Anchoring protein is required for cAMP-dependent stimulation of L-type Ca2+ channels in rabbit portal vein

Stimulation of cardiac L-typeCa²⁺ channels by cAMP-dependentprotein kinase (PKA) requires anchoring of PKA to a specificsubcellular environment by A-kinase anchoring proteins (AKAP). Thisstudy evaluated the possible requirement of AKAP in PKA-dependentregulation of L-type Ca²⁺ channelsin vascular smooth muscle cells using the conventional whole cellpatch-clamp technique. Peak Ba²⁺current in freshly isolated rabbit portal vein myocytes wassignificantly increased by superfusion with either 0.5 µM isoproterenol (131 ± 3% of the control value,n = 11) or 10 µM 8-bromoadenosine3',5'-cyclic monophosphate (8-BrcAMP; 114 ± 1%,n = 8). The PKA-induced stimulatory effects ofboth isoproterenol and 8-BrcAMP were completely abolished by a specificPKA inhibitor KT-5720 (0.2 µM) or by dialyzing cells with Ht 31 (100 µM), a peptide that inhibits the binding of PKA to AKAP. In contrast,Ht 31 did not block the excitatory effect of the catalytic subunit ofPKA when dialyzed into the cells. These data suggest that stimulationof Ca²⁺ channels in vascularmyocytes by endogenous PKA requires localization of PKA through bindingto AKAP.

     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

Role of aspartate residues in Ca(2+) affinity and permeation of the distal ECaC1

The Ca²⁺ affinity andpermeation of the epithelial Ca²⁺ channel (ECaC1) wereinvestigated after expression in Xenopus oocytes. ECaC1displayed anomalous mole-fraction effects. Extracellular Ca²⁺ and Mg²⁺ reversibly inhibited ECaC1 wholecell Li⁺ currents: IC₅₀ = 2.2 ± 0.4 µM (n = 9) and 235 ± 35 µM (n = 10), respectively. These values compare well with theCa²⁺ affinity of the L-type voltage-gated Ca²⁺(Ca_V1.2) channel measured under the same conditions,suggesting that high-affinity Ca²⁺ binding is awell-conserved feature of epithelial and voltage-gated Ca²⁺channels. Neutralization of D550 and E535 in the pore region had nosignificant effect on Ca²⁺ and Mg²⁺ affinities.In contrast, neutralization of D542 significantly decreasedCa²⁺ affinity (IC₅₀ = 1.1 ± 0.2 mM,n = 6) and Mg²⁺ affinity(IC₅₀ > 25 ± 3 mM, n = 4).Despite a 1,000-fold decrease in Ca²⁺ affinity in D542N,Ca²⁺ permeation properties and theCa²⁺-to-Ba²⁺ conductance ratio remainedcomparable to values for wild-type ECaC1. Together, our observationssuggest that D542 plays a critical role in Ca²⁺ affinitybut not in Ca²⁺ permeation in ECaC1.

     

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells

Rapamycin and FK-506 are immunosuppressive drugs thatbind a ubiquitous immunophilin, FKBP12, but immunosuppressivemechanisms and side effects appear to be different. Rapamycin bindsrenal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na⁺ channels in A6cells. Channel NP_o was 0.5 ± 0.08 (n = 6)during the first 5 min but fell close to zero after 20 min. Application of 1 µM rapamycin reactivated Na⁺ channels(NP_o = 0.47 ± 0.1; n=6), but 1 µMFK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor,mimicked the rapamycin-induced reactivation in a nonadditive manner.However, rapamycin did not reactivate Na⁺ channels if cellswere exposed to 1 µM FK-506 before rapamycin. In PKC assays,rapamycin was as effective as the PKC inhibitor; however, epithelialNa⁺ channel (ENaC) phosphorylation was low under baselineconditions and was not altered by PKC inhibitors or activators. Theseresults suggest that rapamycin activates Na⁺ channels bybinding FKBP12 and inhibiting PKC, and, in renal cells, despite bindingthe same immunophilin, rapamycin and FK-506 activate differentintracellular signaling pathways.

     

Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage

Little is known of the functional properties of the mammalian,brain-specific Na⁺/H⁺ exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, andits activity was characterized using the fluorescent pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na⁺ (apparent K_Na = 27 ± 5 mM) and a Hill coefficient near 3 for the intracellularproton concentration with a half-maximal activity at an intracellularpH of 6.93 ± 0.03. NHE5 activity was inhibited by acute exposureto 8-bromo-cAMP or forskolin (which increases intracellular cAMP byactivating adenylate cyclase). The kinase inhibitor H-89 reversed thisinhibition, suggesting that regulation by cAMP involves a proteinkinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did nothave a significant effect on activity. The protein kinase C (PKC)activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKCantagonist chelerythrine chloride blunted this effect. Activity wasalso inhibited by hyperosmotic-induced cell shrinkage but wasunaffected by a hyposmotic challenge. These results demonstrate thatrat brain NHE5 is downregulated by activation of PKA and PKC and bycell shrinkage, important regulators of neuronal cell function.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Effect of neuropeptide Y on hemodynamics of the rabbit lung

Lang, Sally A., and Michael B. Maron.Effect of neuropeptide Y on hemodynamics of the rabbit lung.J. Appl. Physiol. 84(2): 618-623, 1998.We evaluated the effect of neuropeptide Y (NPY) on thehemodynamics of the isolated rabbit lung perfused at constant flow andoutflow pressure. Doses of10⁸ and10 ⁷ M NPY increasedpulmonary arterial pressure (Ppa) from 11.5 ± 1.0 (SE) mmHg to,respectively, 16.4 ± 1.5 and 26.0 ± 3.8 mmHg (P < 0.05, n = 5 mmHg lungs), with 78 ± 4%of the increase at 10⁷ Mresulting from an increased arterial resistance. At the latter dose,pulmonary capillary pressure increased from 5.8 ± 0.9 to 9.4 ± 1.0 mmHg (P < 0.05). Whenadministered in the presence of norepinephrine,10⁸ and10⁷ M NPY(n = 6) produced extreme increases inPpa to 66.1 ± 20.5 and 114.7 ± 25.5 mmHg, respectively, thatwere due primarily to an increased arterial resistance. To determinethe significance of circulating NPY as a pulmonary vasoactive agent, wemeasured plasma NPY-like immunoreactivity in anesthetized rabbits after massively activating the sympathetic nervous system with veratrine. NPY-like immunoreactivity increased from 74 ± 10 to 111 ± 10 (SE) pM (P < 0.05). Thus,although NPY is a potent vasoconstrictor in the rabbit lung, it is notlikely that plasma NPY concentrations rise sufficiently, even aftermassive sympathetic nervous system activation, to produce pulmonaryvasoconstriction in the intact rabbit.

     

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells

We have used fluo3-loaded mouse pancreatic acinar cells to investigate the relationshipbetween Ca²⁺ mobilization andintracellular pH (pH_i). TheCa²⁺-mobilizing agonist ACh (500 nM) induced a Ca²⁺ release in theluminal cell pole followed by spreading of the Ca²⁺ signal toward the basolateralside with a mean speed of 16.1 ± 0.3 µm/s. In the presence of anacidic pH_i, achieved by blockade of theNa⁺/H⁺exchanger or by incubation of the cells in aNa⁺-free buffer, a slowerspreading of ACh-evoked Ca²⁺ waveswas observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s,respectively). The effects of cytosolic acidification on thepropagation rate of ACh-evokedCa²⁺ waves were largely reversibleand were not dependent on the presence of extracellularCa²⁺. A reduction in the spreadingspeed of Ca²⁺ waves could also beobserved by inhibition of the vacuolarH⁺-ATPase with bafilomycinA₁ (11.1 ± 0.6 µm/s), whichdid not lead to cytosolic acidification. In contrast, inhibition of theendoplasmic reticulum Ca²⁺-ATPaseby 2,5-di-tert-butylhydroquinone ledto faster spreading of the ACh-evokedCa²⁺ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatmentof the cells with bafilomycin A₁.Cytosolic alkalinization had no effect on the spreading speed of theCa²⁺ signals. The data suggestthat the propagation rate of ACh-induced Ca²⁺ waves is decreased byinhibition of Ca²⁺ release fromintracellular stores due to cytosolic acidification or toCa²⁺ pool alkalinizationand/or to a decrease in the proton gradient directed from theinositol 1,4,5-trisphosphate-sensitiveCa²⁺ pool to the cytosol.

     

Intracellular signaling leads to the hypertrophic effect of neuropeptide Y

Signal transduction pathways involved in the hypertrophic effectof neuropeptide Y (NPY) were investigated in adult cardiomyocytes. Reduction of transforming growth factor- activity inserum-supplemented media abolished the induction of hypertrophicresponsiveness to NPY. In responsive cells, NPY (100 nM) increasedprotein synthesis, determined as incorporation of[¹⁴C]phenylalanine, by35 ± 15% (P < 0.05, n = 16 cultures). In these cells, NPYactivated pertussis toxin (PTx)-sensitive G proteins andphosphatidylinositol (PI) 3-kinase. PTx and inhibition of PI 3-kinaseabolished the hypertrophic effect of NPY. NPY also activated proteinkinase C (PKC) and mitogen-activated protein (MAP) kinase. Inhibitionof these two kinases attenuated the induction of creatine kinase(CK)-BB but not the growth response to NPY. In conclusion, NPYstimulates protein synthesis in adult cardiomyocytes via activation ofPTx-sensitive G proteins and PI 3-kinase and it induces the fetal-typeCK-BB via activation of PKC and MAP kinase.

     

Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells

The hypothesis that protein kinase C (PKC) isable to regulate the whole cell Ca-activated K(K_Ca) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog1,2-dioctanoyl-sn-glycerol (DOG) at 10 µM attenuated the current-voltage(I-V)relationship of the K_Ca current significantly and reduced the K_Cacurrent at +70 mV by 70 ± 4% (n = 14). In contrast, 10 µM DOG after pretreatment of the cells with 1 µM calphostin C or 1 µM PKC inhibitor peptide, selective PKCinhibitors, and 10 µM1,3-dioctanoyl-sn-glycerol, aninactive diacylglycerol analog, did not significantly alter theK_Ca current. Furthermore, thecatalytic subunit of PKC (PKC_C)at 0.1 U/ml attenuated theI-Vrelationship of the K_Ca currentsignificantly, reduced the K_Cacurrent at +70 mV by 44 ± 3% (n = 17), and inhibited the activity of singleK_Ca channels at 0 mV by 79 ± 9% (n = 6). In contrast, 0.1 U/mlheat-inactivated PKC_C did notsignificantly alter the K_Cacurrent or the activity of singleK_Ca channels. Thus these resultssuggest that PKC is able to considerably attenuate theK_Ca current of freshly isolatedrat tail artery smooth muscle cells independently of effects of PKC onlocal Ca release events, most likely by a direct effect on theK_Ca channel.     

Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells

Mucin secretion by airway goblet cells is under the control ofapical P2Y₂, phospholipaseC-coupled purinergic receptors. In SPOC1 cells, the mobilization ofintracellular Ca²⁺ by ionomycin orthe activation of protein kinase C (PKC) by phorbol 12-myristate13-acetate (PMA) stimulates mucin secretion in a fully additive fashion[L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis.Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):L201-L210, 1997]. This apparent independence between PKC andCa²⁺ in the stimulation of mucinsecretion was tested in streptolysin O-permeabilized SPOC1 cells. Thesecells were fully competent to secrete mucin whenCa²⁺ was elevated from 100 nM to3.1 µM for 2 min following permeabilization; theCa²⁺EC₅₀ was 2.29 ± 0.07 µM.Permeabilized SPOC1 cells were exposed to PMA or 4-phorbol atCa²⁺ activities ranging from 10 nMto 10 µM. PMA, but not 4-phorbol, increased mucin release at allCa²⁺ activities tested: at 10 nMCa²⁺ mucin release was 2.1-foldgreater than control and at 4.7 µM Ca²⁺ mucin release was maximal(3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µMthan at 10 nM Ca²⁺. Hence, SPOC1cells possess Ca²⁺-insensitive,PKC-dependent, and Ca²⁺-dependentPKC-potentiated pathways for mucin granule exocytosis.

     

Control of Ca2+ wave propagation in mouse pancreatic acinar cells

We haveinvestigated control mechanisms involved in the propagation ofagonist-induced Ca²⁺ waves inisolated mouse pancreatic acinar cells. Using a confocal laser-scanningmicroscope, we were able to show that maximal stimulation of cells withacetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initialCa²⁺ release of comparable amountswith both agonists at the luminal cell pole. SubsequentCa²⁺ spreading to the basolateralmembrane was faster with ACh (17.3 ± 5.4 µm/s) than with bombesin(8.0 ± 2.2 µm/s). The speed of bombesin-inducedCa²⁺ waves could be increased upto the speed of ACh-induced Ca²⁺waves by inhibition of protein kinase C (PKC). Activation of PKCsignificantly decreased the speed of ACh-inducedCa²⁺ waves but had only littleeffect on bombesin-evoked Ca²⁺waves. Within 3 s after stimulation, production of inositol1,4,5-trisphosphate [Ins(1,4,5)P₃]was higher in the presence of ACh compared with bombesin, whereasbombesin induced higher levels of diacylglycerol (DAG) than ACh. Thesedata suggest that the slower propagation speed of bombesin-inducedCa²⁺ waves is due to higheractivation of PKC in the presence of bombesin compared with ACh. Thehigher increase in bombesin- compared with ACh-induced DAG productionis probably due to activation of phospholipase D (PLD). Inhibition ofthe PLD-dependent DAG production by preincubation with 0.3% butanolled to an acceleration of the bombesin-induced Ca²⁺ wave. In further experiments,we could show that ruthenium red (100 µM), an inhibitor ofCa²⁺-inducedCa²⁺ release in skeletal muscle,also decreased the speed of ACh-induced Ca²⁺ waves. The effect ofruthenium red was not additive to the effect of PKC activation. Fromthe data, we conclude that, following Ins(1,4,5)P₃-inducedCa²⁺ release in the luminal cellpole, secondary Ca²⁺ release fromstores, which are located in series between the luminal and the basalplasma membrane, modifies Ca²⁺spreading toward the basolateral cell side byCa²⁺-inducedCa²⁺ release. Activation of PKCleads to a reduction in Ca²⁺release from these stores and therefore could explain the slower propagation of Ca²⁺ waves in thepresence of bombesin compared with ACh.

     

Muscle metabolites and performance during high-intensity, intermittent exercise

Six men werestudied during four 30-s "all-out" exercise bouts on anair-braked cycle ergometer. The first three exercise bouts wereseparated by 4 min of passive recovery; after the third bout, subjectsrested for 4 min, exercised for 30 min at 30-35% peakO₂ consumption, and rested for afurther 60 min before completing the fourth exercise bout. Peak powerand total work were reduced (P < 0.05) during bout 3 [765 ± 60 (SE) W; 15.8 ± 1.0 kJ] compared withbout 1 (1,168 ± 55 W, 23.8 ± 1.2 kJ), but no difference in exercise performance was observed betweenbouts 1 and4 (1,094 ± 64 W, 23.2 ± 1.4 kJ). Before bout 3, muscle ATP,creatine phosphate (CP), glycogen, pH, and sarcoplasmic reticulum (SR)Ca²⁺ uptake were reduced, whilemuscle lactate and inosine 5'-monophosphate wereincreased. Muscle ATP and glycogen before bout4 remained lower than values beforebout 1 (P < 0.05), but there were no differences in muscle inosine 5'-monophosphate, lactate, pH, and SR Ca²⁺ uptake. Muscle CP levelsbefore bout 4 had increased aboveresting levels. Consistent with the decline in muscle ATP wereincreases in hypoxanthine and inosine before bouts3 and 4. The decline in exercise performance does not appear to be related to a reduction inmuscle glycogen. Instead, it may be caused by reduced CP availability, increased H⁺ concentration,impairment in SR function, or some other fatigue-inducing agent.

     

Altered Ca(2+) handling by ryanodine receptor and Na(+)-Ca(2+) exchange in the heart from ovariectomized rats: role of protein kinase A

Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (±dP/dt_max) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca²⁺ homeostasis that determines the contractile function. Particular emphasis was given to Ca²⁺ handling by ryanodine receptor (RyR) and Na⁺-Ca²⁺ exchange (NCX). ⁴⁵Ca²⁺ fluxes via the RyR, NCX, and Ca²⁺-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca²⁺ by these Ca²⁺ handling proteins with the overall Ca²⁺ homeostasis by determining the Ca²⁺ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca²⁺ handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca²⁺ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca²⁺ through RyR and removal of Ca²⁺ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx. ovariectomy     

Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation

We examined protein kinase C (PKC)-dependentregulation ofNa⁺-K⁺-ATPasein frog mucociliary cells. Activation of PKC by12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol(diC8) either in intact cells or isolated membranes resulted in aspecific inhibition ofNa⁺-K⁺-ATPaseactivity by ~25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximalinhibition concentration (IC₅₀) = 0.5 ± 0.1 nM and 2.4 ± 0.2 µM, respectively, for TPA anddiC8] and were not influenced byCa²⁺. Analysis of the ouabaininhibition pattern revealed the presence of twoNa⁺-K⁺-ATPaseisoforms with IC₅₀ values forcardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM,respectively. Most importantly, the isoform possessing a higheraffinity for ouabain was almost completely inhibited by TPA, whereasits counterpart was hardly sensitive to the PKC activator. The resultssuggest that, in frog mucociliary cells, PKC regulatesNa⁺-K⁺-ATPaseand that this action is related to the specificNa⁺-K⁺-ATPaseisoform.

     

PKC regulation of cardiac CFTR Cl- channel function in guinea pig ventricular myocytes

The role ofprotein kinase C (PKC) in regulating the protein kinase A(PKA)-activated Cl currentconducted by the cardiac isoform of the cystic fibrosis transmembraneconductance regulator (cCFTR) was studied in guinea pig ventricularmyocytes using the whole cell patch-clamp technique. Althoughstimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu)alone did not activate thisCl current, even whenintracellular dialysis was limited with the perforated patch-clamptechnique, activation of PKC did elicit a significant response in thepresence of PKA-dependent activation of the current by the-adrenergic receptor agonist isoproterenol. PDBuincreased the magnitude of theCl conductance activated bya supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% (n = 9) when added afterisoproterenol and by 36 ± 16% (n = 14) when introduced before isoproterenol. 4-Phorbol12,13-didecanoate, a phorbol ester that does not activate PKC, did notmimic these effects. Preexposure to chelerythrine orbisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl current by 79 ± 7.7% (n = 11) and 52 ± 10%(n = 8), respectively. Ourresults suggest that although acute activation of endogenous PKC alonedoes not significantly regulate cCFTRCl channel activity innative myocytes, it does potentiate PKA-dependent responses, perhapsmost dramatically demonstrated by basal PKC activity, which may play apivotal role in modulating the function of these channels.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts

The ability to image calciumsignals at subcellular levels within the intact depolarizing heartcould provide valuable information toward a more integratedunderstanding of cardiac function. Accordingly, a system combiningtwo-photon excitation with laser-scanning microscopy was developed tomonitor electrically evoked [Ca²⁺]_itransients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca²⁺]_itransients were recorded at depths 100 µm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked[Ca²⁺]_i transients were highly synchronizedamong neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50%(t_90-50%) and from 50 to 10%(t_50-10%) of the peak[Ca²⁺]_i were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2 Hz, 62 ± 3 and94 ± 6 ms (n = 19, P < 0.05 vs.1 Hz) in rhod-2-loaded cardiomyocytes.[Ca²⁺]_i decay was markedly slower infura-2-loaded hearts (t_90-50% at 1 Hz,128 ± 9 ms and at 2 Hz, 88 ± 5 ms;t_50-10% at 1 Hz, 214 ± 18 ms and at2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of[Ca²⁺]_i decline resulted from increasedcytosolic Ca²⁺ buffering, because the kinetics of rhod-2decay resembled those obtained with fura-2 after incorporation of theCa²⁺ chelator BAPTA. Propagating calcium waves and[Ca²⁺]_i amplitude alternans were readilydetected in paced hearts. This approach should be of general utility tomonitor the consequences of genetic and/or functional heterogeneity incellular calcium signaling within whole mouse hearts at tissue depthsthat have been inaccessible to single-photon imaging.

     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_m values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀ (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride