Phorbol Dibutyrate Research Articles

Role of Protein Kinase C in Substance P-Induced Synaptic Depression in the Nucleus Accumbens in vitro

Objectives: This study set out to determine the roles of protein kinase A (PKA) and protein kinase C (PKC) signalling cascades in substance P- (SP-) mediated synaptic depression in the nucleus accumbens. Materials and Methods: We used whole-cell patch recording in rat forebrain slices to study the effects of excitatory and inhibitory modulators of PKA and PKC to determine their effects on SP-induced synaptic depression. Results: We showed that cAMP and PKC, but not PKA, are involved in SP-induced synaptic depression. Bath application of SP (1 µM) depressed evoked excitatory postsynaptic currents (EPSCs) by –27.50 ± 5.6% (n = 8). Pretreatment of slices with 10 µM forskolin or rolipram prevented SP (1 µM) from depressing evoked EPSCs (–0.8 ± 6.7%, n = 6; p > 0.05 and 1.6 ± 5.6%, n = 8; p > 0.05, respectively). Furthermore, 8-bromo cAMP (1 mM) also blocked the effect of SP (–0.5 ± 14.8, n = 4, p > 0.05). However, H-89 (1 µM) did not block the SP-induced synaptic depression (–32.3 ± 4.0%, n = 4, p < 0.05). By contrast, PKC inhibitors bisindolylmaleimide (1 µM; 4.0 ± 5.1%, n = 6; p > 0.05) and calphostin C (400 nM; –6.7 ± 6.5%, n = 4, p > 0.05) both blocked SP-induced synaptic depression. Phorbol dibutyrate caused a synaptic depression of –33.0. ± 5.0% and abolished the effect of SP (1 µM, –5.9 ± 8.6%, n = 4, p > 0.05). Conclusion: Our findings demonstrate that PKC and cAMP are involved in SP-induced synaptic depression while PKA is apparently not involved. Involvement of multiple signalling pathways may reflect the fact that SP uses several intermediates to depress EPSCs.

Phosphorylation of CPI‐17 and MYPT in rabbit bladder smooth muscle: Role of PKC, Rho kinase, or both

Smooth muscle is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase (MLCP). Inhibition of the MLCP following agonist stimulation results in a net increase in MLC phosphorylation due to PKC catalyzed CPI-17 phosphorylation or Rho kinase (ROK) catalyzed phosphorylation of a phosphatase subunit (MYPT). This study was designed to investigate if PKC and ROK inhibit MLCP by two distinct pathways or if there is cross-talk in bladder smooth muscle. This goal was pursued by measuring contraction of rabbit bladder smooth muscle in response to carbachol or phorbol dibutyrate (PDBu) in the absence and presence of inhibitors of PKC (Bisindolylmaleimide-1) or ROK (H-1152). Inhibition of PKC and ROK depressed both carbachol and PDBu induced contractions. PKC inhibition decreases contraction mainly through the inhibition of CPI-17 phosphorylation and to a lesser degree by inhibition of MYPT phosphorylation at both Thr696 and Thr850. ROK inhibition decreases basal levels of MYPT phosphorylation, predominantly at Thr850 which suggests a role for a constitutively active ROK. We propose that PKC modulates contraction directly by phosphorylation of CPI-17 and indirectly by activation of ROK resulting in an increase in MYPT phosphorylation. ROK on the other hand only acts via MPYT phosphorylation in bladder smooth muscle.

Signalling pathways involved in the control of sperm cell volume

The ability to maintain cellular volume is an important general physiological function, which is achieved by specific molecular mechanisms. Hypotonically induced swelling results in the opening of K+ and Cl- ion channels, through which these ions exit with accompanying water loss. This process is known as regulatory volume decrease (RVD). The molecular mechanisms that control the opening of the ion channels in spermatozoa are as yet poorly understood. The present study investigated pathways of osmo-signalling using boar spermatozoa as a model. Spermatozoa were diluted into isotonic and hypotonic Hepes-buffered saline in the presence or absence of effector drugs, and at predetermined intervals volume measurements were performed electronically. Treatment with protein kinase C (PKC) inhibitors staurosporine, bismaleimide I and bismaleimide X led to dose-dependent increases of both isotonic and hypotonic volumes (P<0.05). However, as the isotonic volume was affected more than the hypotonic volume, the kinase inhibitors appeared to improve RVD, whereas activation of PKC with phorbol dibutyrate blocked RVD. The increase in isotonic cell volume induced by bismaleimide X was observed in chloride-containing medium but not in the medium in which chloride was replaced by sulphate, implying that PKC was involved in the control of chloride channel activity, e.g. by closing the channel after volume adjustment. The protein phosphatase PP1/PP2 inhibitors calyculin and okadaic acid increased the isotonic volume only slightly but they greatly increased the relative cell volume and blocked RVD. The activation of RVD processes was found to be cAMP-dependent; incubation with forskolin and papaverine improved volume regulation. Moreover, papaverine was able to overcome the negative effect of protein phosphatase inhibitors. The mechanism of sperm RVD appears to involve (a) alterations in protein phosphorylation/dephosphorylation balance brought about by PKC and PP1 and (b) a cAMP-dependent activating pathway.

Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells

We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained α-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.

(±)3,4-Methylenedioxyamphetamine elicits action potential bursts in a central snail neuron

The effects of (±)3,4-methylenedioxyamphetamine (MDA) were studied in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. The RP4 neuron generated spontaneous action potentials. Extracellular or intracellular application of MDA elicited action potential bursts of the central RP4 neuron. The action potential bursts elicited by MDA were not blocked when neurons were immersed in high-Mg 2+ solution, Ca 2+-free solution, nor after continuous perfusion with atropine, d-tubocurarine, propranolol, prazosin, haloperidol, sulpiride or methiothepin. Notably, the induction of action potential bursts was blocked by pretreatment with protein kinase C (PKC) inhibitors, chelerythrine and Ro 31-8220, but not by protein kinase A (PKA) inhibitors, KT-5720 and H89, nor by the phospholipase C (PLC) inhibitor, U73122. PKC activators, i.e., phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acety- sn-glycerol (OAG; a membrane-permeant DAG analog), facilitate the induction of action potential bursts elicited by MDA. Voltage-clamp studies revealed that MDA decreased the delayed rectifying K + current (I KD) of the RP4 neuron. Further, although Ro 31-8220 did not affect the I KD, Ro 31-8220 decreased the inhibitory effect of MDA on the I KD. These results suggest that the generation of action potential bursts elicited by MDA was not due to (1) the synaptic effects of neurotransmitters, (2) the cholinergic, adrenergic, dopaminergic or serotoninergic receptors of the excitable membrane. Instead, the MDA-elicited action potential bursts are closely related to PKC activity and the inhibitory effects on the I KD.

Protein kinase C (PKC) regulates contractility of myometrium from pregnant women

PREGNANT WOMEN VICTOR FOMIN, ANDRIS KRONBERGS, MATTHEW HOFFMAN, University of Delaware, Biological Sciences, Newark, Delaware, Christiana Hospital, Newark, Delaware OBJECTIVE: Prematurity is one of the most significant problems of human pregnancy. According to the March of Dimes, the rate of premature birth increased between 1981 and 2003 by 30% (from 9.4 to 12.3%). A detailed knowledge of the mechanisms of uterine contraction can help to improve clinical control of abnormal uterine function and reverse this trend. Important regulator of smooth muscle contraction is protein kinase C (PKC). However, the role of PKC in uterine (myometrial) contraction remains unclear. This study was designed to investigate an effect of PKC on contraction and intracellular Ca concentration ([Ca]i) in myometrium from term pregnant women. STUDY DESIGN: The myometrial samples were obtained at the time of cesarean section surgery according to the protocol approved by Christiana Hospital and University of Delaware IRB Committees. The isometric contraction (tension) and [Ca]i were measured simultaneously in fura-2 loaded myometrial strips. RESULTS: The PKC activation with phorbol 12,13-dibutyrate (PDBu) increased tonic contraction time and dosedependently reaching maximum after 2hrs at 10 -6M with no effect on [Ca]i. PDBu first potentiated KCl(50mM) contractile responses followed by their inhibition after 1 hr incubation with no effect on the amplitude of the [Ca]i response. PKCalpha inhibitor Go6976 decreased the PDBu effect on the tonic contraction. The depletion of PKCalpha protein with the specific antisense oligonucleotide decreased the KCl-induced contractile and [Ca]i responses. CONCLUSION: It is suggested that PKC regulates uterine contraction through different signaling pathways. First, PKC (possibly PKCalpha) stimulates KCl-induced contractile responses followed by their inhibition due to yet to be determined mechanisms. PKC also induces myometrial tonic contraction without changes in [Ca]i. (Supported by NIH R55 HD45802).

Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1

Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca(2+) sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were approximately 15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTPgammaS was increased in KO compared with WT as shown using a pull-down assay. Following alpha-toxin permeabilization, no difference in Ca(2+) sensitivity or in Ca(2+) sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased alpha(1)-adrenergic contraction. Following inhibition of PKC, alpha(1)-adrenergic contraction was normalized. PDBu-induced Ca(2+) sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca(2+) sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca(2+) sensitivity.

Characterization and Phospholipase D Mediation of the Angiotensin II Priming Response in Adrenal Glomerulosa Cells

Bovine adrenal glomerulosa cells are primed by an initial treatment with angiotensin II (AngII) to respond with enhanced secretion to a second exposure to AngII or agents that increase calcium influx. We hypothesized that the mechanism of priming involves a persistent increase in diacylglycerol (DAG) generated via sustained activity of phospholipase D (PLD). In this report, we sought to define the time frame of this priming response as well as determine its mechanism using assays of aldosterone secretion, PLD activation, and radiolabeled diacylglycerol levels. We found that in primary cultures priming was observed for up to 50 min after AngII washout, suggesting that the priming window is protracted in these cultures relative to freshly isolated cells. The phorbol ester, phorbol 12,13-dibutyrate (PDBu), was used to investigate the role of sustained PLD activation in the persistent DAG and priming responses. PDBu was able to both prime glomerulosa cells to respond with enhanced secretion to AngII and elicit a persistent increase in DAG after PDBu washout. This persistent increase in DAG levels with an initial exposure to PDBu or AngII was not the result of maintained PLD activity after agent removal because PLD activation returned to basal levels by 30 min after washout. Finally, inhibition of PLD signaling during the initial AngII treatment inhibited the subsequent response to AngII or another agent that increases calcium influx. Thus, our results suggest that persistent DAG resulting from PLD signaling mediates the priming response to AngII or PDBu.

Partial Bladder Outlet Obstruction Selectively Abolishes Protein Kinase C Induced Contraction of Rabbit Detrusor Smooth Muscle

Despite the acute onset, partial bladder outlet obstruction in the rabbit induces detrusor remodeling similar to that in men with benign prostatic hyperplasia in terms of its impact on structural and functional alterations in smooth muscle. We determined if partial bladder outlet obstruction induced remodeling alters the protein kinase C signaling pathway that leads to contraction. Smooth muscle from control animals and those subjected to 2 weeks of partial bladder outlet obstruction were mounted for isometric force recording, measurement of myosin light chain phosphorylation and levels of adducin phosphorylation. Bladder muscle strips were stimulated by phorbol dibutyrate or carbachol in the presence and absence of bisindolylmaleimide-1. Smooth muscle strips from animals subjected to partial bladder outlet obstruction showed little to no increase in stress in response to phorbol dibutyrate and no increase in myosin light chain phosphorylation levels. Muscle strips from control animals produced a robust contraction with concomitant increases in myosin light chain phosphorylation. Inhibition of protein kinase C by bisindolylmaleimide-1 significantly depressed carbachol induced contractions of muscle strips from control animals but it had no effect on carbachol induced contractions of muscle strips from outlet obstructed animals. Phorbol dibutyrate increased phospho-adducin levels in muscle strips from the 2 animal sources, suggesting that protein kinase C could be activated. We propose that partial bladder outlet obstruction does not alter protein kinase C activation, but rather abolishes or uncouples the pathway(s) downstream of protein kinase C, leading to contraction. Loss of this pathway may contribute to the loss of normal voiding behavior and the resultant decompensated state.

Cross-linking of MHC class II molecules interferes with phorbol 12,13-dibutyrate-induced differentiation of resting B cells by inhibiting Rac-associated ROS-dependent ERK/p38 MAP kinase pathways leading to NF-κB activation

In addition to their essential role in antigen presentation, major histocompatibility complex (MHC) class II molecules have been described as the receptor associated with signal transduction regulating B-cell function. In previous experiments, we found that cross-linking of MHC class II molecules with corresponding anti-MHC class II antibodies inhibited NF-κB-activated signaling pathways associated with the proliferation and differentiation of the LPS-stimulated primary and resting B-cell line, 38B9. We also found that exposure to the anti-MHC class II antibody reduced the production of ROS, which function as secondary signal transducers, in the phorbol 12,13-dibutyrate (PDBU)-treated (but not in the LPS-treated) resting B-cell line. In this study, we investigated the molecular mechanisms in the ROS-associated signaling pathway leading to PDBU-induced NF-κB activation that results in B-cell differentiation and speculated that the signaling pathway was inhibited by exposure to the anti-MHC class II antibody. We also found that this inhibition was mediated through down-regulation of the activated Rac/ROS-associated ERK/p38 MAPK signaling pathway in PDBU-treated 38B9 cells. Collectively, these findings suggest that ROS-associated molecules are involved in MHC class II-associated negative signal transduction in resting B cells.

Conjugated Linoleic Acid Reduces Phorbol Ester-Induced Prostaglandin F2α Production by Bovine Endometrial Cells

Recent interest in conjugated linoleic acid (CLA) research stems from the well-documented anticarcinogenic, antiatherogenic, antidiabetic, and antiobesity properties of CLA in animal models. The objective of this study was to examine the effects of 2 CLA isomers (cis-9,trans-11 and trans-10,cis-12) on phorbol 12,13-dibutyrate (PDBu)-induced PGF2α production in cultured bovine endometrial (BEND) cells. Confluent BEND cells were incubated in the absence (control) or presence of 100μM each of linoleic acid, cis-9,trans-11 CLA, or trans-10,cis-12 CLA for 24h. After incubation, cells were rinsed and then stimulated with PDBu (100 ng/mL) for 6h. Compared with untreated cells, PDBu stimulated PGF2α secretion (+25-fold) within 6h. The increases in PGF2α secretion were paralleled by signifi-cant induction of prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA (+63-fold) and protein (+1.6-fold) expression. In spite of stimulatory effects on PGHS-2 and peroxisome proliferator-activated receptor δ (PPARδ) mRNA responses, CLA greatly decreased PGF2α production by PDBu-stimulated BEND cells. There was no evidence for PDBu or CLA modulation of PPARδ protein synthesis in cultured BEND cells. Results indicated that CLA modulation of PGF2α production by BEND cells was not mediated through PGHS-2 or PPARδ gene repression.

Mucosal stimulation activates secretomotor neurons via long myenteric pathways in guinea pig ileum

This study examined whether mucosal stimulation activates long secretomotor neural reflexes and, if so, how they are organized. The submucosa of in vitro full thickness guinea pig ileal preparations was exposed in the distal portion and intracellular recordings were obtained from electrophysiologically identified secretomotor neurons. Axons in the intact mucosa of the oral segment were stimulated by a large bipolar stimulating electrode. In control preparations, a single stimulus pulse evoked a fast excitatory postsynaptic potential (EPSP) in 86% of neurons located 0.7-1.0 cm anal to the stimulus site. A stimulus train evoked multiple fast EPSPs, but slow EPSPs were not observed. To examine whether mucosal stimulation specifically activated mucosal sensory nerve terminals, the mucosa/submucosa was severed from the underlying layers and repositioned. In these preparations, fast EPSPs could not be elicited in 89% of cells. Superfusion with phorbol dibutyrate enhanced excitability of sensory neurons and pressure-pulse application of serotonin to the mucosa increased the fast EPSPs evoked by mucosal stimulation, providing further evidence that sensory neurons were involved. To determine whether these reflexes projected through the myenteric plexus, this plexus was surgically lesioned between the stimulus site and the impaled neuron. No fast EPSPs were recorded in these preparations following mucosal stimulation whereas lesioning the submucosal plexus had no effect. These results demonstrate that mucosal stimulation triggers a long myenteric pathway that activates submucosal secretomotor neurons. This pathway projects in parallel with motor and vasodilator reflexes, and this common pathway may enable coordination of intestinal secretion, blood flow, and motility.

Differential actin isoform reorganization in the contracting A7r5 cell

In the present study, we investigated the reorganization of alpha- and beta-actin in the contracting A7r5 smooth muscle cell. The remodeling of these actin variants was markedly different in response to increasing concentrations of phorbol 12, 13-dibutyrate (PDBu). At the lowest concentrations (< or =10(-7) mol/L), cells showed an approximately 70% loss in alpha-actin stress fibers with robust transport of this isoform to podosomes. By comparison, beta-actin remained in stress fibers in cells stimulated at low concentrations (< or =10(-7) mol/L) of PDBu. However, at high concentrations (> or =10(-6)mol/L) approximately 50% of cells showed transport of beta-actin to podosomes. Consistent with these findings, staining with phalloidin indicated a significant decrease in the whole-cell content of F-actin with PDBu treatment. However, staining with DNase I indicated no change in the cellular content of G-actin, suggesting reduced access of phalloidin to tightly packed actin in the podosome core. Inhibition of protein kinase C (staurosporine, bisindolymaleimide) blocked PDBu-induced (5 x 10(-8) mol/L) loss in alpha-actin stress fibers or reversed podosome formation with re-establishment of alpha-actin stress fibers. By comparison, these inhibitors caused partial loss of beta-actin stress fibers. The results support our earlier conclusion of independent remodeling of alpha- and beta-actin cytoskeletal structure and suggest that the regulation of these structures is different.

Desensitization of somatostatin-induced inhibition of low extracellular magnesium concentration-induced calcium spikes in cultured rat hippocampal neurons

Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg 2+ concentration ([Mg 2+] o)-induced intracellular free Ca 2+ concentration ([Ca 2+] i) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg 2+] o to 0.1 mM elicited repetitive [Ca 2+] i spikes. These [Ca 2+] i spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18–24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca 2+] i spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 μM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCε translocation inhibitor peptide (1 μM). In addition, suramin (3 μM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 μM) completely blocked the low [Mg 2+] o-induced [Ca 2+] i spikes, glutamate-induced [Ca 2+] i transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg 2+] o-induced [Ca 2+] i spikes through the activation of GRK2 and partly a novel PKCε in cultured rat hippocampal neurons.

Dual effect of calmodulin on store-operated Ca2+ -permeable cation channels in rabbit portal vein myocytes.

We have previously described a Ca(2+)-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes, which is activated by agents that deplete internal Ca(2+) stores and also by protein kinase C (PKC). In the present study, we investigated the effect of calmodulin (CaM) on store-operated channels (SOCs) with electrophysiological techniques. Bath application of the CaM inhibitor calmidazolium (CMZ) to quiescent cells produced transient activation of SOC activity in cell-attached patches. CMZ produced a dual effect on cyclopiazonic acid (CPA)-evoked SOCs by initially inducing an increase in mean open probability (NP(o)) and subsequently producing a marked inhibition of SOC activity. In contrast, SOCs activated by the cell-permeable Ca(2+) chelator 1,2-bis (2-aminophenoxy)ethane-N-N,N',N'-tetraacetic acid (BAPTA-AM) were inhibited by CMZ. In inside-out patches where SOCs were activated by CPA or the PKC activator phorbol-12,13-dibutyrate (PDBu), bath application of CaM induced an initial inhibition followed by an increase in SOC activity. In contrast, CaM only enhanced BAPTA-AM-evoked SOC activity in inside-out patches. Bath application of CaM to the cytoplasmic surface of quiescent inside-out patches evoked single channel currents with biophysical properties similar to SOCs. The inhibitory action of CaM on PDBu-induced SOC activity was inhibited by the calmodulin-dependent kinase II (CaM kinase II) inhibitor autocamtide-related inhibitory peptide (AIP) but not by inhibitors of calcineurin or myosin light chain kinase (MLCK). In addition, CaM-evoked channel currents were inhibited by coapplication of purified CaM kinase II but not by inhibitors of CaM kinase II, calcineurin or MLCK. With whole-cell and cell-attached recording, bath application of the CaM kinase II inhibitors KN93 and AIP evoked SOCs in unstimulated myocytes. These results indicate that CaM has pronounced dual inhibitory and excitatory actions on SOCs with the inhibitory effect of CaM mediated by CaM kinase II. Moreover, the present work provides strong evidence that CaM has an important role in activating SOCs, possibly through a direct action on channel/associated proteins.

Protein Kinase D Intracellular Localization and Activity Control Kinase D-interacting Substrate of 220-kDa Traffic through a Postsynaptic Density-95/Discs Large/Zonula Occludens-1-binding Motif

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.

Controle da síntese de prostaglandina F2± no endométrio bovino in vitro

A síntese de prostaglandina F2± (PGF2±) endometrial, resulta de uma complexa cascata de eventos intracelulares que ocorrem de maneira altamente coordenada. A síntese de PGF2± pode ser estimulada na presença da melitina e do forbol 12,13 dibutirato (PDBu). O objetivo do presente estudo foi verificar se a produção basal e a estimulação aguda de PGF2± são dependentes da síntese de proteínas. No experimento 1, explantes obtidos de fêmeas bovinas (n=2), cíclicas, não lactantes, no dia 15 do ciclo estral foram incubados em quadruplicata, com meio de cultivo (KHB) ou KHB suplementado com PDBu 10-6M, melitina 10-6M ou melitina 10-5M. Amostras do meio foram coletadas 0 e 60 minutos após os tratamentos e as concentrações de PGF2± foram mensuradas por radioimunoensaio. Com 60 minutos após os tratamentos houve aumento das concentrações médias de PGF2± (P&lt;0,06) em resposta ao tratamento com PDBu comparado ao grupo KHB e melitina. No experimento 2, explantes endometriais de fêmeas bovinas (n=4), não gestantes, no 17° dia do ciclo estral, pesando de 80 a 100mg foram incubados em KHB suplementado com 0, 50, 100 ou 200mg/mL de CHX e 0 ou 100ng/mL de PDBu em um arranjo fatorial 2 x 4, em quadruplicata. Amostras do meio foram coletadas 0 e 60 minutos após os tratamentos e as concentrações de PGF2± foram mensuradas por radioimunoensaio. A integridade dos explantes endometriais tratados com CHX foi avaliada por cortes histológicos. Foi observado aumento na produção de PGF2± em resposta ao tratamento com PDBu (P&lt;0,01), entretanto, não houve diferenças na produção de PGF2± pelos tecidos tratados com diferentes concentrações de CHX, associadas ou não ao PDBu. A integridade histológica dos explantes foi preservada. Conclui-se que a produção basal e a estimulação aguda da síntese de PGF2± não são dependentes da síntese de novas proteínas.

Endothelin-1 and PKC induce positive inotropy without affecting pHi in ventricular myocytes.

It has been proposed that intracellular alkalinization underlies the enhanced contractility of ventricular myocytes exposed to endothelin (ET)-1. The effects of ET-1 on the contractility and intracellular pH (pH(i)) were examined here in cultured adult rat ventricular myocytes by employing the pH-sensitive fluorescent dye SNARF-1. Variable pH(i) changes were observed on ET-1 stimulation. Most myocytes (n = 20 of 32) did not alkalinize, but showed an approximate 60% increase in twitch amplitude in response to ET-1. In the remaining myocytes (12 of 32), ET-1 induced an increase in pH(i) by 0.05 +/- 0.02 pH units with a similar approximate 60% increase in twitch amplitude. Therefore, there was no strong correlation between ET-1-mediated positive inotropy (enhanced contractility) and intracellular alkalinization. To determine whether ET-1 contractile and pH(i) responses were mediated by protein kinase C (PKC), yellow fluorescent protein (YFP)-fused dominant negative (dn) PKC constructs were used as isoform specific inhibitors. In dn-PKC-epsilon-YFP-expressing myocytes, the ET-1-mediated positive inotropic response was greatly diminished to 13 +/- 15%, but alkalinization was still observed. Expression of dn-PKC-delta-YFP also did not block alkalinization, but in this case the positive inotropic response was still observed. In a previous study, we showed that expression of PKC-delta and PKC-epsilon caused a strong positive inotropy on stimulation with phorbol 12,13-dibutyrate (PDBu). Using this system, PDBu failed to affect pH(i) in the majority of PKC expressing myocytes despite increases in twitch amplitudes of >60%. Overall, the poor correlation of positive inotropic responses and alkalinization was observed for ET-1 with and without dn-PKC constructs and for PDBu with and without wild-type PKC constructs. These results suggest that ET-1 produces positive inotropy via PKC-epsilon by mechanisms other than intracellular alkalinization.

Regulation of α1-adrenoceptor-mediated contractions of uterine arteries by PKC: effect of pregnancy

Protein kinase C (PKC) plays an important role in the regulation of uterine artery contractility and its adaptation to pregnancy. The present study tested the hypothesis that PKC differentially regulates alpha(1)-adrenoceptor-mediated contractions of uterine arteries isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep. Phenylephrine-induced contractions of NPUA and PUA sheep were determined in the absence or presence of the PKC activator phorbol 12,13-dibutyrate (PDBu). In NPUA sheep, PDBu produced a concentration-dependent potentiation of phenylephrine-induced contractions and shifted the dose-response curve to the left. In contrast, in PUA sheep, PDBu significantly inhibited phenylephrine-induced contractions and decreased their maximum response. Simultaneous measurement of contractions and intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in the same tissues revealed that PDBu inhibited phenylephrine-induced [Ca(2+)](i) and contractions in PUA sheep. In NPUA sheep, PDBu increased phenylephrine-induced contractions without changing [Ca(2+)](i). Western blot analysis showed six PKC isozymes, alpha, beta(I), beta(II), delta, epsilon, and zeta, in uterine arteries, among which beta(I), beta(II), and zeta isozymes were significantly increased in PUA sheep. In contrast, PKC-alpha was decreased in PUA sheep. In addition, analysis of subcellular distribution revealed a significant decrease in the particulate-to-cytosolic ratio of PKC-epsilon in PUA compared with that in NPUA sheep. The results suggest that pregnancy induces a reversal of PKC regulatory role on alpha(1)-adrenoceptor-mediated contractions from a potentiation in NPUA sheep to an inhibition in PUA sheep. The differential expression of PKC isozymes and their subcellular distribution in uterine arteries appears to play an important role in the regulation of Ca(2+) mobilization and Ca(2+) sensitivity in alpha(1)-adrenoceptor-mediated contractions and their adaptation to pregnancy.

Vasorelaxant effect of euxanthone in the rat thoracic aorta

This study was undertaken to investigate the effect of euxanthone on isolated rat thoracic aorta. Euxanthone concentration-dependently relaxed high K +-induced sustained contractions with IC 50 values of 32.28 ± 1.73 μM and this inhibition was antagonized by increasing the Ca 2+ concentration in the medium. These results indicated that euxanthone may have calcium antagonistic property. Euxanthone also relaxed norepinephrine (NE)-induced sustained contractions with IC 50 values of 32.50 ± 2.15 μM and this relaxant effect was unaffected by the removal of endothelium or by the presence of propranolol, indomethacin, glibenclamide or N ω-nitro- l-arginine. Moreover, euxanthone inhibited both the phasic and tonic contractions induced by NE in a concentration-dependent manner and showed more potent inhibition on phasic contraction ( P < 0.01). Pre-treatment with euxanthone inhibited vascular contraction induced by phorbol 12, 13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, in either the presence or absence of Ca 2+ in the solution with IC 50 values of 20.15 ± 1.56 and 18.30 ± 1.62 μM, respectively. However, when the tissues were treated with euxanthone after the PDBu-induced contraction had reached a steady state, the tension was not affected by euxanthone. This study also showed that the inhibitory effect of pre-treatment of euxanthone was more potent than the post-treatment after the tension had reached a steady state. These results suggested that the vasorelaxation of euxanthone may be through multiple pathways involved PKC-mediated signal pathway and calcium-independent pathway besides the direct inhibition of calcium influx and its vasorelaxant effect is more active on calcium-independent pathway and more sensitive to the initial stage of contraction.