Coupling In Smooth Muscle Research Articles

Excitation-transcription coupling in smooth muscle is associated with vascular remodeling

Excitation-transcription coupling in smooth muscle is associated with vascular remodeling

Model discrimination for Ca2+ -dependent regulation of myosin light chain kinase in smooth muscle contraction.

Excitation-contraction coupling in smooth muscle is mediated by the Ca2+ - and calmodulin-dependent regulation of myosin light chain kinase. The precise mechanism of this regulation remains controversial, and several mathematical models have been proposed for the interaction of the three species. These models have previously been analyzed at steady state primarily by numerical simulation of differential equations, for which parameter values must be estimated from data. Here, we use the linear framework for timescale separation to demonstrate that models of this general kind can be solved analytically for an equilibrium steady state, without having to determine parameter values. This analysis leads to parameter-independent methods for discriminating between the models, for which we propose experiments that could be performed with existing methods.

Dynamic Assessment of Ca2+ Sensitivity of Isometric Force in Intact Airway Smooth Muscle Using Phase Loop Plots

A transient elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) triggers a force response in excitation‐contraction coupling. The Ca2+ sensitivity of muscle reflects the relationship between [Ca2+]cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca2+]cyt at which 50% force is generated (ED50). In smooth muscle, Ca2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca2+]cyt. However, this conventional measurement overlooks the dynamic nature of Ca2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca2+]cyt and force and determined their temporal dependence using a phase loop plot. We examined the phase loop plot assessment of Ca2+ sensitivity in ASM under the condition in which the conditional assessment indicated an increase (Calyculin A treatment) in Ca2+ sensitivity. Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips were loaded with Fura‐2 AM (Ca2+ fluorescent indicator) and stimulated using 1 mM ACh. Isometric force and [Ca2+]cyt responses were simultaneously measured using a Guth Muscle Research System. ASM strips were exposed to Calyculin A (phosphatase inhibitor; 100 nM for 10 min), which was previously reported to alter static Ca2+ sensitivity. Phase loop plots were calculated from simultaneous [Ca2+]cyt and force responses. The extent of rMLC phosphorylation was determined at different time periods after ACh stimulation using Phos‐tag SDS‐PAGE (SuperSep Phos‐tag; Wako Pure Chemical Industries, Ltd.) and Western blotting. Calyculin A increased ACh‐induced isometric force in ASM strips, but it had no effect on the amplitude of evoked [Ca2+]cyt transients. These results suggested that Calyculin A increased static Ca2+ sensitivity. However, the phase loop plots showed no significant shift in dynamic Ca2+ sensitivity with Calyculin A. These results indicate that the phase loop plot provides a valuable tool to assess dynamic Ca2+ sensitivity of isometric force generation in ASM, which better reflects the complex process of excitation‐contraction coupling in smooth muscle. These results further indicate that dynamic Ca2+ sensitivity of force generation in ASM is independent of rMLC phosphorylation.Support or Funding InformationSupported by NIH grant HL126451 (GCS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role and regulation of Abelson tyrosine kinase in Crk-associated substrate/profilin-1 interaction and airway smooth muscle contraction

BackgroundAirway smooth muscle contraction is critical for maintenance of appropriate airway tone, and has been implicated in asthma pathogenesis. Smooth muscle contraction requires an “engine” (myosin activation) and a “transmission system” (actin cytoskeletal remodeling). However, the mechanisms that control actin remodeling in smooth muscle are not fully elucidated. The adapter protein Crk-associated substrate (CAS) regulates actin dynamics and the contraction in smooth muscle. In addition, profilin-1 (Pfn-1) and Abelson tyrosine kinase (c-Abl) are also involved in smooth muscle contraction. The interplays among CAS, Pfn-1 and c-Abl in smooth muscle have not been previously investigated.MethodsThe association of CAS with Pfn-1 in mouse tracheal rings was evaluated by co-immunoprecipitation. Tracheal rings from c-Abl conditional knockout mice were used to assess the roles of c-Abl in the protein-protein interaction and smooth muscle contraction. Decoy peptides were utilized to evaluate the importance of CAS/Pfn-1 coupling in smooth muscle contraction.ResultsStimulation with acetylcholine (ACh) increased the interaction of CAS with Pfn-1 in smooth muscle, which was regulated by CAS tyrosine phosphorylation and c-Abl. The CAS/Pfn-1 coupling was also modified by the phosphorylation of cortactin (a protein implicated in Pfn-1 activation). In addition, ACh activation promoted the spatial redistribution of CAS and Pfn-1 in smooth muscle cells, which was reduced by c-Abl knockdown. Inhibition of CAS/Pfn-1 interaction by a decoy peptide attenuated the ACh-induced actin polymerization and contraction without affecting myosin light chain phosphorylation. Furthermore, treatment with the Src inhibitor PP2 and the actin polymerization inhibitor latrunculin A attenuated the ACh-induced c-Abl tyrosine phosphorylation (an indication of c-Abl activation).ConclusionsOur results suggest a novel activation loop in airway smooth muscle: c-Abl promotes the CAS/Pfn-1 coupling and actin polymerization, which conversely facilitates c-Abl activation. The positive feedback may render c-Abl in active state after contractile stimulation.

Caffeine relaxes smooth muscle through actin depolymerization

Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.

Reciprocal regulation controlling the expression of CPI-17, a specific inhibitor protein for the myosin light chain phosphatase in vascular smooth muscle cells

Cellular activity of the myosin light chain phosphatase (MLCP) determines agonist-induced force development of smooth muscle (SM). CPI-17 is an endogenous inhibitor protein for MLCP, responsible for mediating G-protein signaling into SM contraction. Fluctuations in CPI-17 expression occur in response to pathological stresses, altering excitation-contraction coupling in SM. Here, we determined the signaling pathways regulating CPI-17 expression in rat aorta tissues and the cell culture using a pharmacological approach. CPI-17 transcription was suppressed in response to the proliferative stimulus with platelet-derived growth factor (PDGF) through the ERK1/2 pathway, whereas it was elevated in response to inflammatory, stress-induced and excitatory stimuli with transforming growth factor-β, IL-1β, TNFα, sorbitol, and serotonin. CPI-17 transcription was repressed by inhibition of JNK, p38, PKC, and Rho-kinase (ROCK). The mouse and human CPI-17 gene promoters were governed by the proximal GC-boxes at the 5'-flanking region, where Sp1/Sp3 transcription factors bound. Sp1 binding to the region was more prominent in intact aorta tissues, compared with the SM cell culture, where the CPI-17 gene is repressed. The 173-bp proximal promoter activity was negatively and positively regulated through PDGF-induced ERK1/2 and sorbitol-induced p38/JNK pathways, respectively. By contrast, PKC and ROCK inhibitors failed to repress the 173-bp promoter activity, suggesting distal enhancer elements. CPI-17 transcription was insensitive to knockdown of myocardin/Kruppel-like factor 4 small interfering RNA or histone deacetylase inhibition. The reciprocal regulation of Sp1/Sp3-driven CPI-17 expression through multiple kinases may be responsible for the adaptation of MLCP signal and SM tone to environmental changes.

Endothelin‐1 increases the frequency of smooth muscle calcium waves in vena cava but not aorta

Vascular contraction is associated with a global increase in smooth muscle Ca2+. The frequency and amplitude of localized intracellular Ca2+ release events, such as IP3 receptor‐mediated Ca2+ waves, can also regulate excitation‐contraction coupling in smooth muscle. While the vasoconstrictor ET‐1 does increase IP3 formation, it is unclear if IP3 receptor‐mediated Ca2+ waves are a regulator of ET‐1‐induced contraction in arteries and veins. We hypothesized that ET‐1 increases the frequency and amplitude of Ca2+ waves in rat aorta (RA) and vena cava (RVC). RA and RVC were mounted en face in an imaging superfusion chamber and incubated with the intensiometric fluorescent Ca2+ indicator Fluo 4‐AM (10 μM). Using spinning‐disc confocal microscopy, image sequences were first recorded with buffer alone, and then maximal contractile concentrations of norepinephrine (NE, 10 μM) (RVC) or phenylephrine (PE, 10 μM) (RA), and finally ET‐1 (100 nM). ET‐1 (100 nM) increased Ca2+ wave frequency significantly in RVC versus control (0.72±0.16 Hz vs. 0.45±0.13 Hz), but not in RA. Neither NE (10 μM) nor PE (10 μM) altered Ca2+ wave frequency or amplitude in RA and RVC. These data indicate that intracellular Ca2+ mobilization by ET‐1 is different in arteries and veins. These data also provide indirect evidence that ET receptors are coupled to IP3‐mediated Ca2+ release mechanisms in the vena cava but not aorta. Supported by NIH P01HL70687.

Toward a Concept of Stretch Coupling in Smooth Muscle: A Thesis by Lars Thuneberg on Contractile Activity in Neonatal Interstitial Cells of Cajal

The hypothesis was put forward by Thuneberg that rhythmically contracting interstitial cells of Cajal (ICC) were sensing stretch of the musculature and that this information was transmitted to smooth muscle cells via peg and socket contacts. The present study provides the evidence for the contractile nature of ICC as perceived by Thuneberg. The contractile activity is shown by video frame subtraction and by tracking areas of interest in sequential video frames. Thuneberg used neonatal ICC in culture maintained between two coverslips thereby allowing growth factors to quickly reach optimal concentrations. Contractions of ICC were seen to precede smooth muscle contractions. In addition, strong contractions were observed solely in branches of ICC. It is hoped that this communication will stimulate discussion about the contractile nature of ICC and that this phenomenon will eventually find its place amongst the physiological properties of the ICC networks of the gut musculature.

Effects of spermine on the relaxation response of rat detrusor smooth muscles

Endogenous polyamines are known to influence excitation-contraction coupling in smooth muscle. This study was designed to determine the effects of the polyamines spermine, spermidine, and putrescine on the contractile responses of rat detrusor smooth muscles. Under physiological conditions, isometric tension recordings were made of isolated bladder strips from excised rat bladder. The effects of spermine, spermidine, and putrescine (1 mM each) on the bladder contractions induced by various agents, i.e., acetylcholine, bethanechol, high-K, and tetraethylammonium (TEA) were measured. A conventional patch clamp technique was used in whole cell mode with single smooth muscle cells of rat bladder. Calcium channel currents were recorded to determine the effects of spermine on channel activities. Polyamines elicited a concentration-dependent relaxations on the contractile agents induced contractures. Spermine showed the most potent relaxation effect of the polyamines examined, and relaxed the contractions induced by the agents. Calcium channel activities were significantly reduced by adding 1 mM spermine to the bath. We concluded that spermine exerts a potent relaxant effect on rat bladder smooth muscle, and this effect appears to be mediated by calcium channel antagonism.

DIFFERENTIAL ROLES OF RYANODINE‐ AND THAPSIGARGIN‐SENSITIVE INTRACELLULAR Ca2+ STORES IN EXCITATION–CONTRACTION COUPLING IN SMOOTH MUSCLE OF GUINEA‐PIG TAENIA CAECI

1. To explore roles of intracellular Ca(2+) stores in excitation-contraction coupling in smooth muscle, we examined the effects of ryanodine, a fixer of ryanodine receptor-Ca(2+) channels to an open state, and thapsigargin, a selective inhibitor of the Ca(2+) pump in the intracellular stores, on smooth muscle contraction in the presence and absence of extracellular Ca(2+) in guinea-pig taenia caeci. 2. In Ca(2+) -free solution, contractions induced by 0.1 mmol/L carbachol and 0.1 mmol/L histamine were reduced to approximately 65% of control by either 1 micro mol/L thapsigargin or 10 micro mol/L ryanodine. In contrast, caffeine-induced contraction was reduced to approximately 40% of control by ryanodine, but was not affected by thapsigargin. 3. In the presence of extracellular Ca(2+), thapsigargin slowly induced a large and sustained contraction. In contrast, ryanodine did not induce an apparent contraction, but increased the sensitivity of contractile responses to receptor agonists (carbachol, AHR-602 and histamine) or depolarizing high K(+) with no changes in the maximal contraction. 4. These results suggest that there are pharmacological and physiological differences between ryanodine- and thapsigargin-sensitive intracellular Ca(2+) stores in excitation-contraction coupling in smooth muscle, which may be responsible for their differential effects on the Ca(2+) -influx pathway.

Hypoxia-induced vascular smooth muscle relaxation: increased ATP-sensitive K+ efflux or decreased voltage-sensitive Ca2+ influx?

controversy continues regarding the cellular mechanisms of hypoxic vasodilation. The vascular endothelium may contribute to hypoxic dilations by releasing relaxing factors that act on the smooth muscle ([4][1], [6][2]–[8][3]). In contrast, numerous studies have suggested a role of the vascular

Excitation–transcription coupling in smooth muscle

Calcium (Ca2+) signals affect virtually every biological process, including both contraction and gene transcription in smooth muscle. Ca2+-regulated gene transcription is known to be important for both physiological and pathological responses in smooth muscle. The aim of this review is to discuss the current understanding of gene transcription regulated by excitation through Ca2+ signalling using a comparison of the two most characterized Ca2+-regulated transcription factors in smooth muscle, Ca2+-cyclic AMP response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT). Recent studies have shown commonalities and differences in the regulation of CREB and NFAT through both voltage- and non-voltage-gated Ca2+ channels that lead to expression of smooth muscle cell specific differentiation markers as well as markers of proliferation. New insights into the regulation of specific genes through companion elements on the promoters of Ca2+-regulated genes have led to new models for transcriptional regulation by Ca2+ that are defined both by the source and duration of the Ca2+ signal and the composition of enhancer elements found within the regulatory regions of specific genes. Thus the combination of signalling pathways elicited by particular Ca2+ signals affect selective promoter elements that are key to the ultimate pattern of gene transcription.

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

The sarco/endoplasmic reticulum (SR/ER) is the primary storage and release site of intracellular calcium (Ca2+) in many excitable cells. The SR is a tubular network, which in smooth muscle (SM) cells distributes close to cellular periphery (superficial SR) and in deeper aspects of the cell (deep SR). Recent attention has focused on the regulation of cell function by the superficial SR, which can act as a buffer and also as a regulator of membrane channels and transporters. Ca2+ is released from the SR via two types of ionic channels [ryanodine- and inositol 1,4,5-trisphosphate-gated], whereas accumulation from thecytoplasm occurs exclusively by an energy-dependent sarco-endoplasmic reticulum Ca2+-ATPase pump (SERCA). Within the SR, Ca2+ is bound to various storage proteins. Emerging evidence also suggests that the perinuclear portion of the SR may play an important role in nuclear transcription. In this review, we detail the pharmacology of agents that alter the functions of Ca2+ release channels and of SERCA. We describe their use and selectivity and indicate the concentrations used in investigating various SM preparations. Important aspects of cell regulation and excitation-contractile activity coupling in SM have been uncovered through the use of such activators and inhibitors of processes that determine SR function. Likewise, they were instrumental in the recent finding of an interaction of the SR with other cellular organelles such as mitochondria. Thus, an appreciation of the pharmacology and selectivity of agents that interfere with SR function in SM has greatly assisted in unveiling the multifaceted nature of the SR.

Identification and Characterization of Zipper-interacting Protein Kinase as the Unique Vascular Smooth Muscle Myosin Phosphatase-associated Kinase

Excitation-contraction coupling in smooth muscle involves activation of myosin light chain (MLC) phosphorylation, which increases activity of the myosin actin-activated ATPase, resulting in contraction. Phosphorylation of MLC phosphatase (SMPP-1M) by Rho-associated kinase or endogenous SMPP-1M-associated kinase inhibits SMPP-1M, enhancing MLC phosphorylation and contraction. However, the precise identity of SMPP-1M-associated kinase remains unclear. Biochemical evidence strongly supports the idea that SMPP-1M-associated kinase is related to the human serine/threonine leucine zipper-interacting protein kinase (hZIPK), which is important in cell apoptosis, and the SMPP-1M-associated kinase has therefore been called ZIP-like kinase (MacDonald, J. A., Borman, M. A., Murani, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419-2424). Whether the vascular smooth muscle SMPP-1M-associated kinase is a truncated version of hZIPK, native hZIPK, or a unique homologue of hZIPK is unclear. Here we show that only native hZIPK mRNA and protein are detectable in human vascular smooth muscle cells (VSMCs). High stringency screening of a human aortic cDNA library for the SMPP-1M-associated kinase identified 18 positive clones, all of which proved to be clones of hZIPK. PCR-based studies of VSMC RNA revealed native hZIPK transcripts but no evidence for splice variants of hZIPK or a ZIP-like kinase. Northern blotting studies of multiple vascular and non-vascular tissue RNAs, including human bladder RNA, showed only 2.3 kb of mRNA predicted for full-length hZIPK. Immunoblotting showed native full-length 52-kDa hZIPK expression in VSMCs. Full-length and N-terminal hZIPK bound the C-terminal domain (amino acids 681-847) of the myosin binding subunit (MBS) of SMPP-1M. hZIPK immunoprecipitated with the MBS of SMPP-1M and dominant negative RhoA inhibited the hZIPK-MBS interaction. These data identify hZIPK as the unique SMPP-1-associated kinase expressed in human vesicular smooth muscle and support a role for Rho in promoting the hZIPK-MBS interaction.

RYR2 proteins contribute to the formation of Ca(2+) sparks in smooth muscle.

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

A new view of K+ -induced contraction in rat aorta: the role of Ca2+ binding.

Strong, K+ -induced contractions of rat aorta in Ca-free, Mg-free media were not accompanied by increased intracellular calcium concentration, [Ca2+](i), whereas such contractions in the presence of the divalent cations were correlated with rising [Ca2+](i) as assessed by fura-2. At the same time, calcium channel blockers, a modulator of Ca2+-binding proteins, and a modulator of actin polymerization, inhibited all types of K+ -induced contractions. Increasing the K+ in isotonic medium evoked a rise of (45)Ca2+ binding to the plasma membrane of freshly isolated aortic cells. Although Ca2+ -dependent events underlie the mechanism of K+ -induced vascular contractions in both the presence and absence of Ca2+, in contrast to the view that [Ca2+](i) is a key regulator of excitation-contraction coupling in smooth muscle, we suggest that the modulation of Mg2+ -dependent Ca2+ binding, probably within/at the L-type calcium channel by K+, is a trigger for aortic contraction. This Ca2+ binding may then activate actin-myosin interaction.

Kinetics and Energetics of Mg2+,ATP-Dependent Ca2+Transport in the Plasma Membrane of Smooth Muscle Cells

Calcium ions play a crucial role in the excitation/contraction coupling in smooth muscles. I would like to interpret the biochemical mechanisms underlying Ca2+ exchange and dynamics of such an exchange in the smooth muscles. Particular emphasis is laid on the examination of kinetic, energetic, and catalytic properties of the membrane-linked energy-dependent Ca2+-transporting systems involved in regulation of the intracellular Ca2+ concentration in smooth muscle cells (SMC). It was suggested that the Mg2+,ATP-dependent plasma membrane calcium pump (Ca2+,Mg2+-ATPase) plays a key role in regulation of the Ca2+ concentration in SMC. The purpose of this review is to analyze some of our own results concerning kinetic, energetic, and catalytic properties of the calcium pump of the SMC plasma membrane. In our experiments, we used different biochemical models (namely, fractions of the membrane subcellular structures, highly purified Ca2+,Mg2+-ATPase of the SMC plasma membrane solubilized and reconstituted in the lyposomes, and suspension of digitonin-treated SMC) and a number of methods (including preparative biochemistry, enzymology, membranology, tracer 45Ca2+ flux analysis, and chemical and enzymological kinetics). We have shown that sodium azide-insensitive Mg2+,ATP-dependent Ca2+ accumulation in ureter smooth muscle microsomes is determined by two components. One component represents the Mg2+,ATP-dependent calcium pump of the sarcoplasmic reticulum functionally potentiated by Ca2+-precipitating permeating anions, oxalate or phosphate and inhibited by thapsigargin or cyclopiazonic acid, the highly selective inhibitors of the calcium pump of sarco(endo)plasmic rerticulum. Another component represents the Mg2+,ATP-dependent calcium pump of the plasma membrane functionally potentiated by phosphate. This pump is not inhibited by thapsigargin and cyclopiazonic acid. The effects of temperature, dielectric permeability (D), and ionic strength on the activity of purified Ca2+,Mg2+-ATPase solubilized from the myometrial sarcolemma were studied. The results suggest that changes in the polarity of the incubation medium markedly affect the activity of transport Ca2+,Mg2+-ATPase, and electrostatic interactions between the enzyme activity center and specific ligands (Mg·ADP-, in particular) significantly contribute to the energetics of ATP hydrolysis. Therefore, our data show that changes in the incubation medium polarity significantly affects the ATP-hydrolase activity of Ca2+,Mg2+-ATPase solubilized from the SMC plasma membranes, and electrostatic interactions between the enzyme active sites and reactants (in particular, Mg·ADP-) contribute to a significant extent to the energetics of ATP hydrolysis. We cannot rule out that under physiological conditions the local D values of the myoplasm may differ from that of water, and, moreover, may change (especially near the membrane surface) depending on the metabolic level of SMC. We suppose that local changes in the cytoplasmic D value will affect the plasma membrane calcium pump and, consequently, the efficiency of control of intracellular Ca2+ homeostasis in smooth muscle. So, our biochemical models are suitable experimental objects for studying the kinetic, energetic, and catalytic properties of the Mg2+,ATP-dependent calcium pump of the SMC plasma membrane. In addition, our data might be useful for screening of the mechanisms underlying the action of different physico-chemical factors involved in modulation of the contraction/relaxation cycle.

Evidence of Gap Junctions in the Stable Nonobstructed Human Bladder

Evidence of Gap Junctions in the Stable Nonobstructed Human Bladder

Distinct contractile systems for electromechanical and pharmacomechanical coupling in smooth muscle.

Electromechanical coupling by KCl depolarization of bladder preparations elicits an initial phasic and subsequent tonic contraction. Using a smooth-muscle myosin heavy chain (SM-MyHC) knock-out mouse model we could previously demonstrate, that phasic and tonic contraction of intact neonatal bladder preparations could be elicited through the recruitment of SM-MyHC and non-muscle myosin heavy chains (NM-MyHC), respectively. Inhibition of myosin light chain kinase (MLCK) by ML-7 eliminated the phasic contraction of wild-type (+/+), rather than tonic contraction of neonatal bladder strips prepared from both +/+ and homozygous SM-MyHC knock-out (-/-) mice. Pharmacomechanical coupling upon PDBu-induced activation of protein kinase C of neonatal bladder preparations elicited tonic contraction of both +/+ and -/- murine. We suggest that: i) electromechanical coupling activates both SM-MyHC and NM-MyHC systems via a ML-7 sensitive and insensitive pathway, respectively. ii) Pharmacomechanical coupling recruits part of the NM-MyHC system rather than SM-MyHC.

Calcium release events in excitation-contraction coupling in smooth muscle.

Although smooth muscle cells are not organized in sarcomeres, as are striated muscles, nevertheless Ca2+ for contraction is released from the sarcoplasmic reticulum (SR) at certain preferred sites. These sites commonly discharge packets of Ca2+ spontaneously and have been called frequent discharge sites (FDSs). Each spontaneous release of a Ca2+ packet usually leads to a burst of openings of Ca2+-activated K+ channels in the cell membrane which produces a spontaneous transient outward current (STOC) in smooth muscle cells under voltage clamp. When fluorescent Ca2+ indicators such as Fluo-3 became available, the spontaneous transient increases in [Ca2+]i produced by Ca2+ packets released from the SR were also detected in cardiac muscle as flashes of fluorescence or 'sparks'. Sparks in smooth muscle consist of smaller Ca2+ packets that can give rise to 'microsparks'. In some smooth muscles which have Ca2+-activated Cl- channels, STICs (spontaneous transient inward currents) are also found to be associated with sparks. FDSs have been found to be important initiating sites for a Ca2+ wave in response to an action potential or in response to receptor activation and possibly other stimuli, such as stretch. In both cases Ca2+-induced Ca2+ release seems to be crucially involved.