Roles of Ca2+ and protein kinase C in the excitatory response to serotonin in embryonic molluscan ciliary cells

We examined the roles of Ca2+ and protein kinase C (PKC) in the cilio-excitatory response to serotonin in pedal ciliary cells from Helisoma trivolvis embryos. Serotonin (5-hydroxytryptamine; 5-HT; 100 micromol/L) induced an increase in ciliary beat frequency (CBF) was abolished by microinjected BAPTA (50 mmol/L), but was only partially inhibited by the phospholipase C inhibitor U-73122 (10 micromol/L). The diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol (100 micromol/L) and 1,2-dioctanoyl-sn-glycerol (100 micromol/L) caused increases in [Ca2+]i that were smaller than those induced by serotonin. In the absence of extracellular Ca2+, 1,2-dioctanoyl-sn-glycerol (100 micromol/L) failed to elicit an increase in both CBF and [Ca2+]i. In contrast, the serotonin-induced increase in CBF persisted in the absence of extracellular Ca2+, although the increase in [Ca2+]i was abolished. PKC inhibitors bisindolylmaleimide (10 and 100 nmol/L) and calphostin C (10 nmol/L) partially inhibited the serotonin-induced increase in CBF, but didn't affect the serotonin-induced change in [Ca2+]i. These findings suggest that an intracellular store-dependent increase in [Ca2+]i mediates the cilio-excitatory response to serotonin. Furthermore, although PKC is able to cause an increase in [Ca2+]i through calcium influx, it contributes to the cilio-excitatory response to 5-HT through a different mechanism.     

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).     

Activation of corticotropin-releasing factor 2 receptor inhibits Purkinje neuron P-type calcium currents via Goα-dependent PKC epsilon pathway

Corticotropin-releasing factor (CRF) receptors have been demonstrated to be widely expressed in the central nervous system and in many peripheral tissues of mammalians. However, it is still unknown whether CRF receptors will function in cerebellar Purkinje neurons. In the present study, we investigated the expression profile of CRF receptors in rat cerebellum and identified a novel functional role of CRFR2 in modulating Purkinje neuron P-type Ca²⁺ currents (P-currents). We found that CRFR2α mRNA, but not CRFR1 and CRFR2β, was endogenously expressed in rat cerebellum. Activation of CRFR2 by UCN2 inhibited P-currents in a concentration-dependent manner (IC₅₀ ~ 0.07 µM). This inhibitory effect was abolished by astressin2B, a CRFR2 antagonist, and was blocked by GDP-β-S, pertussis toxin, or a selective antibody raised against the G_oα. Inhibition of phospholipase C (PLC) blocked the inhibitory action of UCN2. The application of diacylglycerol (DAG) antagonist, 1-hexadecyl-2-acetyl-sn-glycerol, as well as inhibition of either protein kinase C or its epsilon isoform (PKCε) abolished the UCN2 effect while 1-oleoyl-2-acetyl-sn-glycerol (EI-150), a membrane-permeable DAG analogue, occluded UCN2-mediated inhibition. In addition, UCN2 significantly increases spontaneous firing frequency of Purkinje neurons in cerebellar slices. In summary, activation of CRFR2 inhibits P-currents in Purkinje neurons via G_oα-dependent PLC/PKCε pathway, which might contribute to its physiological functions in the cerebellum.     

Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

We examined the effect of angiotensin II (ANG II) on epithelial Na(+) channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NP(o) (a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca(2+)-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H(2)O(2) diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca(2+)-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.     

Modulation of delayed rectifier potassium current by angiotensin II in CATH.a cells

Angiotensin II (Ang II) modulates, via Ang II type 1 (AT(1)) receptors, the activity of brain catecholaminergic neurons. Here we utilized catecholaminergic CATH.a cells to define the effects of Ang II on delayed rectifier K(+) current (I(Kv)), one of the factors that determines changes in neuronal activation. Receptor binding analyses demonstrated the presence of AT(1) receptors in CATH.a cells. Whole cell voltage clamp experiments in these cells revealed that Ang II (100nM) produced a significant inhibition of I(Kv), that was abolished by the AT(1) receptor blocker, losartan (1 microM), or by inhibition of phospholipase C (PLC) with U73122 (10 microM). Furthermore, this action of Ang II was completely abolished by co-inhibition of protein kinase C (PKC) and calcium/calmodulin protein kinase II (CaMKII). These results demonstrate that Ang II produces an inhibition of I(Kv) in CATH.a cells, via an intracellular pathway that includes PLC, PKC, and CaMKII.     

Angiotensin II stimulates cardiac L-type Ca(2+) current by a Ca(2+)- and protein kinase C-dependent mechanism

Angiotensin II (ANG II) evokes positive inotropic responses in various species. However, the effects of this peptide on L-type Ca(2+) currents (I(Ca)) are still controversial. We report in this study that the effects of ANG II on I(Ca) differ depending on the mode of patch-clamp technique used, standard whole cell (WC) or perforated patch (PP). No significant effects of ANG II (0.5 microM) were observed when WC in cells dialyzed with high EGTA was used. However, when the intracellular milieu was preserved using PP, ANG II induced a significant 77 +/- 6% increase in I(Ca) (-2.2 +/- 0.3 in control and -3.9 +/- 0.6 pA/pF in ANG II, n = 8, P < 0.05). When WC was used in cells dialyzed with low Ca(2+) buffer capacity (EGTA 0.1 mM), ANG II was able to induce an increase in I(Ca) (-3.5 +/- 0.3 in control vs. -4.8 +/- 0.4 pA/pF in ANG II, n = 13, P < 0.05). This increase was prevented when the cells were also dialyzed with the protein kinase C (PKC) inhibitor chelerythrine (50 microM) or calphostin C (1 microM). The above results allow us to conclude that strong intracellular Ca(2+) buffering prevents the physiological actions of ANG II on cardiac I(Ca), which are also dependent on activation of PKC.     

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 microM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 microM Gd3+ or 1 microM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 microM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 +/- 2mV(n = 7), as did 30 microM 1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 microM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.     

Different signaling pathways involved in glucose- and cell swelling-induced insulin secretion by rat pancreatic islets in vitro.

BACKGROUND: The objective was to compare signal transduction pathways exploited by glucose and cell swelling in stimulating insulin secretion. METHODS: Isolated rat (Wistar) pancreatic islets were stimulated in vitro by 20 mmol/l glucose or 30% hypotonic medium (202 mOsm/kg) in various experimental conditions. RESULTS: Glucose did not stimulate insulin release in calcium free medium. Cell swelling-induced insulin release in calcium free medium, even in the presence of the membrane permeable calcium chelator BAPTA/AM (10 micromol/l). Protein kinase C (PKC) inhibitor bisindolylmaleimide VIII (1 micromol/l) abolished the stimulation of insulin secretion by glucose but did not affect the swelling-induced insulin release. PKC activator phorbol 12-13-dibutyrate (1 micromol/l) stimulated insulin secretion in medium containing Ca2+ and did not potentiate insulin secretion stimulated by hypotonic extracellular fluid. Dilution of the medium (10-30%) had an additive effect on the glucose-induced insulin secretion. Noradrenaline (1 micromol/l) abolished glucose-induced insulin secretion but did not inhibit hypotonic stimulation either in presence or absence of Ca2+. CONCLUSION: Glucose- and swelling-induce insulin secretion through separate signal transduction pathways. Hyposmotic stimulation is independent from both the extracellular and intracellular Ca2+, does not involve PKC activation, and could not be inhibited by noradrenaline. These data indicate a novel signaling pathway for stimulation of insulin secretion exploited by cell swelling.     

Low- and high-affinity phorbol ester and diglyceride interactions with protein kinase C: 1-O-alkyl-2-acyl-sn-glycerol enhances phorbol ester- and diacylglycerol-induced activity but alone does not induce activity

Phorbol ester-induced conventional protein kinase C (PKCalpha, -betaIota/IotaIota, and -gamma) isozyme activities are potentiated by 1,2-diacyl-sn-glycerol. This has been attributed to a "cooperative" interaction of the two activators with two discrete sites termed the low- and high-affinity phorbol ester binding sites, respectively [Slater, S. J., Milano, S. K., Stagliano, B. A., Gergich, K. J., Ho, C., Mazurek, A., Taddeo, F. J., Kelly, M. B., Yeager, M. D., and Stubbs, C. D. (1999) Biochemistry 38, 3804-3815]. Here, we report that the 1-O-alkyl ether diglyceride, 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), like its 1,2-diacyl counterpart, 1-oleoyl-2-acetyl-sn-glycerol (OAG), also potentiated PKCalpha, -betaI/II, and -gamma activities induced by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). Similar to OAG, HAG was found to bind to the low-affinity phorbol ester binding site and to enhance high-affinity phorbol ester binding, and to decrease the level of Ca(2+) required for phorbol ester-induced activity, while being without effect on the Ca(2+) dependence of membrane association. Thus, similar to OAG, HAG may also potentiate phorbol ester-induced activity by interacting with the low-affinity phorbol ester binding site, leading to a reduced level of Ca(2+) required for the activating conformational change. However, HAG was found not to behave like a 1,2-diacyl-sn-glycerol in that alone it did not induce PKC activity, and also in that it enhanced OAG-induced activity. The results reveal HAG to be a member of a new class of "nonactivating" compounds that modulate PKC activity by interacting with the low-affinity phorbol ester binding site.     

Conducted vasoconstriction in rat mesenteric arterioles: role for dihydropyridine-insensitive Ca(2+) channels

The aim of this study was to evaluate the role of voltage-operated Ca(2+) channels in the initiation and conduction of vasoconstrictor responses to local micropipette electrical stimulation of rat mesenteric arterioles (28 +/- 1 microm, n = 79) in vivo. Local and conducted (600 microm upstream from the pipette) vasoconstriction was not blocked by TTX (1 micromol/l, n = 5), nifedipine, or nimodipine (10 micromol/l, n = 9). Increasing the K(+) concentration of the superfusate to 75 mmol/l did not evoke vasoconstriction, but this depolarizing stimulus reversibly abolished vasoconstrictor responses to current stimulation (n = 7). Addition of the T-type Ca(2+) antagonist mibefradil (10 micromol/l, n = 6) to the superfusate reversibly blocked local and conducted vasoconstriction to current stimulation. With the use of RT-PCR techniques, it was demonstrated that rat mesenteric arterioles <40 microm do not express mRNA for L-type Ca(2+) channels (alpha(1C)-subunit), whereas mRNA coding for T-type subunits was found (alpha(1G)- and alpha(1H)-subunits). The data indicate that L-type Ca(2+) channels are absent from rat mesenteric arterioles (<40 microm). Rather, the vasoconstrictor responses appear to rely on other types of voltage-gated, dihydropyridine-insensitive Ca(2+) channels, possibly of the T-type.     

ANG II increases 2-deoxyglucose uptake in mouse embryonic stem cells

It is now suggested that all components of the renin-angiotensin system are present in many tissues, including the embryo and may play a major role in embryo development and differentiation. However, little is known regarding whether ANG II regulates glucose transport in mouse embryonic stem (ES) cells. Thus, the effects of ANG II on [3H]-2-deoxyglucose (2-DG) uptake and its related signal pathways were examined in mouse ES cells. ANG II significantly increased cell proliferation and 2-DG uptake in concentration- and time-dependent manner (>18 h, >10(-8) M) and increased mRNA and protein level of GLUT1 by 31+/-7% and 22+/-5% compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of ANG II on 2-DG uptake. ANG II-induced increase of 2-DG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD 123319, an ANG II type 2 (AT2) receptor blocker. In addition, ANG II-induced stimulation of 2-DG uptake was attenuated by phospholipase C (PLC) inhibitors, neomycin and U 73122 and ANG II increased inositol phosphates (IPs) formation by 37+/-8% of control. Protein kinase C (PKC) inhibitors, staurosporine, bisindolylmaleimide I, and H-7 also blocked ANG II-induced stimulation of 2-DG uptake. Indeed, ANG II activated a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PKC. A 23187 (Ca2+ ionophore) increased 2-DG uptake and nifedifine (L-type Ca2+ channel blocker) blocked it. In conclusion, ANG II increased 2-DG uptake by PKC activation via AT1 receptor in mouse ES cells.     

Complex regulation of store-operated Ca2+ entry pathway by PKC-epsilon in vascular SMCs

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.     

Phosphorylation of S1505 in the cardiac Na+ channel inactivation gate is required for modulation by protein kinase C

Inactivation of both brain and cardiac Na+ channels is modulated by activation of protein kinase C (PKC) but in different ways. Previous experiments had shown that phosphorylation of serine 1506 in the highly conserved loop connecting homologous domains III and IV (LIII/IV) of the brain Na+ channel alpha subunit is necessary for all effects of PKC. Here we examine the importance of the analogous serine for the different modulation of the rH1 cardiac Na+ channel. Serine 1505 of rH1 was mutated to alanine to prevent its phosphorylation, and the resulting mutant channel was expressed in 1610 cells. Electrophysiological properties of these mutant channels were indistinguishable from those of wild-type (WT) rH1 channels. Activation of PKC with 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced WT Na+ current by 49.3 +/- 4.2% (P < 0.01) but S1505A mutant current was reduced by only 8.5 +/- 5.4% (P = 0.29) when the holding potential was -94 mV. PKC activation also caused a -17-mV shift in the voltage dependence of steady-state inactivation of the WT channel which was abolished in the mutant. Thus, phosphorylation of serine 1505 is required for both the negative shift in the inactivation curve and the reduction in Na+ current by PKC. Phosphorylation of S1505/1506 has common and divergent effects in brain and cardiac Na+ channels. In both brain and cardiac Na+ channels, phosphorylation of this site by PKC is required for reduction of peak Na+ current. However, phosphorylation of S1506 in brain Na+ channels slows and destabilizes inactivation of the open channel. Phosphorylation of S1505 in cardiac, but not S1506 in brain, Na+ channels causes a negative shift in the inactivation curve, indicating that it stabilizes inactivation from closed states. Since LIII/IV containing S1505/S1506 is completely conserved, interaction of the phosphorylated serine with other regions of the channel must differ in the two channel types.     

Protein kinase C and mitogen-activated protein kinase cascade in mouse cumulus cells: cross talk and effect on meiotic resumption of oocyte

Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cumulus cells are involved in FSH-induced meiotic resumption of cumulus-enclosed oocytes (CEOs), but their regulation and cross talk are unknown. The present experiments were designed to investigate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in induced meiotic resumption of mouse oocytes. PKC activators, phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced the meiotic resumption of CEOs and activation of MAPK in cumulus cells, whereas this effect could be abolished by PKC inhibitors, calphostin C and chelerythrine, or MEK inhibitor U0126. These results suggest that PKC might induce the meiotic reinitiation of CEOs by activating MAPK in cumulus cells. Both PKC inhibitors and U0126 inhibited the FSH-induced germinal vesicle breakdown (GVBD) of oocytes and MAPK activation in cumulus cells, suggesting that PKC and MAPK are involved in FSH-induced GVBD of mouse CEOs. Protein synthesis inhibitor cycloheximide (CHX) inhibited FSH- or PMA-induced oocyte meiotic resumption, but not the MAPK activation in cumulus cells. FSH and PKC activators induced the GVBD in denuded oocytes cocultured with cumulus cells in hypoxanthine (HX)-supplemented medium, and this effect could be reversed by U0126. Thus, when activated by FSH and PKC, MAPK may stimulate the synthesis of specific proteins in cumulus cells followed by secretion of an unknown positive factor that is capable of inducing GVBD in oocytes.     

Prolactin secretion in permeable GH3 pituitary cells is stimulated by Ca2+ and protein kinase C activators

We have used GH3 cells permeabilized by electric field discharge to examine the effects of Ca2+ and protein kinase C activators (phorbol ester and diacylglycerol) on prolactin (PRL) release. Ca2+ was found to stimulate PRL release approximately 4 fold at 3 microM Ca2+ with a half-maximal response at approximately .5 microM estimated free Ca2+. 12-O-tetradecanoyl phorbol-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol stimulated PRL release throughout a range of Ca2+ concentrations (1 nM -3 microM), but stimulation was greater at higher Ca2+ concentrations (.1 microM to 1 microM). Both agents decreased by 1.8 fold the apparent [Ca2+] at which half-maximal stimulation of secretion occurred. Quin 2 was used to measure the free [Ca2+] of intact and permeable cells; PRL secretion at a free [Ca2+] corresponding to resting cytoplasmic [Ca2+] was 10% of maximal, while secretion at the [Ca2+] corresponding to the Ca2+ spike induced by thyrotropin-releasing hormone was approximately 25% of maximal.     

AT(1) and AT(2) receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts

We assessed ANG II type 1 (AT(1)) and type 2 (AT(2)) receptor (R) expression and functional recovery after ischemia-reperfusion with or without AT(1)R/AT(2)R blockade in isolated working rat hearts. Groups of six hearts were subjected to global ischemia (30 min) followed by reperfusion (30 min) and exposed to no drug and no ischemia-reperfusion (control), ischemia-reperfusion and no drug, and ischemia-reperfusion with losartan (an AT(1)R antagonist; 1 micromol/l), PD-123319 (an AT(2)R antagonist; 0.3 micromol/l), N(6)-cyclohexyladenosine (CHA, a cardioprotective adenosine A(1) receptor agonist; 0.5 micromol/l as positive control), enalaprilat (an ANG-converting enzyme inhibitor; 1 micromol/l), PD-123319 + losartan, ANG II (1 nmol/l), or ANG II + losartan. Compared with controls, ischemia-reperfusion decreased AT(2)R protein (Western immunoblots) and mRNA (Northern immunoblots, RT-PCR) and impaired functional recovery. PD-123319 increased AT(2)R protein and mRNA and improved functional recovery. Losartan increased AT(1)R mRNA (but not AT(1)R/AT(2)R protein) and impaired recovery. Other groups (except CHA) did not improve recovery. The results suggest that, in isolated working hearts, AT(2)R plays a significant role in ischemia-reperfusion and AT(2)R blockade induces increased AT(2)R protein and cardioprotection.     

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and G?6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.     

Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver

Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors G?6850 (1 micromol/liter) and G?6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells. However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. G?6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.