Smooth muscle is responsible for the contractility of hollow organs, such as blood vessels, the gastrointestinal tract, the bladder, or the uterus. Its structure differs greatly from that of skeletal muscle, although it can develop isometric force per cross-sectional area that is equal to that of skeletal muscle. However, the speed of smooth muscle contraction is only a small fraction of that of skeletal muscle.

Structure: The most striking feature of smooth muscle is the lack of visible cross striations (hence the name smooth). Smooth muscle fibers are much smaller (2-10 m in diameter) than skeletal muscle fibers (10-100 m ). It is customary to classify smooth muscle as single-unit and multi-unit smooth muscle (Fig. SM1). The fibers are assembled in different ways. The muscle fibers making up the single-unit muscle are gathered into dense sheets or bands. Though the fibers run roughly parallel, they are densely and irregularly packed together, most often so that the narrower portion of one fiber lies against the wider portion of its neighbor. These fibers have connections, the plasma membranes of two neighboring fibers form gap junctions that act as low resistance pathway for the rapid spread of electrical signals throughout the tissue. The multi-unit smooth muscle fibers have no interconnecting bridges. They are mingled with connective tissue fibers.

Fig. SM1. Single-unit and multi-unit smooth muscle.

Electron micrographs of smooth muscle reveal that the actin filaments are organized through attachment to the dense bodies that contain a-actinin, a Z-band protein in skeletal muscle. Thus, it is assumed that the dense bodies function as Z-lines. The ratio of thin to thick filaments is much higher in smooth muscle (~15:1) than in skeletal muscle (~6:1). Smooth muscle is rich in intermediate filaments that contain two specific proteins, desmin and vimentin.

Innervation and stimulation: Smooth muscle is primarily under the control of autonomic nervous system, whereas skeletal muscle is under the control of the somatic nervous system. The single-unit smooth muscle has pacemaker regions where contractions are spontaneously and rhythmically generated. The fibers contract in unison, that is the single unit of smooth muscle is syncytial. The fibers of multi-unit smooth muscle are innervated by sympathetic and parasympathetic nerve fibers and respond independently from each other upon nerve stimulation.

Nerve stimulation in smooth muscle causes membrane depolarization, like in skeletal muscle. Excitation, the electrochemical event occurring at the membrane is followed by the mechanical event, contraction. In the case of smooth muscle, this excitation-contraction coupling is termed electromechanical coupling; the link for the coupling is Ca2+ that permeates from the extracellular space into the intracellular water of smooth muscle. There is another excitation mechanism in smooth muscle, which is independent of the membrane potential change; it is based on receptor activation by drugs or hormones followed by muscle contraction. This is termed pharmacomechanical coupling. The link is Ca2+ that is released from an internal source, the sarcoplasmic reticulum.

The role of mechanical events of smooth muscle in the wall of hollow organs is twofold: 1) Its tonic contraction maintains organ dimensions against imposed load. 2) Force development and muscle shortening, like in skeletal muscle.

Myofibril proteins: In general, smooth muscle contains much less protein (~110 mg/g muscle) than skeletal muscle (~200 mg/g). Notable is the decreased myosin content, ~20 mg/g in smooth muscle versus ~80 mg/g in skeletal muscle. On the other hand, the amounts of actin and tropomyosin are the same in both types of muscle. Smooth muscle does not contain troponin, instead of it there are two other thin filament proteins, caldesmon and calponin.

The amino acid sequence of smooth muscle actin is very similar to that of its skeletal muscle counterpart, and it seems likely that their three-dimensional structures are also similar. Smooth muscle actin combines with either smooth or skeletal muscle myosin. However, there is a major difference in the activation of myosin ATPase by actin, smooth muscle myosin has to be phosphorylated for actin-activation to occur.

The size and shape of the smooth muscle myosin molecule is similar to that of the skeletal muscle myosin (Fig. M1). There is a small difference in the light chain composition; out of the four light chains of the smooth muscle myosin two have molecular weight of 20,000 and two of 17,000. The 20,000 light chain is phosphorylatable. Upon phosphorylation of the light chain the actin-activated smooth muscle myosin ATPase increases about 50-fold, to about 0.16 mol ATP hydrolyzed per mol of myosin head per sec, at physiological ionic strength and temperature. (Under the same conditions, the actin-activated skeletal muscle myosin ATPase is 10 -20 mol/mol/sec). The ionic strength dependence of smooth muscle myosin Ca2+-activated ATPase also differs from that of skeletal muscle myosin (Fig. M5), increasing ionic strength increases the smooth muscle myosin ATPase but decreases the skeletal muscle myosin ATPase.

Four smooth muscle specifc myosin heavy chain isoforms are known ( described in Quevillon-Cheruel et al., 1999). Two isoforms (named SMB and SMA) are defined by the presence or the absence of an insert of seven amino acids in the N-terminal globular head region. The two others (SM1 and SM2) differ at their C-termini by 43 versus 9 amino acids. To understand the role of the C-terminal extremities of SM1 and SM2 in smooth muscle thick filament assembly, various fragments of these myosins, such as the rod region, the rod with no tailpiece, or light meromyosins were prepared as recombinant proteins in bacterial cells (Rowner et al., 2001; Quevillon-Cheruel et al.,1999). The results showed that the smooth muscle myosin tailpieces differentially affect filament assembly and suggested that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.

Although the mechanism of thick filament assembly for purified smooth muscle myosins in vitro has been described, the regulation of thick filament formation in intact muscle is poorly understood. Cross-sectional density of the thick filaments measured electron microscopically in intact airway smooth muscle (Herrera et al., 2002) showed that the density increased substantially (144%) when the muscle was activated. In resting muscle, in the absence of Ca2+, the filament density decreased by 35%. It appears that in smooth muscle filamentous myosin exists in equilibrium with monomeric myosin; activation favors filament formation.

Kathleen Trybus pioneered in expressing and purifying smooth muscle myosin subfragments using the baculovirux /insect cell expression system. This procedure and the methods needed to characterize the new proteins (gel assays, ATPase activity determinations, transient state kinetic parameters, and the vitro motility assay) are described in her review (Trybus, 2000). Studies on engineered smooth muscle myosin and heavy meromyosin showed: the interaction between the regulatory light chain domains on two heads is critical for regulation of smooth muscle myosin (Li et al., 2000; Sweeney et al., 2000), a long, weakly charged actin-binding loop is required for phosphorylation-dependent regulation of smooth muscle myosin (Rovner, 1998), and coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance (Lauzon et al., 2001).

In vitro, both caldesmon and calponin are inhibiting the actin-activated ATPase activity of phosphorylated smooth muscle myosin. In case of calponin, this inhibitory activity is reversed by the binding of Ca2+-calmodulin or by phosphorylation. Calponin is a 34-kDa protein containing binding sites for actin, tropomyosin and Ca2+-calmodulin. Caldesmon is a long, flexible, 87-kDa protein containing binding sites for myosin, as well as actin, tropomyosin, and Ca2+-calmodulin. Electron microscopy and three-dimensional image reconstruction of isolated smooth muscle thin filaments revealed that calponin and caldesmon are located peripherally along the long-pitch actin helix (Hodgkinson et al., 1997; Lehman et al., 1997). The physiological role of caldesmon or calponin is not known.

Phosphorylation and Dephosphorylation of the 20-kDa Myosin Light Chain

Myosin light chain kinase and myosin light chain phosphatase: Smooth muscle (as well as skeletal and cardiac muscle) contains myosin light chain kinase (MLCK), activated by Ca2+-calmodulin, the enzyme which transfers the terminal phosphate group of ATP to serine (and/or threonine) hydroxyl groups of phosphorylatable light chain (LC) according to the following reaction:

LC-OH + MgATP2- ® LC-O-PO32- + MgADP- + H+ (1)

Dephosphorylation is brought about by smooth muscle myosin light chain phosphatase (MLCP) according to the following reaction:

LC-O-PO32- + H2O ® LC-OH + HPO42- (2)

The properties of MLCK are reviewed by Stull et al. (1996) and the properties of MLCP are reviewed by Hartshorne et al. (1998).

It is generally believed that LC phosphorylation-dephosphorylation controls the contraction-relaxation cycle of smooth muscle:

For a long time, research focused on the role of MLCK in smooth muscle contractility, but recently the interest shifted to MLCP. It turned out that MLCP is composed of three subunits: a catalytic subunit of 37-38-kDa of the type 1 phosphatase, a subunit of about 20-kDa whose function is not known, and a larger 110-130-kDa subunit that targets MLCP to myosin. The phosphatase activity of the catalytic subunit is low and it is enhanced significantly by addition of the targeting subunit. Upon phosphorylation of serine and threonine residues in the targeting subunit, its activating effect on the catalytic subunit is lost, and thereby the MLCP holoenzyme is inhibited.

Recent reports (Feng et al., 1999; Kaibuchi et al., 1999; Nagumo et al., 2000; Somlyo and Somlyo, 2000; Sward et al., 2000) indicate that in smooth muscle a Rho-regulated system of MLCP exists. Rho-kinase is the major player in this system, the enzyme phosphorylates the 130-kDa myosin binding subunit of MLCP and thereby inhibits MLCP activity. Due to the antagonism between MLCK and MLCP, inhibition of MLCP results in an increase in the phosphoryl content of LC with concomitant increase in muscle force. Under these conditions, submaximal Ca2+-levels are sufficient for maximal force, a phenomenon called increased Ca2+-sensitivity (Somlyo and Somlyo, 1994). Specific inhibitors for rho-kinase Y-27632 (Feng et al., 1999; Kaibuchi et al., 1999), and HA-1077 (Nagumo et al., 2000; Sward el al., 2000) are available.

MLCP activity can also be inhibited by a 17-kDa myosin phosphatase inhibitor protein, called CPI-17, (Kitazawa et al., 2000) , which inhibits the catalytic subunit of MLCP and the holoenzyme MLCP. Phosphorylation of CPI-17 at Thr38 increases its inhibitory potency 1000-fold. The solution NMR structure of CPI-17 has been determined.(Ohki et al., 2001), it forms a novel four-helix. Phosphorylation of Thr38 induces a conformational change involving displacement of one helix without significant movement of the other three helices. Rho-kinases and PKC are responsible for the phosphorylation of CPI-17.

A rich array of second messengers regulate MLCP activity under physiological and pathological conditions (Solaro, 2000) through phosphorylation of either the targeting subunit of MLCP or CPI-17.

Myosin light chain phosphorylation in intact smooth muscle: 32P-labeling of the muscle is a reliable method for such studies. When a dissected smooth muscle, e.g. artery or a uterine strip, is incubated at 37oC in physiological salt solution containing radioactive inorganic phosphate, the 32P permeates the plasma membrane and enters the intracellular space of the muscle. Through the oxidative phosphorylation mechanism the 32P incorporates into the terminal P group of ATP:

ADP + 32P ® ADP32P

Transfer of the terminal 32P of ATP to LC-OH by MLCK (equation 1) yields the radioactive LC-O-32PO32- species that can be isolated and quantified. The isolation involves two-dimensional (2D) gel electrophoresis and the quantification requires measuring the specific radioactivity of the terminal P of ATP.

Smooth muscle contraction is correlated with LC phosphorylation (reviewed by Bárány and Bárány, 1996c). Fig. SM2 illustrates an experiment: Two carotid arteries were dissected from freshly killed pigs and labeled with 32P. One artery was contracted with KCl for 30 sec then frozen in liquid nitrogen, while the other artery was frozen in the resting state. The arteries were pulverized, washed with perchloric acid to precipitate the muscle proteins and remove 32P-containing phosphate metabolites from the muscle. The washed residue was neutralized with a NaOH solution then dissolved in sodium dodecyl sulfate (SDS). After centrifugation at high speed to remove insoluble particles, the protein content of the supernatant was determined and aliquots of 360 mg protein were subjected to 2D polyacrylamide gel electrophoresis. This procedure separates the proteins according to their charge (pH 4-6) in the first dimension and according to their size (SDS ) in the second dimension. After staining, the profile of the arterial proteins appeared, shown in the upper row of Fig. SM2. LC, is in the lower middle part of the gel, it contains multiple spots. The LC spots were scanned, the staining intensities are shown in the lower row of the Figure. The radioactive spots on the gel were detected by autoradiography, the middle row of Fig. SM2 shows the black spots on the film corresponding to the radioactive spots on the gel.

Visual inspection of the radioactive LC spots in the Figure shows much more radioactivity in LC from the contracting muscle (right) than from the resting muscle (left). One can calculate the incorporation of the 32P-phosphate into LC as follows. First one has to determine the specific radioactivity of the terminal P of ATP from the muscle. The ATP is in the perchloric acid extract of the frozen and pulverized muscle, described before, and Bárány and Bárány (1996c) describe the determination of the specific radioactivity. The next step is the determination of the radioactivity in LC: the gel spots are excised, digested with H2O2, and after the gel is dissolved, radioactivity (counts per minute) is measured. The extent of LC phosphorylation can be calculated from the radioactivity in the LC spots and in the terminal phosphate of ATP, from the total protein applied onto the gel, and from the LC content of the total protein (Bárány and Bárány, 1996c). Such a calculation shows that under conditions of Fig. SM2, the LC of the resting muscle contained 0.25 mol 32P-phosphate/mol LC, whereas the LC of the contracting muscle contained 0.70 mol. Thus, 0.45 mol 32P-phosphate was transferred by MLCK from the terminal phosphate of ADP32P to free LC-OH groups as the result of muscle contraction.


Fig. SM2. Light chain phosphorylation during smooth muscle contraction as studied by 2D gel electrophoresis. (Bárány and Bárány, 1996a, with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press). Left, 32P-labeled porcine carotid arterial muscle was frozen at rest. Right, 32P-labeled porcine carotid arterial muscle was frozen 30 sec after 100 mM KCl challenge. Upper panel shows the Coomassie blue staining pattern of the arterial proteins; middle panel shows the corresponding autoradiograms; bottom panel shows the corresponding densitometric scans of LC.

Isoforms of the 20-kDa myosin light chain: Protein isoforms have the same size but different charge. They are generated either by protein modification or genetic alteration. Protein phosphorylation is the physiological protein modification, because phosphorylation of a protein increases its negative charge. Thus, LC has at least two isoforms, a non-phosphorylated and a phosphorylated one. Genetic alteration changes the amino acid composition of a protein, thereby providing at least two isoforms. For instance, completely dephosphorylated LC exhibits two spots on 2D gels (Fig. SM3) with a percentage distribution of 85% and 15%, corresponding to the major and minor LC isoforms.

Fig. SM3. Myosin light chain isoforms as analyzed by 2D gel electrophoresis. LC was dephosphorylated by homogenizing porcine carotid arteries in 150 mM NaCl and 1 mM EGTA, followed by incubation at 25oC for 2 hours. Top, stained gel, LC spots are numbered as 2 and 4, corresponding to their isoform number. Bottom, densitometric tracing of the LC spots.

If both major and minor LC are phosphorylated, that results in four isoforms (two non-phosphorylated and two phosphorylated). In Fig. SM2 on top level four LC isoforms are seen; on middle level, the autoradiograms reveal that three spots are radioactive. The non-radioactive, most basic spot, corresponds to the major isoform of LC (Fig. SM3).

Figure SM4 illustrates the formation of LC isoforms as a result of phosphorylation. The major isoform (LCa) when mono-phosphorylated (PLCa) moves into Spot 3, and when it is di-phosphorylated (2PLCa) moves into Spot 2. The same Spot 2 also contains the non-phosphorylated minor isoform (LCb), thus the comigration of the di-phosphorylated LC isoform with the minor isoform makes Spot 2 radioactive. This explains why out of the four LC spots three are phosphorylated. The mono-phosphorylated minor isoform (PLCb) moves into Spot 1, which is the most acidic spot.

Fig. SM4. Scheme for the explanation of four stained and three radioactive LC spots, shown on Fig. SM2. (Bárány and Bárány, 1996a, with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press).

The last row of Fig. SM2 shows the muscle contraction-induced changes in the staining intensity of LC spots. In the resting muscle the last peak, the non-phosphorylated major isoform contains as much as 63% of the total intensity, which decreases to 35% in the contracting, muscle. At the same time, the 23% intensity in the preceding peak of the resting muscle increases to 49% upon contraction. This is an example of a simple (but not sensitive) method to follow changes in LC phosphorylation during contraction, without using 32P-labeling of the muscle.

Phosphorylation site: The amino acid sequence of LC exhibits a similarity among LCs from various smooth muscles. Such a conservative sequence suggests a functional significance for the protein. The phosphorylation sites are located at the amino terminal part of the LC molecule, shown in Fig. SM5. Serine 19 is the site that is phosphorylated by MLCK in the intact muscle. Threonine 18 is phosphorylated by MLCK rarely. Beside MLCK, protein kinase C (PKC) also phosphorylates LC; the sites involve Serine 1, Serine 2, and Threonine 9.

Fig. SM5. Phosphorylation sites of LC.

Two-dimensional tryptic peptide mapping: Phosphopeptide maps differentiate MLCK-catalyzed LC phosphorylation from that catalyzed by PKC (Erdodi et al., 1998). Fig. SM6 illustrates the experiment: With ADP32P as a substrate, pure LC was phosphorylated either by MLCK (middle panel), or PKC (right panel). Actomyosin that contains endogenous LC, MLCK, and PKC, was also phosphorylated (left panel). The 32P-LC was isolated by 2D gel electrophoresis, digested by trypsin, and the peptides were separated by 2D peptide mapping. The map of LC phosphorylated by MLCK exhibits four peptides: A, B, both containing serine residues, corresponding to the Ser-19 site, and C, D, both containing threonine, corresponding to the Thr-18 site. When LC is phosphorylated by PKC, the map exhibits two peptides: E, containing serine, corresponding to Ser-1 or Ser-2 site, and F, containing threonine, corresponding to theThr-9 site. When LC is phosphorylated in actomyosin, peptides characteristic for both MLCK and PKC phosphorylation are present.

Fig. SM6. Autoradiograms of 2D phosphopeptide maps of LC tryptic digests.

Based on two-dimensional peptide mapping, one can identify the enzymes that phosphorylate LC during smooth muscle contraction (Fig. SM7). The phosphopeptide pattern of LC from K+-contracted artery (left part) is practically identical with that of LC{MLCK} pattern shown in Fig. SM6; thus, the conclusion is clear, MLCK is the main enzyme that phosphorylates LC during the K+-contraction. In contrast, when the muscle is contracted with phorbol dibutyrate (PDBu), an activator of PKC, the LC pattern (right part) resembles that of Actomyosin shown in Fig. SM6, thus, both enzymes, MLCK and PKC phosphorylate LC during the PDBu-contraction. One can determine the contribution of each enzyme to the phosphorylation, either by densitometry of the spots, or by counting the radioactivity in the spots, The results showed about 35% contribution by PKC.

Fig. SM7. Phosphopeptide maps of LC from K+-contracted muscle versus PDBu-treated muscle.

The role of Ca2+ in light chain phosphorylation: As in skeletal muscle, Ca2+ also plays a central role in the contractility of smooth muscle. In skeletal muscle TN-C is the target of the myoplasmic Ca2+, whereas in smooth muscle Ca2+ activates MLCK. Actually, the Ca2+ complexed to calmodulin is the activator of the enzyme. In agreement with the in vitro studies, intact smooth muscle cease contracting when Ca2+ is omitted from the bathing solution, or when it is complexed with EGTA. Furthermore, inhibitors of calmodulin, such as trifluoperazine or chlorpromazine inhibit smooth muscle contraction.

In the resting muscle there is about 0.1 µM Ca2+, upon stimulation the Ca2+ concentration increases about 100-fold through electromechanical or pharmacomechanical coupling. It is conventional to use fluorescent indicators to follow changes in the intracellular Ca2+ concentration immediately after the stimulation and during the plateau of the mechanical activity. Large variations are reported, depending on the nature of the smooth muscle, the tissue preparation, or the drug used. However, all investigators agree that in order to elicit relaxation the Ca2+ level in the sarcoplasm must be returned near to the resting value. Two mechanisms participate in decreasing the Ca2+ level: 1) The plasma membrane Ca2+ transporting ATPase pumps Ca2+ from the inside into the extracellular space. 2) The sarco(endo)plasmic reticulum Ca2+ transporting ATPase pumps Ca2+ into the SR.

Stretch-induced light chain phosphorylation: As discussed before, smooth muscle can be stimulated electrically or by chemical agents. Here we describe the mechanochemical activation of smooth muscle. Stretching of arterial or uterine muscles induced light chain phosphorylation to the same extent as was observed in muscles contracted by K+ or norepinephrine (Bárány and Bárány, 1996c). Muscles which were stretched 1.6 times their resting length did not develop tension, but contracted normally when the stretch was released and the muscles were allowed to return to their rest length. Importantly, this contraction was spontaneous, indicating that the stretch-induced activation carries all the information necessary for normal contraction. Mobilization of Ca2+ was necessary for the stretch-induced light chain phosphorylation and contraction to occur. When EGTA (the strong Ca2+ complexing agent) was added to the muscle bath both the stretch-induced phosphorylation and the stretch-release-induced tension were inhibited; however, upon removal of EGTA by washings, both processes were fully restored. Treatment of the muscle with chlorpromazine (the calmodulin inhibitor) also abolished both the stretch-induced LC phosphorylation and the stretch-release-induced tension development. These results suggest the presence of mechanosensitive receptors in smooth muscle that are interacting with Ca2+ release channels in SR.

Further comments are warranted on the finding that 1.6 times stretched muscles, which are unable to contract (because there is no overlap between actin and myosin filaments), are able to fully phosphorylate their LC. Accordingly, smooth muscle contraction and LC phosphorylation are not coupled. Time course experiment also demonstrated that LC phosphorylation precedes tension development. Thus, LC phosphorylation plays a role in the activation process but not in the contraction per se. Furthermore, K+-contracted muscle maintains its tension for a prolonged time although its LC becomes dephosphorylated. This is another example for the lack of coupling between phosphate content of LC and contractility of muscle.

Phosphorylation of Heat-Shock Proteins

Low molecular weight heat schock proteins are phosphorylated in smooth muscle: A 27-28-kDa protein is phosphorylated in various intact smooth muscles and smooth muscle cells (reviewed in Bárány and Bárány, (1996c). Cyclic nucleotide-dependent vasorelaxation is associated with the phosphorylation of a 20-kDa heat shock protein, called HSP20 (Beal et al., 1997; Rembold et al., 2000). It was found that HSP20 is an actin-associated protein (Brophy et al.,1999; Rembold et al. 2000) suggesting that smooth muscle relaxation may be brought about by the binding of the phosphorylated HSP20 to the actin filaments.

Signal Transduction

The binding of an agonist (e.g. norepinephrine or oxytocin) to the surface receptor of smooth muscle induces a signal that spreads from the outside to the inside of the plasma membrane and activates several effectors that ultimately initiate contraction. There are three components of this system that we discuss: 1) Inositol 1,4,5-trisphosphate, 2) G-proteins, 3) Phosphoinositide-specific phospholipase C.

Inositol 1,4,5-trisphosphate: The inositol ring contains six hydroxyl residues, most of them can be phosphorylated by specific kinases. Inositol 1-monophosphate is the constituent of phosphatidylinositol (PI) one of the phospholipids in animal cell membranes. PI 4-kinase and PI (4) P 5-kinase to generate PI (4) P and PI (4,5) P2, respectively, sequentially phosphorylate PI. Inside the cell membrane resides a phosphoinositide specific phospholipase C, one of its hydrolytic product is inositol 1,4,5-trisphosphate (IP3), (see Fig. SM 8).

Fig. SM8. D-myo-inositol 1,4,5-trisphosphate. (Bárány and Bárány, 1996b,with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press). The arrow indicates the site of the ester link with diacylglycerol in phosphatidylinositol. The negative charge of the phosphate group is not indicated.

G-proteins: The guanine nucleotide binding proteins (G-proteins) are heterotrimers consisting of a-, b- and g-subunits. The a-subunits appear to be most diverse and are believed to be responsible for the specificity of the interaction of different G-proteins with their effectors. Fig. SM9 depicts a simple model for the activation of G-proteins. In the basal state, the a-subunit contains bound GDP and association of a- and bg-subunits is highly favored, keeping the G-protein in the inactive form. Stimulation of the G-protein results when it binds GTP rather than GDP. Receptors interact most efficiently with the heterotrimeric form of the G-protein and accelerate activation by increasing the rate of dissociation of GDP and enhancing the association of GTP. Activation of G-protein coupled receptor results in the dissociation of heterotrimeric G-proteins into a-subunits and bg-dimers. Finally, the G-protein a-subunit has an intrinsic hydrolytic activity that slowly converts GTP to GDP and returns the G-protein to its inactive form.

Fig. SM9. Model for the activation of G-proteins. ( Bárány and Bárány, 1996b, with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press).

Phosphoinositide-specific phospholipase C: This term refers to a family of enzymes all specific for the phosphoinositide moiety of the phosphatidylinositol, but differing in their specificity depending on the number of the phosphoryl groups on the inositol ring. The b-, g- and d-isoforms of PI-phospholipase C (PI-PLC) show the greatest specificity for the trisphosphorylated phospholipid (PIP2)). There are two basic mechanisms by which agonists activate PIP2 hydrolysis (Fig. SM10). In case of hormones, neurotransmitters, and certain other agonists, the signal is transduced to b-isozymes of PI-PLC. The upper left row of Fig. SM10 shows the most common pathway for PI-PLCb-isoform activation, initiated by stimulation of a a1-adrenergic receptor (a1-R) with norepinephrine (NE), and involving Gaq-proteins. The lower left row shows the activation of PI-PLC-b isoforms, initiated by acetylcholine (ACH) stimulation of M2-muscarinic receptor (M2-R), and mediated by the b g-subunit of the pertussis toxin-sensitive G-protein (GI). Concerning the other basic activating mechanism, e.g. in the case of growth factors, activation of their receptors results in enhanced tyrosine kinase activity. The right part of Fig. SM10 shows the activation of PI-PLC-g isoforms, initiated by the binding of epidermal growth factor (EGF) to its receptors, and executed by the tyrosine phosphorylation (YP) of PI-PLC-g . In all three examples, the activated PI-PLC hydrolyzes PIP2 to form the messengers IP3 and diacylglycerol (DAG). IP3 releases Ca2+ from the sarcoplasmic reticulum and thereby initiates smooth muscle contraction. DAG activates protein kinase C, the exact result of this activation is not known at the cellular level.

Fig. SM 10. Pathways for activation of PI-PLC isoforms. (Bárány and Bárány, 1996b, with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press). .

The Contractile Event of Smooth Muscle

A scheme for smooth muscle contraction is shown in Fig. SM11. Contraction is initiated by the increase of Ca2+ in the myoplasm; this happens in the following ways:

  1. Ca2+ may enter from the extracellular fluid through channels in the plasmalemma. These channels open, when the muscle is electrically stimulated or the plasmalemma is depolarized by excess K+.
  2. Due to agonist induced receptor activation, Ca2+ may be released from the sarcoplasmic reticulum (SR). In this pathway, the activated receptor interacts with a G-protein (G) which in turn activates phospholipase C (PLC). The activated PLC hydrolyzes phosphatidyl inositol bisphosphate; one product of the hydrolysis is inositol 1,4,5-trisphosphate (IP3). IP3 binds to its receptor on the surface of SR, this opens Ca2+ channels and Ca2+ from SR is entering the myoplasm.
  3. Ca2+ combines with calmodulin (CaM) and the Ca2+ -CaM complex activates MLCK, which in turn phosphorylates LC. The phosphorylated myosin filament combines with the actin filament and the muscle contracts.

Fig. SM11. A scheme for smooth muscle contraction. ( Bárány, 1996, with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press).

Two books (Bárány, 1996; Kao and Carsten, 1997) and a special journal issue (Murphy, 1999) are recommended for further studying the mechanism of smooth muscle contraction.and relaxation.

Monomer (G) to Polymer (F) Transformation of Actin in Smooth Muscle

Mehta and Gunst (1999) and Jones et al (1999) reported the existence of G-actin in smooth muscle, based on the method of DNase I inhibition and phalloidin staining, respectively. Subsequently, Bárány, et al (2001) showed the exchange of the actin-bound nucleotide in intact smooth muscle. This was based on the separation of the actin bound nucleotides from the cytoplasmic nucleotides with 50% ethanol (Fig. SM12).

Fig. SM12. Extraction of nucleotides and radioactivity from 32P-labeled arterial smooth muscles (From Bárány et al., 2001).The percentage of the total absorbance and counts eluted from the muscles in 8 extractions is shown on the ordinate.

32P-labeled arterial smooth muscles were extracted in the refrigerator several times for elutions of the nucleotides (Absorbance at 260 nm) and the 32P-label (Counts). The absorbance and the counts are very high in the first extract and drop in the successive five extractions virtually to zero. In the 7th and 8th elution the muscles were extracted with 1.5% perchloric acid (PCA). New absorbance and radioactivity appeared, corresponding to the release of the bound-nucleotide and the associated 32P-label from actin.

The composition  of the PCA extract is shown on Fig. SM13.

Fig. SM13. Dowex -1 chromatography of Extracts No. 7 and 8, shown in Fig. SM12.(From Bárány et al., 2001). Squares correspond to Counts per ml and triangles correspond to Absorbance.

Three radioactive peaks are seen, the first from Pi, the second from ADP, and the third from ATP. For ADP and ATP the absorbance is also shown; 85% of the total absorbance is from ADP and 15% from ATP. Since F-actin contains bound-ADP and G-actin contains bound-ATP, the data indicate that 85% of the total actin in this muscle preparation was in the polymeric and 15% in the monomeric form. Determination of the Pi concentration by a colorimetric method showed that the molar ratio of Pi to ADP was close to one.

In order to quantify the extent of exchange of the actin-bound nucleotide and Pi, one has to determine their specific activity (counts /min/mol nucleotide or Pi) and compare it with those of the specific activities (s.a.) of the gamma- and beta-phosphates of the cytoplasmic ATP and that of PCr (Bárány et al., 2001). With this knowledge one can calculate the percentage exchange for each of the actin components; for instance , the percentage exchange of the actin-bound- ADP is:

(s.a. of actin-ADP/s.a. of beta-P of cytoplasmic ATP) x 100

Fig. SM14 compares the exchange of the actin-bound ADP between smooth and skeletal muscles. The exchange is rapid in smooth muscle, half-time about 15 min, whereas the exchange is slow in skeletal muscle, about 15% in three hours, in agreement with the studies of Martonosi et al., 1960) in live animals.

Fig. SM14. Time course of the exchange of the actin-bound ADP in smooth (porcine carotid artery) and skeletal (rat vastus lateralis) muscle. (From Bárány et al., 2001).

 Characteristics of the exchange of the actin-bound nucleotide in smooth muscle:

ATP is a prerequisite for the exchange to take place. If ATP synthesis is inhibited by azide or iodoacetamide the exchange is also inhibited. If ATP sysnthesis is reduced, by incubation of the muscles with deoxyglucose, instead of glucose, the exchange is also reduced.

Ca2+ is not required for the exchange, i.e. full exchange is observed in the muscle in the presence of EGTA.

Several smooth muscles, arteries, uteri, urinary bladder, and stomach exhibiited the exchange of the actin-bound nucleotide and phosphate, suggesting that the exchange is a property of every smooth muscle.

Upon contraction of smooth muscle, the exchange of the bound-nucleotide and phosphate decreased and upon relaxation from the contracted state it increased, suggesting that polymerization-deplolymerization of actin is a part of the contraction-relaxation cycle of smooth muscle.


Bárány, M. (1996). Biochemistry of Smooth Muscle Contraction. Academic Press.

Bárány, K. and Bárány, M. (1996a). Myosin light chains. In Biochemistry of Smooth Muscle Contraction (M. Bárány , Ed.), pp. 21-35, Academic Press.

Bárány, M. and Bárány, K. (1996b). Inositol 1,4,5-trisphosphate production. In Biochemistry of Smooth Muscle Contraction (M. Bárány, Ed.), pp. 269-282, Academic Press.

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