Selective inhibition of inward rectifier K+ channels (Kir2.1 or Kir2.2) abolishes protection by ischemic preconditioning in rabbit ventricular cardiomyocytes

Volume regulatory Cl- channels are key regulators of ischemic preconditioning (IPC). Because Cl- efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that I(K1) channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly (P<0.0001) reduced the percent cell death in nontransfected cardiomyocytes [IPC(CM) 18.0+/-2.1% versus control (C(CM)) 48.3+/-1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPC(CM)+AdEGFPKir2.1DN 45.8+/-2.3% (mean+/-SEM) and IPC(CM)+AdEGFPKir2.2DN 47.9+/-1.4% versus IPC(CM); P<0.0001] in cultured cardiomyocytes (n=8 hearts). Transfection of cardiomyocytes with AdEGFPKir2.1DN or AdEGFPKir2.2DN did not affect cell death in control (nonpreconditioned) cardiomyocytes (both C(CM)+ AdEGFPKir2.1DN 45.8+/-0.7% and C(CM)+AdEGFPKir2.2DN 46.2+/-1.3% versus C(CM); not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after I(K1) blockade with 20 micromol/L BaCl2 plus 1 micromol/L nifedipine (to prevent Ba2+ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that I(K1) channels play an important role in IPC protection.     

乙醇及其代谢产物对新生大鼠原代培养心房肌细胞乙酰胆碱敏感型钾通道Kir3.1蛋白表达的影响

目的 明确乙醇及其代谢产物乙醛对新生SD大鼠原代心房肌细胞乙酰胆碱敏感型钾通道Kir3.1蛋白表达的影响,并探讨该通道及乙醛在急性乙醇中毒引发心律失常的过程中所发挥的效应.方法 采用胰蛋白酶及Ⅱ型胶原酶提取并培养150只新生SD大鼠原代心房肌细胞,并采用免疫荧光法进行肌钙蛋白Ⅰ鉴定.采用细胞计数套件-8(CCK-8)法分别测定200 ～ 800 mmol/L乙醇及50～ 500 μmol/L乙醛处理24 h后细胞的生存率,以确定导致新生SD大鼠原代心房肌细胞凋亡所需的乙醛及乙醇浓度.建立以最大非凋亡浓度(200 mmol/L)的乙醇处理24h及相应剂量(100 μmol/L)乙醛处理24 h的新生SD大鼠原代心房肌细胞模型.采用Western blot法检测乙醇处理组、乙醛处理组及对照组心房肌细胞Kir3.1蛋白的表达情况,并对结果进行统计学分析.结果 (1)成功培养新生SD大鼠原代心房肌细胞,细胞免疫荧光鉴定所培养细胞为心肌细胞,且90％以上细胞肌钙蛋白Ⅰ染色阳性.(2) CCK-8法测定显示400 mmol/L以上的乙醇处理组的心肌细胞生存率明显低于对照组(P＜0.05),而200 mmol/L及以下的乙醇处理组心肌细胞生存率与对照组相比差异无统计学意义(P ＞0.05);400 μmol/L以上的乙醛处理组的心肌细胞生存率明显低予对照组(P＜0.05),而350 μmol/L及以下的乙醛处理组心肌细胞生存率与对照组相比差异无统计学意义(P＞0.05).(3)Western blot检测显示200 mmol/L乙醇及100 μmol/L的乙醛处理24h的新生SD大鼠原代房心肌细胞Kir3.1蛋白表达分别高于对照组(44.52±23.07)％及(45.04±22.01)％ (P ＜0.01),而乙醇组与乙醛组之间Kir3.1蛋白表达差异无统计学意义(P＞0.05).结论 急性乙醇及乙醛处理均能够明显上调乙酰胆碱敏感型钾通道Kir3.1蛋白的表达.     

Phospholemman phosphorylation mediates the protein kinase C-dependent effects on Na+/K+ pump function in cardiac myocytes

Because phospholemman (PLM) regulates the Na(+)/K(+) pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na(+)](i) decline (fluorescence measurements) and current (I(pump)) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, I(pump) (both at 10 and 100 mmol/L Na(+) in the pipette solution) and maximal NKA-mediated Na(+) extrusion rate (V(max)) from 7.9+/-1.1 to 12.7+/-1.9 mmol.L(-1) per minute without altering NKA affinity for internal Na(+) (K(0.5)). In PLM knockout mice, PDBu had no effect on either V(max) or K(0.5). After pretreatment with isoproterenol (ISO) (1 mumol/L), PDBu still increased the NKA V(max) and PLM phosphorylation at Ser63 and Ser68. Conversely, after pretreatment with PDBu, ISO further increased the Na(+) affinity of NKA and phosphorylation at Ser68, as it did alone without PDBu. The final NKA activity was independent of the application sequence. The NKA activity in PLM knockout myocytes, after normalizing the protein level, was similar to that after PDBu and ISO treatment. We conclude that (1) PLM mediates the PKC-dependent activation of NKA function in cardiac myocytes, (2) PDBu and ISO effects are additive in the mouse (affecting mainly V(max) and K(0.5), respectively), and (3) PDBu and ISO combine to activate NKA in wild-type to the level found in the PLM knockout mouse.     

Type 2 iodothyronine deiodinase expression is upregulated by the protein kinase A-dependent pathway and is downregulated by the protein kinase C-dependent pathway in cultured human thyroid cells.

Type 1 and 2 iodothyronine deiodinases (D1 and D2) catalyze thyroxine (T4) activation. In human thyroid, unlike rodents', both D1 and D2 are expressed. We have investigated the effects of thyrotropin (TSH), dibutyryl cyclic adenosine monophosphate [(Bu)2cAMP] (an activator of protein kinase A [PKA]), 12-O-tetradecanoylphorbor 13-actate (TPA) (an activator of protein kinase C [PKC]), T4, and triiodothyronine (T3) on the D2 mRNA levels and activity in cultured human thyroid cells. D2 mRNA levels were increased by TSH and (Bu)2cAMP, and the increment was faster and greater than that of D1 mRNA levels. The increment of the maximum velocity (Vmax) value for D2 by (Bu)2cAMP stimulation was similar to that of D2 mRNA levels, suggesting that (Bu)2cAMP enhances D2 activity mainly at the pretranslational level. Cycloheximide, a protein synthesis inhibitor, partially inhibited the increase of D2 mRNA levels by (BU)2cAMP, suggesting that de novo protein synthesis-dependent pathways are involved. TPA suppressed the D2 mRNA levels in the presence of (Bu)2cAMP. However, T3 and T4 did not significantly change the D2 mRNA levels and activity. In conclusion, D2 expression in human thyroid cells is more rapidly and strongly upregulated by the PKA pathway than D1 expression, and is downregulated by the PKC pathway.     

Beta-adrenergic regulation of the muscarinic-gated K+ channel via cyclic AMP-dependent protein kinase in atrial cells

Cholinergic and beta-adrenergic stimulations of ionic currents are major physiological mechanisms in the regulation of heart rate and contractility. Muscarinic receptor stimulation is known to reduce beta-adrenergic effects on calcium current via reduction of cyclic AMP. Whether the beta-adrenergic stimulation affects the muscarinic response is not known. I report here that the beta-adrenergic agonist isoproterenol enhanced the muscarinic-activated K+ channel activity in rat atrial cells. Application of cyclic AMP-dependent protein kinase or its catalytic subunit to the cytoplasmic side of the membrane augmented the acetylcholine-activated K+ channel activity twofold to threefold. Increases in channel activity produced by isoproterenol or cyclic AMP-dependent protein kinase were associated with fourfold to fivefold and approximately twofold increases in the mean open and closed time durations, respectively. Alkaline phosphatase treatment reversed these effects. These results suggest that cyclic AMP-dependent phosphorylation of the K+ channel or associated regulatory proteins modulates the gating kinetics of the channel. This mechanism may be important in the regulation of pacemaker activity and, thus, the heart rate during beta-adrenergic stimulation.     

Changes in activity and regulation of the cardiac Ca2+ channel (L-type) by protein kinase C in chronic alcohol-exposed rats

Background: It has been reported recently that long‐term alcohol exposure in rats increases the number of dihydropyridine binding sites in cardiac membrane preparations. We fed Sprague Dawley® rats a liquid diet that contained ethanol as 36% of total calories for 4 to 6 months and studied how alcohol exposure affected the activity and regulation of the cardiac Ca²⁺ channel. Methods: Dihydropyridine‐sensitive cardiac Ca²⁺ channel activity was measured as the rate of Mn²⁺ quench of the cytosolic fura‐2 signal in electrically stimulated myocytes. Results: In control rat myocytes, pretreatment with phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), reduced the rate of Mn²⁺ quench to 68% of the untreated cell response. Pretreatment with GF109203X, a protein kinase C inhibitor, enhanced the rate of influx by 56%, whereas Gö6976, an inhibitor of PKC α, β, and γ, did not affect the rate of influx. By contrast, PMA did not affect the rate of Mn²⁺ quench in alcoholic myocytes; however, the PKC inhibitor GF109203X still enhanced the rate of Mn²⁺ quench by 33%. Similar to control myocytes, no effect was observed after pretreatment with Gö6976 in the alcoholic cells. In both Western blot and immunoprecipitation experiments, PKC ? expression in alcohol‐exposed myocytes was reduced to 68% of the control. However, the ratio of membrane/cytosolic distribution of PKC ? in alcoholic myocytes was increased from 1.6 to 2.6. No change was detected in the expression of PKC α and PKC δ. PKC activity, measured in the presence of Gö6976, which inhibits PKC α, β, and γ, was reduced in alcoholic myocytes to 57% of the control, but the proportion of PKC activity in the particulate fraction was increased from 26% in the control myocytes to 36% in the alcoholic myocytes. Conclusions: Altered expression and activity of PKC may be associated with changes in the regulation of the cardiac Ca²⁺ channel found in the hearts of rats chronically exposed to alcohol. Specifically, we found that the novel class of PKC isozymes is responsible for regulating the cardiac Ca²⁺ channel in control cardiomyocytes, and that the loss of PMA modulation found in the alcoholic cells may be due, in part, to reduced expression and altered distribution of PKC ?.     

Epithelial K channel expressed in Xenopus oocytes is inactivated by protein kinase C.

K homeostasis is maintained in higher animals by epithelia of the kidney and intestine. Little is known regarding the molecular regulation of K secretion. We injected Xenopus oocytes with mRNA from teleost intestine, a K-secreting epithelium with apical membrane K channels. Oocytes expressed a conductance that displayed whole-cell current properties with the following characteristics: marked selectivity for K over Na and Cl, voltage-independent kinetics, Ca insensitivity, tonic activation, and inward rectification in symmetrical K. Barium, quinine, and tetraethylammonium blocked the conductance, whereas apamin, charybdotoxin, and 4-aminopyridine did not. The K conductance was rapidly (t1/2 = 10 min) and completely inactivated by 4 beta-phorbol 12-myristate 13-acetate but not by 4 alpha-phorbol 12,13-didecanoate. Sucrose density gradient fractionation revealed that mRNA required for expression is in the 1- to 2-kilobase size range, suggesting the possibility that a single subunit encodes the channel. The K conductance expressed from injection of size-fractionated mRNA was identical in all respects to that seen using unfractionated mRNA, including response to 4 beta-phorbol 12-myristate 13-acetate. The results suggest that protein kinase C regulates K secretion in epithelia by modulation of apical K channels.     

Single L-type Ca(2+) channel regulation by cGMP-dependent protein kinase type I in adult cardiomyocytes from PKG I transgenic mice

OBJECTIVE: Calcium entry via the L-type Ca(2+) channel (LTCC) is crucial for excitation-contraction (EC) coupling and activation of Ca(2+)-dependent signal transduction pathways in cardiac myocytes. Both nitric oxide (NO), signaling via cGMP, and acetylcholine, signaling via the muscarinic receptor, have been identified as negative regulators of beta-adrenoreceptor-stimulated LTCC activity in cardiac myocytes. METHODS: To examine the potential role of cGMP-dependent protein kinase type I (PKG I) in the inhibitory effects of NO/cGMP and the muscarinic receptor on LTCC activity, we generated transgenic (TG) mice overexpressing PKG I selectively in cardiac myocytes under the control of the alpha-myocin heavy chain promoter. Single LTCC-gating properties were assessed in isolated ventricular myocytes from adult wild-type (WT) and PKG I transgenic (TG) mice. RESULTS: Basal LTCC activity (peak average current, mean open probability, mean availability) was significantly decreased by the nitric oxide donor DEA-NO (0.1 micromol/l) and the cGMP-analog 8-Br-cGMP (1 mmol/l) in TG but not in WT cardiac myocytes. Conversely, muscarinic (carbachol, 1 micromol/l) stimulation had no significant effect on basal LTCC activity in either WT or TG cardiac myocytes. beta-Adrenergic stimulation with isoproterenol (1 micromol/l) increases single LTCC activity in WT and TG cardiac myocytes to the same extent. The inhibitory effects of DEA-NO and 8-Br-cGMP on isoproterenol activation of the LTCC current were significantly enhanced in TG as compared to WT cardiac myocytes. By contrast, carbachol inhibition of isoproterenol-stimulated single LTCC activity was not enhanced in TG cardiac myocytes. CONCLUSION: Transgenic overexpression of PKG I augments NO/cGMP inhibition but not muscarinic inhibition of single LTCC activity, indicating that PKG I is a downstream target for NO/cGMP, but not the muscarinic receptor in adult cardiac myocytes.     

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.     

Rapid component I(Kr) of the guinea-pig cardiac delayed rectifier K(+) current is inhibited by beta(1)-adrenoreceptor activation, via cAMP/protein kinase A-dependent pathways

OBJECTIVE: The antiarrhythmic potential of betablockers contributes to their beneficial effects in the treatment of cardiac diseases, although the molecular basis of their class II antiarrhythmic action has not been clarified yet. METHODS: To investigate a putative functional link between beta-adrenoreceptors and the fast component of cardiac delayed rectifier K(+) channels (I(Kr)), whole-cell patch-clamp experiments were performed with isolated guinea-pig ventricular myocytes. Tail currents of I(Kr) were measured at -40 mV after short (200 ms) test pulses to +40 mV. RESULTS: After application of the unspecific beta-receptor agonist isoproterenol (10 microM) for 12 min, the I(Kr) tail current was decreased by 72%, with an IC(50) of 1.4 microM. The specific beta(1)-blocker CGP207120A (10 microM) significantly attenuated the isoproterenol effect (net 24% decrease). The specific beta(1)-agonist xamoterol (10 microM), could mimic the isoproterenol effect (58% decrease). Modulators of beta(2)- or beta(3)-adrenoreceptors were far less effective. When isoproterenol or xamoterol were combined with KT5720 (2.5 microM), a specific inhibitor of protein kinase A (PKA), their effects were drastically reduced, indicating that PKA presumably mediates the beta(1)-adrenergic inhibition of I(Kr). Tail current reductions by cAMP, forskolin, PKA catalytic subunit and a combination of PKA holoenzyme and cAMP support an involvement of PKA in the regulation of I(Kr). CONCLUSIONS: The functional link between I(Kr) and the beta(1)-adrenergic receptor involving PKA may play an important role in arrhythmogenesis and contribute to the antiarrhythmic action of clinically used beta(1)-blockers.     

Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the "translocation" of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.     

Paracrine hypertrophic factors from cardiac non-myocyte cells downregulate the transient outward current density and Kv4.2 K+ channel expression in cultured rat cardiomyocytes.

OBJECTIVES: Cardiac hypertrophy is characterized by a prolongation of action potential duration (APD) and a reduction of outward K+ currents, primarily the transient outward current (Ito). Since the interaction between cardiac non-myocyte cells (NMCs) and cardiomyocytes (MCs) plays a critical role during the process of myocardial hypertrophy, in the present study, we investigated the effects of NMCs on cell growth and K+ channel expression in cultured newborn rat ventricular cells. METHODS: Single MCs were isolated from day-old Wistar rat ventricles and cultured for a period of five days. The effects of NMCs were examined by MC-NMC co-culture or incubating pure MCs in NMC-conditioned growth medium (NCGM). Whole-cell voltage-clamp recording and Western blot analysis using a polyclonal antibody against rat Kv4.2 channel protein were performed. RESULTS: A marked increase in surface area and total cell protein concentration of MCs was observed in the MC-NMC co-culture. In the pure MC culture, this hypertrophic effect could be mimicked by a 72-h addition of NCGM, with a significant prolongation of APD25 (APD at 25% repolarization) and a 42% decrease in Ito density (at +30 mV). The rates of inactivation and recovery from inactivation of Ito were unchanged. In the NCGM-treated MC culture, Western blots of MC proteins also showed a 36% reduction of the Kv4.2 K+ channel protein level. In addition, the NCGM-induced MC hypertrophy was partially inhibited by anti-insulin-like growth factor-1 (IGF-1) antibody, while it revealed no effects on Ito density and Kv4.2 channel expression. CONCLUSIONS: These findings first demonstrate that some paracrine hypertrophic factors released from cardiac NMCs, although unidentified, downregulate cardiac K+ channel expression.     

Low expression of 3 beta-hydroxy-5-ene steroid dehydrogenase gene in human fetal adrenals in vivo; adrenocorticotropin and protein kinase C-dependent regulation in adrenocortical cultures.

Human fetal adrenals are very active in steroid production. They make large amounts of dehydroepiandrosterone sulfate which is further converted to estrogens in placenta. Fetal adrenals cannot make cortisol efficiently from cholesterol or pregnenolone, but they can convert progesterone to cortisol. To clarify the molecular basis of the very low activity of 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) in human fetal adrenals we studied the expression of 3 beta HSD gene in fetal adrenals in vivo and in culture conditions. Human adult adrenals, placenta and a testicular Leydig cell tumor clearly expressed 3 beta HSD gene when studied by Northern blotting, but fetal adrenals and ovaries had no detectable 3 beta HSD mRNA by this method. Polymerase chain reaction analysis of cDNA samples derived from different human tissues revealed 3 beta HSD gene expression in placenta, adult adrenal and adult ovarian granulosa cells after 25 cycles of amplification. Fetal adrenal samples became positive only after additional amplification cycles, which verifies the very low expression of 3 beta HSD gene in fetal adrenals. In cell culture conditions both ACTH and a protein kinase C regulator 12-O-tetradecanoyl phorbol-13-acetate induced 3 beta HSD gene expression. We conclude: 1) the very low activity of 3 beta HSD in human fetal adrenals is due to the low expression of this gene; 2) both cAMP and protein kinase C-dependent mechanisms regulate 3 beta HSD gene expression in adrenocortical cells.     

Tyrosine receptor kinase B (TrkB) protein expression in the human endometrium

Tyrosine kinase receptor B (TrkB) gene expression, a neurotrophic factor receptor expressed in the brain and ovary, has recently been identified in deep infiltrating endometriosis by gene array. TrkB is thought to be important in resistance to anchorage independent apoptosis (ANOIKIS) and thus could be important in the pathogenesis of endometriosis. However, TrkB protein expression in the eutopic endometrium of women with and without endometriosis is unknown. Therefore, we examined TrKB protein expression in the endometrium by Western blot (n = 50) and immunohistochemistry (n = 17). Immunoblots of endometrial biopsies were prepared from women with endometriosis (n = 21) vs. healthy controls (n = 29) undergoing benign gynecological surgery at McMaster University Medical Centre. TrkB protein expression was significantly higher in immunoblots from women with endometriosis compared to women without endometriosis. In samples of archived paraffin-embedded endometrial tissue TrkB was localized to the cytoplasm of epithelial cells of the eutopic endometrium from women with endometriosis (n = 7) and without endometriosis (n = 10). We conclude that TrkB protein is expressed in human endometrium. Our results also suggest that TrkB expression may be greater in women with endometriosis compared to women without endometriosis.     

Phospholipase D activity in human platelets is inhibited by protein kinase A, involving inhibition of phospholipase D1 translocation

Phospholipase D hydrolyzes phosphatidylcholine to phosphatidic acid and choline. It is established that thrombin induces PLD activation in human platelets. We found recently that two isoforms of PLD, PLD1 and PLD2 are present in platelets and these become translocated to the plasma membrane area upon thrombin activation. Since cAMP is a negative regulator in platelets, we measured the effect of the platelet antagonists PGE(1) and forskolin (both of which raise intracellular cAMP levels) on PLD activity in resting and thrombin-activated platelets. We found that both PGE(1) and forskolin inhibited thrombin-induced PLD activation by 40-50%, but interestingly PGE(1) caused a modest elevation of PLD activity in resting platelets. Further studies using inhibitors as well as specific activators of protein kinases A and G suggest a role for PKA in the inhibition of thrombin-activated PLD. We found that PGE(1), forskolin and the PKA activator inhibited PLD1 translocation in thrombin-stimulated platelets, and that the PKG-activator had no effect. The present results suggest a role for PKA in the regulation of thrombin-induced PLD1 activity and translocation in platelets.     

Involvement of guanylyl cyclase, protein kinase A and Na+ K+ ATPase in relaxations of bovine isolated bronchioles induced by GEA 3175, an NO donor

The present study was designed to investigate the role of the sodium potassium adenosine triphosphatase (the Na(+)K(+) ATPase) in relaxation of bovine isolated bronchioles by a new NO donor, GEA 3175 (3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulphonyl]amino]-)hydroxide)). Bronchioles were mounted in a wire myograph for isometric tension recordings and contracted with 5-hydroxytryptamine (5-HT) or a K(+) rich solution. Concentration-dependent relaxations evoked by GEA 3175 were inhibited by ouabain or K(+) free solution. The guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 microM) and ouabain (10 nM) reduced GEA 3175-evoked relaxations to the same extent without any additive effect. Iberiotoxin (10 nM), an inhibitor of large conductance Ca(2+)-activated K(+) channels inhibited GEA 3175-evoked relaxations to the same extent as ouabain. Combining ouabain and iberiotoxin completely abolished GEA 3175 relaxation. An inhibitor of protein kinase G (PKG), Rp-beta-phenyl-1,N(2)-etheno-8-bromo-guanosine-3'-5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPs), slightly reduced GEA 3175-induced relaxations. An inhibitor of cyclic AMP-dependent kinase (PKA), Rp-adenosine-3'-5'-cyclic phosphorothioate (Rp-cAMPs), inhibited the GEA 3175-induced relaxations to the same extent as ouabain. Inhibition of both PKG and PKA abolished GEA 3175 relaxation. The study provides evidence that the NO donor GEA 3175 causes guanylyl cyclase-dependent relaxations, taking place through cyclic GMP and cyclic AMP-dependent protein kinases followed by opening of large conductance Ca(2+)-activated K(+) channels and activation of smooth muscle Na(+)K(+) ATPase.     

Renal phenotype in mice lacking the Kir5.1 (Kcnj16) K+ channel subunit contrasts with that observed in SeSAME/EAST syndrome

The heteromeric inwardly rectifying Kir4.1/Kir5.1 K(+) channel underlies the basolateral K(+) conductance in the distal nephron and is extremely sensitive to inhibition by intracellular pH. The functional importance of Kir4.1/Kir5.1 in renal ion transport has recently been highlighted by mutations in the human Kir4.1 gene (KCNJ10) that result in seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME)/epilepsy, ataxia, sensorineural deafness, and renal tubulopathy (EAST) syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. Here, we investigated the role of the Kir5.1 subunit in mice with a targeted disruption of the Kir5.1 gene (Kcnj16). The Kir5.1(-/-) mice displayed hypokalemic, hyperchloremic metabolic acidosis with hypercalciuria. The short-term responses to hydrochlorothiazide, an inhibitor of ion transport in the distal convoluted tubule (DCT), were also exaggerated, indicating excessive renal Na(+) absorption in this segment. Furthermore, chronic treatment with hydrochlorothiazide normalized urinary excretion of Na(+) and Ca(2+), and abolished acidosis in Kir5.1(-/-) mice. Finally, in contrast to WT mice, electrophysiological recording of K(+) channels in the DCT basolateral membrane of Kir5.1(-/-) mice revealed that, even though Kir5.1 is absent, there is an increased K(+) conductance caused by the decreased pH sensitivity of the remaining homomeric Kir4.1 channels. In conclusion, disruption of Kcnj16 induces a severe renal phenotype that, apart from hypokalemia, is the opposite of the phenotype seen in SeSAME/EAST syndrome. These results highlight the important role that Kir5.1 plays as a pH-sensitive regulator of salt transport in the DCT, and the implication of these results for the correct genetic diagnosis of renal tubulopathies is discussed.