Erk1-Mediated Development of Left Ventricular Cardiac Hypertrophy in a P21 Activated Kinase-1 (Pak1) Knockout Mouse Model (original) (raw)
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The Journal of Physiology, 2011
Non-technical summary Parasympathetic nerve activation of M3 and M2 muscarinic acetylcholine receptors initiates and modulates calcium release from the sarcoplasmic reticulum to control airway smooth muscle contraction. Here we investigate M2 acetylcholine receptors that also contribute to contraction through depolarization and recruitment of voltage-dependent calcium channels (VDCCs). We find that the calcium-and voltage-activated potassium channel (BK channel) and its β1 accessory subunit are important proteins that oppose M2-mediated contraction of airway smooth muscle. BK channels contribute to a negative baseline membrane voltage from which M2-mediated depolarization only weakly activates VDCCs. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type VDCCs to contraction that otherwise does not occur with M2 receptor antagonist or with β1 containing BK channels. These findings provide a better understanding of how cholinergic second messenger signalling impinges on voltage-dependent mechanisms and excitation-contraction coupling of smooth muscle.
The Journal of biological chemistry, 2008
Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of ...
The FASEB Journal, 2007
The unique voltage-and Ca 2؉ -dependent K ؉ (BK) channel, prominently expressed in airway smooth muscle cells, has been suggested as an important effector in controlling airway contractility. Its deletion in mice depolarized resting membrane potential of tracheal cells, suggesting an increased openprobability of voltage-gated Ca 2؉ channels. While carbachol concentration-dependently increased the tonic tension of wild-type (WT) trachea, mutant trachea showed a different response with rapid tension development followed by phasic contractions superimposed on a tonic component. Tonic contractions were substantially more dependent on L-type Ca 2؉ current in mutant than in WT trachea, even though L-type Ca 2؉ channels were not up-regulated. In the absence of L-type Ca 2؉ current, half-maximal contraction of trachea was shifted from 0.51 to 1.7 M. In agreement, cholinergic bronchoconstriction was reduced in mutant lung slices, isolated-perfused lungs and, most impressively, in mutant mice analyzed by body plethysmography. Furthermore, isoprenaline-mediated airway relaxation was enhanced in mutants. In-depth analysis of cAMP and cGMP signaling revealed up-regulation of the cGMP pathway in mutant tracheal muscle. Inhibition of cGMP kinase reestablished normal sensitivity toward carbachol, indicating that up-regulation of cGMP signaling counterbalances for BK channel ablation, pointing to a predominant role of BK channel in regulation of airway toneReduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca 2؉ -activated K ؉ channel.
BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle
American Journal of Physiology-lung Cellular and Molecular Physiology, 2006
channel 1-subunit regulation of calcium handling and constriction in tracheal smooth muscle. The large-conductance, Ca 2ϩ -activated K ϩ (BK) channels are regulators of voltage-dependent Ca 2ϩ entry in many cell types. The BK channel accessory 1-subunit promotes channel activation in smooth muscle and is required for proper tone in the vasculature and bladder. However, although BK channels have also been implicated in airway smooth muscle function, their regulation by the 1-subunit has not been investigated. Utilizing the gene-targeted mice for the 1-subunit gene, we have investigated the role of the 1-subunit in tracheal smooth muscle. In mice with the 1-subunit-knockout allele, BK channel activity was significantly reduced in excised tracheal smooth muscle patches and spontaneous BK currents were reduced in whole tracheal smooth muscle cells. Knockout of the 1-subunit resulted in an increase in resting Ca 2ϩ levels and an increase in the sustained component of Ca 2ϩ influx after cholinergic signaling. Tracheal constriction studies demonstrate that the level of constriction is the same with knockout of the 1-subunit and BK channel block with paxillin, indicating that BK channels contribute little to airway relaxation in the absence of the 1-subunit. Utilizing nifedipine, we found that the increased constriction caused by knockout of the 1-subunit could be accounted for by an increased recruitment of L-type voltage-dependent Ca 2ϩ channels. These results indicate that the 1-subunit is required in airway smooth muscle for control of voltage-dependent Ca 2ϩ influx during rest and after cholinergic signaling in BK channels.
Membrane stretch augments the cardiac muscarinic K+ channel activity
The Journal of Membrane Biology, 1995
Arachidonic acid has been shown to activate K+-selective, mechanosensitive ion channels in cardiac, neuronal and smooth muscle cells. Since the cardiac G protein (Gx)-gated, muscarinic K + (KAch) channel can also be activated by arachidonic acid, we investigated whether the KAc h channel was also sensitive to membrane stretch. In the absence of acetylcholine (ACh), KAC h channels were not active, and negative pressure failed to activate these channels. With ACh (10 gM) in the pipette, applying negative pressure (0 to-80 mm Hg) to the membrane caused a reversible, pressure-dependent increase in channel activity in cell-attached and insideout patches (100 BM GTP in bath). Membrane stretch did not alter the sensitivity of the Kac h channel to GTP. When G K was maximally activated with 100 gM GTPyS in inside-out patches, the KAc h channel activity could be further increased by negative pressure. Trypsin (0.5 mg/ ml) applied to the membrane caused activation of the KAC h channel in the absence of ACh and GTP; KAC h channel activity was further increased by stretch. These results indicate that the atrial muscarinic K + channels are modulated by stretch independently of receptor/G protein, probably via a direct effect on the channel protein/ lipid bilayer.
Pfl�gers Archiv European Journal of Physiology, 1991
Large-conductance Ca2+-activated K + channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 gM), an agonist which releases Ca 2 § from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 pM), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cellattached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca 2 § concentration at the cytoplasmic side above 10-7 tool/1. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (Ki) of 0.2 mmol/1 was found for the ATP-induced inhibition. ATP reduced the Ca 2 § sensitivity of the channel, shifting the Ca 2 + activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca 2 § K § channel in airway smooth muscle. This ATP modulation of Ca 2 § K + channels might serve as an important mechanism linking energy status and the contractile state of the cells.
Journal of Biological Chemistry, 2003
The repolarization phase of cardiac action potential is prone to aberrant excitation that is common in cardiac patients. Here, we demonstrate that this phase is markedly sensitive to Ca 2؉ because of the surprising existence of a Ca 2؉-activated K ؉ currents in cardiac cells. The current was revealed using recording conditions that preserved endogenous Ca 2؉ buffers. The Ca 2؉-activated K ؉ current is expressed differentially in atria compared with ventricles. Because of the significant contribution of the current toward membrane repolarization in cardiac myocytes, alterations of the current magnitude precipitate abnormal action potential profiles. We confirmed the presence of a small conductance Ca 2؉-activated K ؉ channel subtype (SK2) in human and mouse cardiac myocytes using Western blot analysis and reverse transcription-polymerase chain reaction and have cloned SK2 channels from human atria, mouse atria, and ventricles. Because of the marked differential expression of SK2 channels in the heart, specific ligands for Ca 2؉-activated K ؉ currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes. Cardiac action potentials (APs) 1 are shaped predominantly by the interplay between transient inward Na ϩ , Ca 2ϩ , and outward K ϩ currents (1). While the repolarization phase of the AP can be wrought by the kinetics of the principal currents, small and sustained outward currents also define this phase, rendering this region prone to irregular membrane excitation.
Voltage effects on muscarinic acetylcholine receptor-mediated contractions of airway smooth muscle
Physiological Reports
Studies have shown that the activity of muscarinic receptors and their affinity to agonists are sensitive to membrane potential. It was reported that in airway smooth muscle (ASM) depolarization evoked by high K + solution increases contractility through direct effects on M3 muscarinic receptors. In this study, we assessed the physiological relevance of voltage sensitivity of muscarinic receptors on ASM contractility. Our findings reveal that depolarization by high K + solution induces contraction in intact mouse trachea predominantly through activation of acetylcholine release from embedded nerves, and to a lesser extent by direct effects on M3 receptors. We therefore devised a pharmacological approach to depolarize tissue to various extents in an organ bath preparation, and isolate contraction due exclusively to ASM muscarinic receptors within range of physiological voltages. Our results indicate that unliganded muscarinic receptors do not contribute to contraction regardless of voltage. Utilizing low K + solution to hyperpolarize membrane potentials during contractions had no effect on liganded muscarinic receptor-evoked contractions, although it eliminated the contribution of voltage-gated calcium channels. However, we found that muscarinic signaling was potentiated by at least 42% at depolarizing voltages (average À12 mV) induced by high K + solution (20 mmol/L K +). In summary, we conclude that contractions evoked by direct activation of muscarinic receptors have negligible sensitivity to physiological voltages. However, contraction activated by cholinergic stimulation can be potentiated by membrane potentials occurring beyond the physiological range of ASM.
Functional effects of KCNQ K+ channels in airway smooth muscle
Frontiers in Physiology, 2013
KCNQ (K v 7) channels underlie a voltage-gated K + current best known for control of neuronal excitability, and its inhibition by G q/11 -coupled, muscarinic signaling. Studies have indicated expression of KCNQ channels in airway smooth muscle (ASM), a tissue that is predominantly regulated by muscarinic receptor signaling. Therefore, we investigated the function of KCNQ channels in rodent ASM and their interplay with G q/11 -coupled M 3 muscarinic receptors. Perforated-patch clamp of dissociated ASM cells detected a K + current inhibited by the KCNQ antagonist, XE991, and augmented by the specific agonist, flupirtine. KCNQ channels begin to activate at voltages near resting potentials for ASM cells, and indeed XE991 depolarized resting membrane potentials. Muscarinic receptor activation inhibited KCNQ current weakly (∼20%) at concentrations half-maximal for contractions. Thus, we were surprised to see that KCNQ had no affect on membrane voltage or muscle contractility following muscarinic activation. Further, M 3 receptor-specific antagonist J104129 fumarate alone did not reveal KCNQ effects on muscarinic evoked depolarization or contractility. However, a role for KCNQ channels was revealed when BK-K + channel activities are reduced. While KCNQ channels do control resting potentials, they appear to play a redundant role with BK calcium-activated K + channels during ASM muscarinic signaling. In contrast to effect of antagonist, we observe that KCNQ agonist flupirtine caused a significant hyperpolarization and reduced contraction in vitro irrespective of muscarinic activation. Using non-invasive whole animal plethysmography, the clinically approved KCNQ agonist retigabine caused a transient reduction in indexes of airway resistance in both wild type and BK β1 knockout (KO) mice treated with the muscarinic agonist. These findings indicate that KCNQ channels can be recruited via agonists to oppose muscarinic evoked contractions and may be of therapeutic value as bronchodilators.
Pflugers Archiv : European journal of physiology, 2014
Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large-conductance Ca(2+)-activated K(+) (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9 ± 1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca(2+) from ryanodine receptors. C...