Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles

To determine whether densities ofcalmodulin (CaM) and CaM-binding proteins are related to phasic andtonic behavior of smooth muscles, we quantified these proteins in theopossum esophageal body (EB) and lower esophageal sphincter (LES),which represent phasic and tonic smooth muscles, respectively. Gelelectrophoresis, immunoprecipitation, Western blot, and hemagglutininepitope-tagged CaM (HA-CaM) overlay assay with quantitative scanningdensitometry and phosphorylation measurements were used. Total proteincontent in the two smooth muscles was similar (~30 mg protein/gfrozen tissue). Total tissue concentration of CaM was significantly(25%) higher in EB than in LES (P < 0.05).HA-CaM-binding proteins were qualitatively similar in LES and EBextracts. Myosin, myristoylated alanine-rich C kinase substrateprotein, Ca²⁺/CaM kinase II, and calponin contents werealso similar in the two muscles. However, content and total activity ofmyosin light chain kinase (MLCK) and content of caldesmon (CaD) werethree- to fourfold higher in EB than in LES. Increased CaM and MLCKcontent may allow for a wide range of contractile force varying fromcomplete relaxation in the basal state to a large-amplitude,high-velocity contraction in EB phasic muscle. Increased content ofCaD, which provides a braking mechanism on contraction, may furthercontribute to the phasic contractile behavior. In contrast, low CaM,MLCK, and CaD content may be responsible for a small range ofcontractile force seen in tonic muscle of LES.

     

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and 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.     

Differences in contractile protein content and isoforms in phasic and tonic smooth muscles

The basis of tonic vs. phasic contractile phenotypes of visceralsmooth muscles is poorly understood. We used gel electrophoresis andquantitative scanning densitometry to measure the content and isoformcomposition of contractile proteins in opossum lower esophagealsphincter (LES), to represent tonic muscle, and circular muscle of theesophageal body (EB), to represent phasic smooth muscle. The amount ofprotein in these two types of muscles is similar: ~27 mg/g of frozentissue. There is no difference in the relative proportion of myosin,actin, calponin, and tropomyosin in the two muscle types. However, theEB contains ~2.4-times more caldesmon than the LES. The relativeratios of - to -contractile isoforms of actin are 0.9 in the LESand 0.3 in EB. The ratio between acidic (LC17a) and basic (LC17b)isoforms of the 17-kDa essential light chain of myosin is 0.7:1 in theLES, compared with 2.7:1 in the EB. There is no significant differencein the ratios of smooth muscle myosin SM1 and SM2 isoforms in the two muscle types. The level of the myosin heavy chain isoform, which contains the seven-amino acid insert in the myosin head, is about threefold higher in the EB compared with LES. In conclusion, the esophageal phasic muscle in contrast to the tonic LES contains proportionally more caldesmon, LC17a, and seven-amino acid-inserted myosin and proportionally less -actin. These differences may providea basis for functional differences between tonic and phasic smoothmuscles.

     

Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins

Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20, and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins.     

The mechanism of calcium control of smooth muscle tonic contraction

It has been shown in the experiments carried out on a fraction of inverted vesicles of myometrium sarcolemma that ATP-dependent Ca2+ transport system prevents dissipation of the calcium gradient directed from the intervesicular space outward with subsequent establishment of the stationary level of cation content inside the membrane vesicles (a blocker of electro-controlled calcium channels diltiasems was present in the incubation medium). Ortovanadatean inhibitor of the sarcolemma calcium pump suppressed Ca2+ stationary exchange in the vesicles fraction. The value of calcium stationary content in the vesicle membrane was regulated both by a change of the calcium pump activity (by varying Mg2+ concentration in the ATP-containing incubation medium), and by modification of calcium permeability of the vesicles (by varying concentration of ionophore A-23187 in this medium). In the presence of diltiasem and ortovanadate the Ca2+ basal current entering the myocytes from hyperpotassium washing solution activated the smooth muscle tonic contraction. In the absence of ortovanadate no contractile response was observed. On the basis of the evidence obtained a mechanism of calcium control of myometrium tonic contraction is proposed. According to this mechanism the Ca2+ current entering the unexcited myocytes under physiological conditions is efficiently compensated by the calcium pump of the sarcolemma. The inhibition of the latter (or an increase of the sarcolemma basal calcium permeability) provides further slow transition of the stationary value of Ca2+ concentration in the myoplasm to a new higher level and activation of the smooth muscle contraction accordingly.     

Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

Protein phosphorylation during spontaneous contraction of smooth muscle

The relationship between spontaneous contraction and protein phosphorylation of rat uterine smooth muscle was studied. Myometrial strips from estrogen-dominated rats were incubated in [³²P]orthophosphate and then frozen at various levels of isometric tension. Proteins were separated by gel electrophoresis and the incorporation of ³²P was measured. Contraction was associated with the phosphorylation of one major protein (20,000 M_r). This phosphorylation preceded maximal tension development and dephosphorylation preceded complete spontaneous relaxation. Two-dimensional gel electrophoresis indicates that the 20,000-M_r protein is the myosin light chain which has been implicated in the regulation of smooth muscle contraction.     

Spontaneously tonic smooth muscle has characteristically higher levels of RhoA/ROK compared with the phasic smooth muscle

The internal anal sphincter (IAS) tone is important for the rectoanal continence. The RhoA/Rho kinase (ROK) pathway has been associated with the agonist-induced sustained contraction of the smooth muscle, but its role in the spontaneously tonic smooth muscle is not known. Present studies compared expression of different components of the RhoA/ROK pathway between the IAS (a truly tonic SM), the rectal smooth muscle (RSM) (a mixture of phasic and tonic), and anococcygeus smooth muscle (ASM) (a purely phasic SM) of rat. RT-PCR and Western blot analyses were performed to determine RhoA, ROCK-II, CPI-17, MYPT1, and myosin light-chain 20 (MLC20). Phosphorylated CPI-17 at threonine-38 residue (p(Thr38)-CPI-17), MYPT1 at threonine-696 residue (p(Thr696)-MYPT1), and MLC20 at threonine-18/serine-19 residues (p(Thr18/Ser19)-MLC20) were also determined in the basal state and after pretreatment with the ROK inhibitor Y 27632. In addition, we compared the effect of Y 27632 on the basal isometric tension and ROK activity in the IAS vs. the RSM. Our data show the highest levels of RhoA, ROCK-II, CPI-17, MLC20, and of phospho-MYPT1, -CPI-17, and -MLC20 in the IAS followed by in the RSM and ASM. Conversely, MYPT1 levels were lowest in the IAS and highest in the ASM. In the IAS, Y 27632 caused a concentration-dependent decrease in the basal tone, levels of phospho-MYPT1, -CPI-17, and -MLC20, and ROK activity. We conclude that RhoA/ROK plays a critical role in the basal tone in the IAS by the inhibition of MLC phosphatase via the phosphorylation of MYPT1 and CPI-17.     

Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles

Laser flash photolysis of caged adenosine triphosphate (ATP), in the presence of Ca2+, was used to examine the time course of isometric force development from rigor states in glycerinated tonic (rabbit trachealis) and phasic (guinea-pig ileum and portal vein) smooth muscles. Photolytic liberation of ATP from caged ATP initiated force development, at 20 degrees C, with half-time (t1/2) of 5.4 s in trachealis and 1.2-2.2 s in the phasic muscles. Prior to photolysis, some muscles were phosphorylated with ATP plus okadaic acid (an inhibitor of myosin light-chain phosphatase) or thiophosphorylated with ATP gamma S to fully activate the regulatory system, before turning on the contractile apparatus. In these prephosphorylated muscles, force development, after caged ATP photolysis, was more rapid than in the unphosphorylated muscles, but the t1/2 values for trachealis (0.8-1.1 s) were still longer than for ileum and portal-vein muscles (0.20-0.25 s). The results suggest that both the contractile machinery and the regulatory system are slower in the tonic than in the phasic smooth muscles. The time course of force development for each muscle type was sigmoidal, with an initial delay (td) of approximately 10% of the t1/2 value. Some possible chemical and mechanical origins of the delay are discussed.     

The role of sarcolemma calcium pump in regulating tonic smooth muscle contraction

Vanadate (10(-4)-10(-3) M) effectively blocks Mg2+, ATP-dependent Ca2+ transport in sarcolemmal vesicles and induces a slowly tonic contraction of the smooth muscle. This contraction was observed both with and without nifedipine (10(-5) M) evoking complete inhibition of hyperpotassium contracture, the Ca2+ removal from the solution washing the muscular preparation stimulating the tone decrease. There is a close correlation between the dose-dependent effects of vanadate on the Ca pump activity and tension. It is concluded that in smooth muscles, at least in myometrium, the sarcolemmal Ca-pump is involved into the control of the tonic tension.     

Rapid myosin phosphorylation transients in phasic contractions in chicken gizzard smooth muscle

In intact smooth muscle strips from chicken gizzard, electrical stimulation and carbachol elicited brief, phasic contractions which were associated with a very rapid, transient phosphorylation of the 20 kDa myosin light chains. The phosphorylation transients reached their peak after 3 s and 6 s and preceded that of force. Phosphorylation was not significantly different from basal levels after 10 s and 30 s while force still amounted to 50% of the peak value. The rate of tension decline could be increased by cessation of stimulation or by addition of atropine, even at apparently basal phosphorylation levels suggesting a phosphorylation independent regulation.     

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.     

HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction

We have investigated the role of heat shock protein 27 (HSP27) phosphorylation and the association of HSP27 with contractile proteins actin, myosin, and tropomyosin. Smooth muscle cells were labeled with [(32)P]orthophosphate. C2-ceramide (0.1 microM), an activator of protein kinase C (PKC), induced a sustained increase in HSP27 phosphorylation that was inhibited by calphostin C. C2-ceramide-induced (0.1 microM) sustained colonic smooth muscle cell contraction was accompanied by significant increases in the association of HSP27 with tropomyosin and in the association of HSP27 with actin. The significant increases occurred at 30 s after stimulation and were sustained at 4 min. Contraction was also associated with strong colocalization of HSP27 with tropomyosin and with actin as observed after immunofluorescent labeling of tropomyosin, actin, and HSP27 followed by confocal microscopy. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that phosphorylation of HSP27 could affect myosin association with actin. In conclusion 1) HSP27 phosphorylation appears to be necessary for reorganization of HSP27 inside the cell and seems to be directly correlated with the PKC signal transduction pathway, and 2) agonist-induced phosphorylation of HSP27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin.     

Parathyroid hormone and parathyroid hormone-related protein inhibit phasic contraction of pig duodenal smooth muscle

Parathyroid hormone (PTH) and a newly discovered PTH-related protein (PTHrP), which has amino-terminal homology with PTH, are potent relaxants of rat gastrointestinal tissues. Since their gastrointestinal relaxant effects have been described only in the rat, we examined their actions in another mammalian species in order to evaluate whether the relaxant property was more generally applicable. Longitudinal smooth muscle strips were obtained from the pig duodenum. The mucosa was removed, the strips were mounted in a tissue chamber, and changes in phasic contraction were detected with a force-displacement transducer and recorded using a polygraph. Acetylcholine-induced phasic contraction was inhibited rapidly in a dose-related manner by [Nle8,18,Tyr34]-bPTH-(1-34)-amide, or hPTHrP-(1-34). The IC50 values for these peptides were 2.6 nM and 6.1 nM, respectively. The maximal effect of both peptides was observed at 60 nM with an 84% decrease of the acetylcholine-induced contraction. At 400 nM, the PTH antagonist, [Nle8,18,Tyr34]-bPTH-(3-34)-amide, had no effect by itself. However, the same 400 nM concentration of this peptide totally blocked the decrease in phasic contraction induced by 10 nM of the bPTH-(1-34) analogue or hPTHrP-(1-34). Our results show that receptors for PTH or PTHrP are present in the muscular layer of the pig duodenum and that activation of these receptors inhibits the phasic contraction of the tissue. Furthermore, the ability of PTH-related peptides to relax gastrointestinal smooth muscle is not restricted to the rat.