the selective PKC antagonist, chelerythrine (10(-6) M), the inhibitory effect of PDBu or BRL (10(-6) M) was significantly reduced. Direct measurement of PDE activity demonstrated the ability of BRL and PDBu (10(-6) M) to augment the activity of these enzymes. Preincubation of ICBs with rolipram (10(-5) M) or chelerythrine (10(-6) M) caused significant reversal of both BRL- and PDBu-induced increases in PDE activity. These results indicate that stimulation of PKC and PDE4 activity is part of the complex mechanism whereby kappa-opioid receptor agonists reduce levels of cAMP in the rabbit ICB. This mechanism of action could contribute to the ability of kappa-opioid agonists to suppress aqueous flow rate and to lower intraocular pressure.