Characterization of the A-Type potassium current in murine gastric fundus smooth muscles

2021 ◽  
Author(s):  
Gregory C. Amberg ◽  
Ji Yeon Lee ◽  
Sang Don Koh ◽  
Kenton M. Sanders
Keyword(s):  
Smooth Muscle ◽  
Time Constant ◽  
Smooth Muscles ◽  
Cell Voltage ◽  
Gastric Fundus ◽  
Membrane Potentials ◽  
Tonic Smooth Muscle ◽  
Recovery From Inactivation ◽  
Kv4 Channels

Transient outward, or "A-type" currents are rapidly inactivating voltage gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole-cell voltage-clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5nA at 0 mV. Inactivation was rapid with a time constant of 71ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide and phrixotoxon-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after TEA displayed half inactivation at -48.1±1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

scholarly journals Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

2015 ◽  
Vol 308 (7) ◽  
pp. G605-G612 ◽  
Author(s):  
Satish Rattan ◽  
Mehboob Ali
Keyword(s):  
Smooth Muscle ◽  
Kinase Inhibitor ◽  
Smooth Muscles ◽  
Rho Kinase ◽  
Actin Binding ◽  
Control Mechanisms ◽  
Molecular Control ◽  
Tonic Smooth Muscle ◽  
Before And After ◽  
Protein Kinase C Inhibitor

Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22.

The Sarcoplasmic Reticulum of Smooth Muscle Fibers

1982 ◽  
Vol 37 (5-6) ◽  
pp. 481-488 ◽  
Author(s):  
L. Raeymaekers
Keyword(s):  
Smooth Muscle ◽  
Calcium Oxalate ◽  
Striated Muscle ◽  
Smooth Muscles ◽  
Morphological Characterization ◽  
Calcium Oxalate Crystals ◽  
Additional Amount ◽  
Skinned Smooth Muscle ◽  
Ca Uptake

Abstract The ability of the sarcoplasmic (endoplasmic) reticulum (SR, ER) of smooth muscle cells to accumulate Ca was demonstrated by measuring the uptake of 45Ca in fibers which were chemically skinned with saponin, and by electron cytochemistry of the accumulated Ca. The Ca uptake was dependent on ATP and it was stimulated by oxalate, as it is the case in SR of striated muscle. Electron microscopy of the skinned smooth muscle preparations revealed the presence of calcium oxalate deposits in the reticulum. The SR vesicles were isolated from several smooth muscles. The purification was carried out by taking advantage of the density increase of the SR vesicles after loading with calcium in the presence of oxalate. Among the muscles investigated the smooth muscle of the pig stomach was found to be the most suitable and it was selected for further biochemical and morphological characterization of the SR vesicles. These vesicles, which contain calcium oxalate crystals, were able to accumulate an additional amount of Ca. The Ca uptake was supported by several energy yielding substrates. Their order of potency was ATP > dATP ≃ UTP > ITP > GTP ≃ CTP. The rate of Ca uptake was two orders of magnitude slower than that in SR of skeletal muscle. The measurement of the level of phosphorylated Ca transport intermediate showed that this difference is due to smaller number of calcium transport sites per vesicle. The distribution of intramembrane particles in freeze-fractured specimens is in agreement with this conclusion.

Effects of prostaglandin F2α on membrane currents in rabbit middle cerebral arterial smooth muscle cells

2003 ◽  
Vol 284 (3) ◽  
pp. H1018-H1027 ◽  
Author(s):  
Nari Kim ◽  
Jin Han ◽  
Euiyong Kim
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Membrane Potentials ◽  
Delayed Rectifier ◽  
Direct Effects ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Arterial Smooth Muscle Cells

Although PGF2αaffects contractility of vascular smooth muscles, no studies to date have addressed the electrophysiological mechanism of this effect. The purpose of our investigation was to examine the direct effects of PGF2α on membrane potentials, Ca2+-activated K+ (KCa) channels, delayed rectifier K+ (KV) channels, and L-type Ca2+channels with the patch-clamp technique in single rabbit middle cerebral arterial smooth muscle cells (SMCs). PGF2αsignificantly hyperpolarized membrane potentials and increased the amplitudes of total K+ currents. PGF2αincreased open-state probability but had little effect on the open and closed kinetics of KCa channels. PGF2αincreased the amplitudes of KV currents with a leftward shift of the activation and inactivation curves and a decrease in the activation time constant. PGF2α decreased the amplitudes of L-type Ca2+ currents without any significant change in threshold or apparent reversal potentials. This study provides the first finding that the direct effects of PGF2α on middle cerebral arterial SMCs, at least in part, could attenuate vasoconstriction.

scholarly journals Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle.

1988 ◽  
Vol 92 (6) ◽  
pp. 713-729 ◽  
Author(s):  
B Himpens ◽  
G Matthijs ◽  
A V Somlyo ◽  
T M Butler ◽  
A P Somlyo
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Muscle Tension ◽  
Smooth Muscles ◽  
Free Calcium ◽  
Myosin Light Chain Phosphorylation ◽  
Tonic Smooth Muscle

The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.

scholarly journals Voltage-activated K+ conductances in freshly isolated embryonic chicken osteoclasts

1989 ◽  
Vol 86 (17) ◽  
pp. 6821-6825 ◽  
Author(s):  
J H Ravesloot ◽  
D L Ypey ◽  
T Vrijheid-Lammers ◽  
P J Nijweide
Keyword(s):  
Time Constant ◽  
Membrane Potentials ◽  
Maximal Conductance ◽  
Exponential Process ◽  
Embryonic Chicken ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
K Concentration ◽  
Cs Ions ◽  
Full Activation

Patch-clamp measurements on freshly isolated embryonic chicken osteoclasts revealed three distinct types of voltage-dependent K+ conductance. The first type of conductance, present in 72% of the cells, activated at membrane potentials less negative than -30 to -20 mV and reached full activation at +40 mV. It activated with a delay, reached a peak value, and then inactivated with a time constant of approximately 1.5 s. Inactivation was complete or almost so. Recovery from inactivation, at -70 mV, had a time constant of roughly 1 s. The conductance could be blocked, at least partly, by 4 mM 4-aminopyridine. The second type of conductance (present in all cells) activated at membrane potentials more negative than -40 to -80 mV and reached full activation at -130 mV. Activation potential and maximal conductance were dependent on the extracellular K+ concentration. Inactivation of the conductance first became apparent at membrane potentials more negative than -100 mV and was a two-exponential process. The conductance could be blocked by external 5 mM Cs+ ions. The third type of conductance (present in all cells) activated at membrane potentials more positive than +30 mV. Generally, the conductance did not inactivate.

Roles of CaM kinase II and phospholamban in SNP-induced relaxation of murine gastric fundus smooth muscles

2006 ◽  
Vol 291 (2) ◽  
pp. C337-C347 ◽  
Author(s):  
Minkyung Kim ◽  
In Soo Han ◽  
Sang Don Koh ◽  
Brian A. Perrino
Keyword(s):  
Smooth Muscle ◽  
Soluble Guanylate Cyclase ◽  
Muscle Relaxation ◽  
Smooth Muscles ◽  
Cyclopiazonic Acid ◽  
Gastric Fundus ◽  
Smooth Muscle Relaxation ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
No Donor

The mechanisms by which nitric oxide (NO) relaxes smooth muscles are unclear. The NO donor sodium nitroprusside (SNP) has been reported to increase the Ca2+ release frequency (Ca2+ sparks) through ryanodine receptors (RyRs) and activate spontaneous transient outward currents (STOCs), resulting in smooth muscle relaxation. Our findings that caffeine relaxes and hyperpolarizes murine gastric fundus smooth muscles and increases phospholamban (PLB) phosphorylation by Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) suggest that PLB phosphorylation by CaM kinase II participates in smooth muscle relaxation by increasing sarcoplasmic reticulum (SR) Ca2+ uptake and the frequencies of SR Ca2+ release events and STOCs. Thus, in the present study, we investigated the roles of CaM kinase II and PLB in SNP-induced relaxation of murine gastric fundus smooth muscles. SNP hyperpolarized and relaxed gastric fundus circular smooth muscles and activated CaM kinase II. SNP-induced CaM kinase II activation was prevented by KN-93. Ryanodine, tetracaine, 2-aminoethoxydiphenylborate, and cyclopiazonic acid inhibited SNP-induced fundus smooth muscle relaxation and CaM kinase II activation. The Ca2+-activated K+ channel blockers iberiotoxin and apamin inhibited SNP-induced hyperpolarization and relaxation. The soluble guanylate cyclase inhibitor 1 H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one inhibited SNP-induced relaxation and CaM kinase II activation. The membrane-permeable cGMP analog 8-bromo-cGMP relaxed gastric fundus smooth muscles and activated CaM kinase II. SNP increased phosphorylation of PLB at Ser16 and Thr17. Thr17 phosphorylation of PLB was inhibited by cyclopiazonic acid and KN-93. Ser16 and Thr17 phosphorylation of PLB was sensitive to 1 H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one. These results demonstrate a novel pathway linking the NO-soluble guanylyl cyclase-cGMP pathway, SR Ca2+ release, PLB, and CaM kinase II to relaxation in gastric fundus smooth muscles.

scholarly journals Characterization of Cecal Smooth Muscle Contraction in Laying Hens

2021 ◽  
Vol 8 (6) ◽  
pp. 91
Author(s):  
Katrin Röhm ◽  
Martin Diener ◽  
Korinna Huber ◽  
Jana Seifert
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Laying Hens ◽  
Ex Vivo ◽  
Isometric Force ◽  
Smooth Muscles ◽  
Smooth Muscle Contraction ◽  
Force Transducer ◽  
Different Levels

The ceca play an important role in the physiology of the gastrointestinal tract in chickens. Nevertheless, there is a gap of knowledge regarding the functionality of the ceca in poultry, especially with respect to physiological cecal smooth muscle contraction. The aim of the current study is the ex vivo characterization of cecal smooth muscle contraction in laying hens. Muscle strips of circular cecal smooth muscle from eleven hens are prepared to investigate their contraction ex vivo. Contraction is detected using an isometric force transducer, determining its frequency, height and intensity. Spontaneous contraction of the chicken cecal smooth muscle and the influence of buffers (calcium-free buffer and potassium-enriched buffer) and drugs (carbachol, nitroprusside, isoprenaline and Verapamil) affecting smooth muscle contraction at different levels are characterized. A decrease in smooth muscle contraction is observed when a calcium-free buffer is used. Carbachol causes an increase in smooth muscle contraction, whereas atropine inhibits contraction. Nitroprusside, isoprenaline and Verapamil result in a depression of smooth muscle contraction. In conclusion, the present results confirm a similar contraction behavior of cecal smooth muscles in laying hens as shown previously in other species.

scholarly journals Subcellular-membrane characterization of [3H]ryanodine-binding sites in smooth muscle

1993 ◽  
Vol 290 (1) ◽  
pp. 259-266 ◽  
Author(s):  
Z D Zhang ◽  
C Y Kwan ◽  
E E Daniel
Keyword(s):  
Smooth Muscle ◽  
Ryanodine Receptor ◽  
Binding Sites ◽  
Vas Deferens ◽  
Smooth Muscles ◽  
Rat Vas Deferens ◽  
Detailed Characterization ◽  
Release Channel ◽  
Subcellular Membrane

The plant alkaloid ryanodine, known to interact selectively with the intracellular Ca(2+)-release channel in skeletal and cardiac muscles, has been repeatedly reported to affect smooth-muscle contractile functions that are consistent with its intracellular action at the Ca(2+)-release channel sites. Direct evidence for the binding of [3H]ryanodine to smooth-muscle membranes is sparse. Following our recent detailed characterization of functional effects of ryanodine and a preliminary report on the presence of [3H]ryanodine binding sites in rat vas deferens smooth muscle, we now report in this study a detailed characterization of binding of [3H]ryanodine to smooth muscle at the subcellular-membrane level. The ryanodine receptor in rat vas deferens muscle layer is primarily of smooth-muscle origin and is localized at the subcellular membrane site that is consistent with its role as a Ca(2+)-release channel in the sarcoplasmic reticulum (SR). Ryanodine binding to its receptor is Ca(2+)-dependent, with half-maximal binding occurring within the physiologically relevant cytosolic Ca2+ concentration. It is also sensitive to many factors, including change in Mg2+ concentration, ionic strength and osmolarity across the membrane vesicles. Agents known to inhibit (Ruthenium Red, Mg2+) or enhance (caffeine, Na+, K+) the Ca(2+)-induced Ca2+ release also inhibit or enhance the binding of ryanodine. Quantitative differences in ryanodine receptors exist among smooth muscles and do not seem to parallel their SR contents. Results from the present study indicate both the need and the basis for future investigations of the functional role of the ryanodine receptor in different smooth muscles.

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