Michael I. Kotlikoff

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter‐organelle crosstalk

We have investigated the mechanism of mitochondrial–nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m‐chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψm) and electron transport‐coupled ATP synthesis, which was also accompanied by an elevated steady‐state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor‐1 (RyR‐1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+‐responsive factors calineurin, calcineurin‐dependent NFATc (cytosolic counterpart of activated T‐cell‐specific nuclear factor) and c‐Jun N‐terminal kinase (JNK)‐dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF‐κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady‐state [Ca2+]i were normalized in genetically reverted cells which contain near‐normal mtDNA levels. We propose that the mitochondria‐to‐nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Receptor‐activated calcium influx in human airway smooth muscle cells.

1. Fluorescence measurements of intracellular calcium concentrations ([Ca2+]i) were made on cultured human airway smooth muscle cells using the dye Fura‐2. The response to either histamine (100 microM) or bradykinin (1 microM) was biphasic, with a transient increase in [Ca2+]i followed by a sustained [Ca2+]i increase lasting many minutes. The average steady‐state (plateau) [Ca2+]i following agonist activation was 267 +/‐ 5 nM, whereas the average basal [Ca2+]i was 148 +/‐ 4 nM. 2. The sustained rise in [Ca2+]i required the continued presence of either histamine or bradykinin and was dependent on extracellular Ca2+. The magnitude of the transient rise in [Ca2+]i was not dependent on extracellular Ca2+. Sustained, receptor‐activated rises in [Ca2+]i were rapidly abolished by chelation of extracellular Ca2+, or addition of non‐permeant polyvalent cations, whereas these agents had minor effects in the absence of agonist. These data indicate that the sustained increase in [Ca2+]i was dependent on receptor‐activated Ca2+ influx. 3. Receptor‐activated Ca2+ influx was not affected by treatment with organic Ca2+ channel antagonists (nifedipine (10 microM), nisoldipine (10 microM) or diltiazem (10 microM] or agonists (Bay K 8644 (500 nM to 10 microM) or Bay R 5417 (500 nM]. The magnitude of the sustained rise was also not affected by pre‐treatment with ouabain (100 microM) indicating little involvement of Na(+)‐Ca2+ exchange in the influx mechanism. 4. Receptor‐activated Ca2+ influx could be completely inhibited by several polyvalent cations (Co2+, Mn2+, Ni2+, ‐Cd2+ or La3+). Quantitative estimates of the potency of block were obtained for Ni2+ and La3+. These measurements indicate that the pKi for Ni2+ was 3.6 and for La3+ was 3.5. 5. Both Mn2+ and Co2+ ions caused a time‐dependent quench of intracellular Fura‐2; however, permeation of neither ion was increased following receptor activation, indicating that the influx pathway is not permeable to these cations. 6. Fura‐2 was used to monitor the rate of Ba2+ entry into airway smooth muscle cells by monitoring the Ca(2+)‐Fura‐2 and Ba(2+)‐Fura‐2 isosbestic points as well as the 340 and 380 nm signals. Cell activation did not increase the rate of Ba2+ entry indicating that the Ca2+ influx pathway was poorly permeant to Ba2+ ions. Ba2+ (2 mM) was able to inhibit Ca2+ entry as shown by its effects on the Ba(2+)‐independent, Ca(2+)‐dependent wavelength (371 nm). 7. The voltage dependence of Ca2+ influx was examined before and after agonist‐induced activation. The effect of KCl‐induced depolarization prior to cell activation was to cause a slight increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Beta-adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms.

Stimulation of calcium-activated potassium (KCa) channels in airway smooth muscle cells by phosphorylation-dependent and membrane-delimited, G protein actions has been reported (Kume, H. A. Takai, H. Tokuno, and T. Tomita. 1989. Nature [Lond.]. 341:152-154; Kume, H., M. P. Graziano, and M. I. Kotlikoff. 1992. Proc. Natl. Acad. Sci. USA. 89:11051-11055). We show that beta-adrenergic receptor/channel coupling is not affected by inhibition of endogenous ATP, and that activation of KCa channels is stimulated by both alpha S and cAMP-dependent protein kinase (PKA). PKA stimulated channel activity in a dose-dependent fashion with an EC50 of 0.12 U/ml and maximum stimulation of 7.38 +/- 2.04-fold. Application of alpha S to patches near maximally stimulated by PKA significantly increased channel activity to 15.1 +/- 3.65-fold above baseline, providing further evidence for dual regulatory mechanisms and suggesting that the stimulatory actions are independent. Analysis of channel open-time kinetics indicated that isoproterenol and alpha S stimulation of channel activity primarily increased the proportion of longer duration events, whereas PKA stimulation had little effect on the proportion of short and long duration events, but resulted in a significant increase in the duration of the long open-state. cAMP formation during equivalent relaxation of precontracted muscle strips by isoproterenol and forskolin resulted in significantly less cAMP formation by isoproterenol than by forskolin, suggesting that the degree of activation of PKA is not the only determinant of tissue relaxation. We conclude that beta-adrenergic stimulation of KCa channel activity and relaxation of tone in airway smooth muscle occurs, in part, by means independent of cyclic AMP formation.

Delayed rectifier potassium channels in canine and porcine airway smooth muscle cells.

1. In order to define the ion channels underlying the inactivating, calcium‐insensitive current in airway smooth muscle cells, unitary potassium currents were recorded from canine and porcine trachealis cells, and compared with macroscopic currents. On‐cell and inside‐out single‐channel currents were compared with whole‐cell recordings made in dialysed cells. 2. Depolarizing voltage steps evoked outward unitary currents. In addition to a large conductance, calcium‐activated potassium channel (KCa), a lower conductance potassium channel was identified. This channel has a conductance of 12.7 pS (on‐cell; 1 mM‐K+ in the pipette). 3. The lower conductance channel (Kdr) was not sensitive to cytosolic Ca2+ concentration and unitary current openings occurred following a delay after the voltage step. The time course of activation of the current composed of averaged single‐channel events was very similar to that of the whole‐cell, delayed rectifier potassium current (IdK), recorded under conditions of low intracellular calcium (Kotlikoff, 1990). 4. Kdr channels also inactivated with kinetics similar to those of the macroscopic current. Averaged single‐channel records revealed a current that inactivated with kinetics that could be described by two exponentials (tau 1 = 0.14 s, tau 2 = 1.1 s; at 5 mV). These values corresponded well with previously determined values for time‐dependent inactivation of IdK. Inactivation of Kdr channels was markedly voltage dependent, and was well fitted by a Boltzmann equation with V50 = ‐53 mV; this was similar to measurements of the macroscopic current, although the V50 value was shifted to more positive potentials in whole‐cell measurements. When only the inactivating component of the macroscopic current was considered, the voltage dependence of inactivation of the single‐channel current and macroscopic current were quite similar. 5. Single‐channel kinetics indicated that Kdr channels occupy one open and two closed states. The mean open time was 1.7 ms. Inactivation results in a prominent increase in the long closed time, with little effect on the mean open time or short closed time. 6. The Kdr channel was not blocked by tetraethylammonium (TEA; 1 mM), charybdotoxin (ChTX; 100 nM) or glibenclamide (20 microM), but was blocked by 4‐aminopyridine (4‐AP; 1 mM). Similarly, 4‐AP blocked the inactivating component of the macroscopic current, but a non‐inactivating current remained. KCa currents were blocked by TEA (0.5‐1 mM) and charybdotoxin (40 nM), but were insensitive to to 4‐AP (1 mM) and glibenclamide (20 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells

1. In order to determine the physiological role of specific potassium currents in airway smooth muscle, potassium currents were measured in freshly dissociated ferret trachealis cells using the nystatin‐permeabilized, whole‐cell method, at 35 degrees C. 2. The magnitude of the outward currents was markedly increased as bath temperature was increased from 22 to 35 degrees C. This increase was primarily due to the increase in maximum potassium conductance (gK,max), although there was also a small leftward shift in the relationship between gK and voltage at higher temperatures. The maximum conductance and the kinetics of current activation and inactivation were also temperature dependent. At 35 degrees C, gating of the current was steeply voltage dependent between ‐40 and 0 mV. Current activation was well fitted by fourth‐order kinetics; the mean time constants of activation (30 mV clamp step) were 1.09 +/‐ 0.17 and 1.96 +/‐ 0.27 ms at 35 and 22 degrees C, respectively. 3. Outward currents using the nystatin method were qualitatively similar to delayed rectifier currents recorded in dialysed cells with high calcium buffering capacity solutions. 4‐Aminopyridine (4‐AP; 2 mM), a specific blocker of delayed rectifier potassium channels in this tissue, inhibited over 80% of the outward current evoked by voltage‐clamp steps to between ‐10 and +20 mV (n = 6). Less than 5% of the outward current was blocked over the same voltage range by charybdotoxin (100 nM; n = 15), a specific antagonist of large‐conductance, calcium‐activated potassium channels in this tissue. 4. The degree to which delayed rectifier and calcium‐activated potassium conductances control resting membrane potential was examined in current‐clamp experiments. The resting membrane potential of current clamped cells was ‐33.6 +/‐ 1.0 mV (n = 62). Application of 4‐AP (2 mM) resulted in a 14.4 +/‐ 1.0 mV depolarization (n = 8) and an increase in input resistance. Charybdotoxin (100 nM) had no effect on resting membrane potential (n = 6). 5. Force measurements were made in isolated strips of trachealis muscle to determine the effect of pharmacological blockade of individual potassium conductances on resting tone. In the presence of tetrodotoxin (1 microM) and atropine (1 microM), 4‐AP increased baseline tension in a dose‐dependent manner, with an EC50 of 1.8 mM (n = 13); application of 5 mM 4‐AP increased tone to 86.8 +/‐ 8.1% of that produced by 1 microM methacholine, and this tone was almost completely inhibited by nifedipine (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstitution of β-Adrenergic Modulation of Large Conductance, Calcium-activated Potassium (Maxi-K) Channels in XenopusOocytes IDENTIFICATION OF THE cAMP-DEPENDENT PROTEIN KINASE PHOSPHORYLATION SITE

The human large conductance, calcium-activated potassium (maxi-K) channel (α and β subunits) and β2-adrenergic receptor genes were coexpressed inXenopus oocytes in order to study the mechanism of β-adrenergic modulation of channel function. Isoproterenol and forskolin increased maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and both channel subunits by 33 ± 5% and 35 ± 8%, respectively, without affecting current activation or inactivation. The percentage of stimulation by isoproterenol and forskolin was not different in oocytes coexpressing the α and β subunits versus those expressing the only the α subunit, suggesting that the α subunit is the target for regulation. The stimulatory effect of isoproterenol was almost completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK) regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide had little effect, indicating that cellular coupling of β2-adrenergic receptors to maxi-K channels involves endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation sites (serine 869) on the α subunit almost completely inhibited β-adrenergic receptor/channel stimulatory coupling, whereas forskolin still stimulated currents moderately (16 ± 4%). These data demonstrate that physiological coupling between β2 receptors and maxi-K channels occurs by the cAMP-PK mediated phosphorylation of serine 869 on the α subunit on the channel.

Voltage‐dependent calcium currents and cytosolic calcium in equine airway myocytes.

1. The relationship between voltage‐dependent calcium channel current (I(Ca)) and cytosolic free calcium concentration ([Ca2+]i) was studied in fura‐2 AM‐loaded equine tracheal myocytes at 35 degrees C and 1.8 mM Ca2+ using the nystatin patch clamp method. The average cytosolic calcium buffering constant was 77 +/‐ 3 (n = 14), and the endogenous calcium buffering constant component is likely to be between 15 and 50. 2. I(Ca) did not evoke significant calcium‐induced calcium release (CICR) since (i)[Ca2+]i scaled with the integrated I(Ca) over the full voltage range of evoked calcium currents, (ii) increases in [Ca2+]i associated with I(Ca) were consistent with cytoplasmic buffering of calcium ions entering through voltage‐dependent calcium channels (VDCCs) only, (iii) there was a fixed instantaneous relationship between transmembrane calcium flux (J(Ca)) and the change in cytosolic free calcium concentration (delta [Ca2+]i) during I(Ca), (iv) caffeine (8 mM) triggered 8‐fold higher calcium transients than I(Ca), and (v) I(Ca) evoked following release of intracellular calcium by caffeine resulted in an equivalent delta[Ca2+]i‐J(Ca) relationship. 3. The time constant (T) for the decay in [Ca2+]i was 8.6 +/‐ 1.5 s (n = 8) for single steps and 8.6 +/‐ 1.1 s (n = 13) following multiple steps that increased [Ca2+]i to much higher levels. Following application of caffeine (8 mM), however, [Ca2+]i decay was enhanced (T = 2.0 +/‐ 0.2 s, n = 3). The rate of [Ca2+]i decay was not voltage dependent, was not decreased in the absence of extracellular Na+ ions, and no pump current was detected. 4. We conclude that under near physiological conditions, neither CICR nor Na(+)‐Ca2+ exchange play a substantial role in the regulation of I(Ca)‐induced increases in [Ca2+]i, and that, even following release of intracellular calcium by caffeine, Na(+)‐Ca2+ exchange does not play an appreciable role in the removal of calcium ions from the cytosol.

Potassium channels in airway smooth muscle : a tale of two channels

Potassium channels are an important determinant of smooth muscle excitability and force generation. Two potassium channels have been fully described in airway smooth muscle: large conductance, calcium-activated potassium channels and voltage-dependent delayed rectifier channels. This article will review the biophysics and pharmacology of these channels and discuss what is currently known with respect to their regulation and physiological significance.

Signalling pathway for histamine activation of non‐selective cation channels in equine tracheal myocytes

The signalling pathway underlying histamine activation of non‐selective cation channels was investigated in single equine tracheal myocytes. Application of histamine (100 μM) activated the transient calcium‐activated chloride current (ICl(Ca)) and sustained, low amplitude non‐selective cation current (ICat). The H1 receptor antagonist pyrilamine (10 μM) blocked activation of ICl(Ca) and ICat. Simultaneous application of histamine (100 μM) and caffeine (8 mm) during H1 receptor blockade activated ICl(Ca), but not ICat. Neither the H2 receptor antagonist cimetidine (20 μM) nor the H3 receptor antagonist thioperamide (20 μM) prevented activation of ICl(Ca) and ICat. Intracellular dialysis of anti‐Gαi/Gαo antibodies completely blocked activation of ICat by histamine, whereas ICl(Ca) was not affected. By contrast, anti‐Gαq/Gα11 antibodies greatly inhibited ICl(Ca), but did not alter activation of ICat. 1‐Oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 20–100 μM) did not induce any current or affect currents activated by histamine or methacholine (mACH). Simultaneous application of OAG and caffeine activated ICl(Ca), but not ICat, indicating that a rise in [Ca2+]i and stimulation of diacylglycerol‐sensitive protein kinase C (PKC) is not sufficient to activate ICat. The phospholipase C inhibitor U73122 (2 μM) blocked histamine activation of ICl(Ca) and ICat, but simultaneous exposure of myocytes to histamine and caffeine restored both ICl(Ca) and ICat in the presence of U73122. Histamine and mACH activated currents with equivalent I–V relationships. The currents activated by these agonists were not additive; following activation of ICat by mACH, histamine failed to induce an additional membrane current. Similarly, mACH did not induce an additional current after full activation of ICat by histamine. We conclude that H1 histamine receptors activate ICat through coupling to Gi/Go proteins. Activation of ICat also requires intracellular calcium release, mediated by H1 receptors coupling to Gq/G11 proteins. This coupling is analogous to the activation of ICat by co‐stimulation of M2 and M3 receptors.

Hypoxia inhibits the Na+/Ca2+ exchanger in pulmonary artery smooth muscle cells

The cellular mechanisms underlying hypoxic pulmonary vasoconstriction are not fully understood. We examined the effect of hypoxia on Ca2+ efflux from the cytosol in single Fura‐2‐loaded pulmonary artery myocytes. During mild hypoxia (pO2=50–60 Torr), peak [Ca2+]i was increased and the rate of Ca2+ removal from the cytosol was markedly slowed after stimuli that elevated [Ca2+]i. Removal of extracellular Na+ potentiated the peak [Ca2+]i rise and slowed the Ca2+ decay rate in cells recorded under normoxic conditions; it did not further slow the Ca2+ decay rate or potentiate the [Ca2+]i increase in hypoxic cells. An Na+/Ca2+ exchange current was recorded in isolated pulmonary artery myocytes. Switching from Li+ to Na+ (130 mM) revealed an inward current with reversal potential consistent with the Na+/Ca2+ exchange current in cells in which [Ca2+]i was clamped at 1 μΜ; similar currents, although smaller, were observed with normal resting [Ca2+]i using the perforated patch clamp technique. The Na+/Ca2+ exchange current was markedly inhibited in myocytes exposed to mild hypoxia. RT‐PCR revealed the expression of specific alternatively spliced RNAs of NCX1 in rat pulmonary arteries. These findings provide an enhanced understanding of the molecular mechanisms underlying hypoxic sensing in pulmonary arteries.—Wang, Y.‐X., Dhulipala, P. K., Kotlikoff, M. I. Hypoxia inhibits the Na+/Ca2+ exchanger in pulmonary artery smooth muscle cells. FASEB J. 14, 1731–1740 (2000)