Smooth Muscle I and II
S.M. Karnam, Ph.D.
Suggested Reading: Berne and Levy (5th Edition, 2003), pp 246-262
Linda S. Costanzo (3nd Edition, 2006), pp 38-43
Learning Objectives: After studying this material, the student should:
a) Analyze the contractile apparatus of smooth muscle and differences with striated
muscle
b) Identify the mechanism of smooth muscle contraction: the role of Ca2+ and
myosin light chain phosphorylation in mediating smooth muscle contraction
c) Differentiate the mechanism of Ca2+-independent contraction (Ca2+ sensitization)
d) Describe the mechanism of muscle relaxation
I. Structure of smooth muscle
Smooth muscle is widespread: it is a major component of the walls of the hollow
organs including airways, gut, urogenital tract and vasculature. Similarities and
dissimilarities with striated muscle are obvious.
The ultimate function of smooth muscle is to develop force or contract, to provide
motility or to alter the dimensions of an organ. For some of the functions contractions
must be phasic (contraction followed by relaxation) to allow the lumen to refill between
contractions. For others, contractions must be tonic (sustained contraction) to resist
continual distending forces.
In most organs, the orientation of smooth muscle is circular or circumferential so
that contraction decreases the intraluminal volume/diameter and thereby increasing
resistance to flow. In the gut, there is a second layer of muscle cells oriented in the
longitudinal axis (oral-to-anal) termed the longitudinal muscle layer. Network of
neurons, glial cells and interstitial cells of Cajal separate the circular and longitudinal
muscle layers.
Smooth muscle cells
Some structural properties are common to all smooth muscle cells: they are single
(uninuclear) cells; they have no transverse striations; they have no T-tubules; they have
abundant caveolae; and they have numerous cell-to-cell junctions. Other aspects of the
muscle structure vary from organ to organ and are related to the characteristic functions
of each muscle.
Smooth muscle cells are spindle-shaped. The characteristic shape seems dictated by
the insertion of the contractile apparatus over the entire cell surface and is essential for
mechanical properties of these cells. Most individual smooth muscle cells are very small
in comparison to skeletal muscle cells.
Figure 1. Organization of the contractile and cytoskeletal apparatus in smooth
muscle cells. Thin actin filaments emerge from the poles of the cytoplasmic dense bodies
and interdigitate with thick myosin filaments. Dense bands in the plasma membrane are
connected to dense bodies in the cytoplasm by intermediate filaments. When juxtaposed
dense bands from adjacent cells can form close intermediate junction (provides
mechanical
coupling).
Gap
junction
provides
electrical
coupling.
a) Caveolae are flask-shaped invaginations of the cell membrane. Caveolae
increase the surface area of the cell. More than one-third of the plasma membrane at the
cell surface is in the form of caveolae, while the rest constitutes the cell surface proper.
The exact function of caveolae, however, is not known.
b) Dense bands are structures associated with the cell membrane where
contractile apparatus and cytoskeleton are anchored to the cell surface. Dense bands are
sometimes coupled to each other in adjacent cells. Dense bodies are scattered throughout
the cytoplasm. The linkage with actin filaments is very evident both in dense bands and
dense bodies.
c) Cell-to-cell junctions are very abundant in smooth muscle, and they serve at
least two fundamental functions: mechanical coupling (transmission of force between
cells) and ionic coupling (transmission of excitation from cell to cell). The mechanical
coupling is supported by intermediate junctions. Ionic or electrical coupling is provided
by gap junctions.
d) Sarcoplasmic reticulum is well developed in smooth muscle. The smooth
sarcoplasmic reticulum can sequester calcium ions from the cytoplasm and is regarded as
a major storage site for calcium. The sarcoplasmic reticulum has inositol trisphosphate
(IP3) receptors and ryanodine receptors, which, when activated by appropriate second
messengers, allow the release of intracellular calcium needed for contraction. The
sarcoplasmic reticulum also contains a calcium ATPase or calcium pump that functions
to sequester calcium into sarcoplasmic reticulum following contraction.
e) Myofilaments: The three classes of filaments found in smooth muscle are
actin (thin, 6-8 nm) filaments, myosin (thick, 14 nm) filaments and intermediate (10 nm)
filaments.
Thin filaments contain actin polymer and tropomyosin as in striated muscle.
Compared to striated muscle, there is very large number of actin filaments in smooth
muscle. Smooth muscle lacks the regulatory protein, troponin, but contains two
unique proteins, calponin and caldesmon.
Thick filaments are large aggregates of myosin molecules and composed of two
heavy chain subunits (~ 200 kDa each) and two each of two types of light chains (two
20-kDa regulatory light chains and two 17-kDa essential light chains). The myosin
heavy chain dimers form globular head and coiled tail regions. The head region
contains distinct sites for actin binding, ATP hydrolysis and association of light chain
subunits.
Intermediate filaments-a cytoskeletal component present in most cell types-are
abundant in smooth muscle, and are far more abundant than in cardiac and skeletal
muscle. They are intermediate in size between and thick and thin filaments. The
main component of intermediate filaments in visceral muscles is desmin and in
vascular muscles is vimentin. Intermediate filaments are anchored in the dense
bodies and bands, and link to the cytoskeleton. These provide mechanical coupling
between force generating proteins (actin and myosin) and the cell membrane thereby
causing deformation and shortening of smooth muscle cells.
Figure 2. Contractile proteins in smooth muscle. Contractile proteins in
smooth muscle are organized into thick and thin filaments as schematically illustrated.
Thin filaments are composed of tropomyosin, caldesmon, and/or calponin bound to two
intertwined strands of polymerized actin. Myosin, composed of two heavy chains and two
pairs of light chains – essential light chains (ELC: 17-kDa) and regulatory light chains
(MLC: 20-kDa)- polymerizes into thick filaments.
II. Innervation of smooth muscle
The role of nerves in smooth muscle is somewhat special, because many muscles
have myogenic activity and can contract independently of nervous influence. Muscle
cells are often electrically coupled, so excitation can spread quickly from innervated cell
to cells that are not directly innervated.
There is wide diversity in the type and degree of innervation of smooth muscle
tissue in the body. For example, both parasympathetic and sympathetic branches of the
autonomic nervous system, as well as post-ganglionic nerves from the enteric nervous
system innervate smooth muscle of the gastrointestinal tract. On the other hand, many
blood vessels receive only sympathetic innervation. Neurotransmitters can be classified
in terms of their ability to cause contraction (excitatory transmitters) and relaxation
(inhibitory transmitters) of smooth muscle cells.
There are two categories of smooth muscle based on how the muscle cells are
stimulated to contract.
A. Single-unit (unitary) smooth muscle is found arranged in sheets and all the
cells in a sheet are able to contract as a single unit. Muscle of the stomach and
intestine behave like unitary type smooth muscle. Unitary smooth muscle is selfexcitable (exhibit spontaneous electrical activity) or myogenic and does not
require nervous stimulation for contraction. This type of smooth muscle is
sparsely innervated and muscle cells are electrically coupled by gap junctions so
that excitation can rapidly spread among cells resulting in unitary contraction.
Autonomic nervous system can modify the rate and strength of contractions.
Other factors that influence contractions are hormones and mechanical stretch.
Figure 3. Schematic of unitary (A) and multiunit (B) smooth muscle.
difference between the two types of muscle cells in innervation and contact.
Note the
B. Multiunit smooth muscle is found in the walls of large blood vessels, in the
large airways of respiratory tract and in the eye muscles. Like striated muscle,
multiunit smooth muscle is neurogenic, that is, it requires nerve stimulus to
initiate contraction. Unlike skeletal muscle there are no neuromuscular junctions.
Neurotransmitters are released into extracellular fluid surrounding the smooth
muscle cells. Certain hormones and drugs can also influence contractions of
multiunit smooth muscle.
III. Electrical properties of smooth muscle
A. Resting Membrane Potential
The resting membrane potential, defined as the steady-state potential at which the
net flow of current (i.e., ions) across the plasma membrane is zero, varies from about -40
to -80 mV in muscle cells of the gut. Differences in resting membrane potential exist
between muscle cells in different regions of gastrointestinal tract, such as the fundus,
corpus, and antrum of the stomach
B. Gated Ion-Selective Channels
In addition to passive ion-selective channels, the plasma membrane contains ionselective channels that can be regulated by membrane potential (i.e., voltage-gated
channels) and by various humoral, hormonal, or neural agents (i.e., ligand-gated
channels). Ligands can activate channels directly and through G proteins in the
membrane. Ligands can also activate, inhibit, or modulate voltage-gated channels
through second messengers.
Voltage-gated Ca2+ channels carry the long-lasting inward Ca2+ current (L-type
Ca channels) and are activated rapidly by depolarization of the plasma membrane but
are inactivated more slowly. Inactivation occurs as a result of Ca2+ influx and membrane
depolarization. Most of the voltage-gated Ca2+ channels are sensitive to the drug
dihydropyridine and are thus, known as dihydropyridine-sensitive Ca2+ channels.
Dihydropyridines are well known in pharmacology as L-type Ca2+ channel blockers.
2+
Several types Voltage-Gated Potassium Ion Channels have been identified in
gastric and intestinal smooth muscle.83–95 The channels differ in their conductance, ranges
of voltage activation, and Ca2+ sensitivities. The most widely distributed is a highconductance, Ca2+-activated, voltage-sensitive K+ channel. During resting conditions,
when cytosolic Ca2+ concentrations are low (<10-7 M), relatively few channels are open.
On stimulation, the increase in calcium induces activation of large numbers of K+
channels, which carry an outward current, that drives the membrane potential to its
resting state. Ca2+ channels and Ca2+-activated K+ channels constitute the electrical
apparatus that sustains rhythmicity in smooth muscle. Activation of Ca2+ channels
induces an inward flow of Ca2+ ions that depolarizes the membrane and increases
cytosolic calcium. Depolarization and an increase in cytosolic calcium inactivate the
Ca2+ channels and activate the K+ channels, by inducing an outward flow of K+ ions.
Suppression of the inward flow of Ca2+ ions and enhancement of the outward flow of K+
ions restore the resting membrane potential.
C. Interstitial Cells of Cajal (ICC): Electrical pacemakers and mediators of
neurotransmission in gastrointestinal tract
In gastrointestinal muscle phasic contractions are caused by electrical activity
termed slow waves, which arise from defined pacemaker regions. Pacemaker areas
contain a variety of cell types, including enteric neurons, glial cells, smooth muscle cells,
immune cells and interstial cells of Cajal (ICC).
ICC are a distinctive population of enteric cells that express c-kit, the protooncogene that encodes the receptor tyrosine kinase, Kit. The closeness of nerve fibers to
ICC, and ICC to smooth muscle cells led Cajal to propose that ICC were functionally
interposed between nerve terminals and smooth muscle cells. ICC and smooth muscle
cells are electrically coupled. The ICC are spontaneously active, generating and
propagating slow-wave depolarizations, and thus regulating phasic contractions in the
gastrointestinal tract.
Figure 4. Profile of a typical slow wave. A rapid upstroke is followed by partial
repolarization, a plateau potential of variable duration and complete repolarization.
A typical slow wave consists of the following sequence: rapid depolarization (i.e.,
upstroke), partial repolarization, a sustained plateau lasting several seconds, and complete
repolarization to the resting membrane potential. Slow waves originate in pacemaker
regions and propagate rapidly throughout its thickness. Propagation is rapid and is
facilitated by the network of ICC and the abundance of gap junctions between muscle
cells.
IV. Excitation-contraction coupling
One of the significant characteristics distinguishing smooth muscle from skeletal
muscle is the existence of multiple pathways for the activation and subsequent regulation
of contractile process. There are many differences between striated and smooth muscle
contraction. These include: a) mechanism of excitation-contraction coupling, b)
differences in the calcium regulation of actin-activated myosin-ATPase activity, 3)
differences in calcium sensitivity.
A. Crossbridge cycling
Contraction is initiated by an increase in intracellular free calcium (either
released from intracellular stores or by influx from extracellular stores) and ATP is
utilized to convert chemical energy into mechanical energy. The force and shortening of
the muscle are dependent on the interaction of thick and thin filaments, the formation and
cycling of crossbridges, and the sliding filament mechanism of contraction.
In its simplest form, the sliding filament mechanism requires:
1. Two types of filaments, one containing myosin and the other containing actin.
2. Overlap of myosin-containing thick filaments with actin-containing thin filaments as a
function of degree of shortening.
3. Neither type of filament is changing its length, regardless of the contractile state of the
muscle.
4. The force necessary for filaments to move relative to each other is provided by
crossbridges projecting from the thick filaments. The crossbridges have following
properties:
a) crossbridges go through cycles of attachment, force-production/movement and
detachment.
b) each crossbridge produces same amount of force
c) force production is proportional to the number of active crossbridges
B. Regulation of contraction by myosin light chain phosphorylation.
Myosin from smooth muscle differs from striated muscle myosin in that smooth
muscle myosin-ATPase activity cannot be actin-activated unless their 20-kDa myosin
light chains (MLC) are phosphorylated.
The discoveries that phosphorylation of smooth muscle myosin results in marked
stimulation of actin-activated myosin-ATPase activity, and that myosin light chain
(MLC) kinase, the enzyme responsible for myosin phosphorylation, requires calcium and
calmodulin for activity led to the development of a model for regulation of smooth
muscle contraction whereby calcium-dependent phosphorylation of myosin light chain
initiates smooth muscle contraction.
In this scheme activators of smooth muscle contraction lead to increase in
cytosolic calcium and the formation of calcium-calmodulin complexes that bind to and
activate the enzyme MLC kinase. Activated MLC kinase catalyzes the phosphorylation
of MLC, which results in dramatic increases in actin-activated myosin-ATPase activity of
the smooth muscle myosin and thereby initiates crossbridge cycling and mechanical
output. Decreases in cytosolic calcium brought by calcium extrusion or uptake into the
sarcoplasmic reticulum, result in inactivation of MLC kinase, MLC dephosphorylation by
myosin light chain phosphatase, and muscle relaxation. Thus smooth muscle contraction
is regulated by the relative activities of MLC kinase and MLC phosphatase
A considerable body of evidence supports a central role for MLC
phosphorylation-dephosphorylation in the regulation of contraction. This evidence is
briefly summarized as follows:
1. Correlation between MLC phosphorylation and actin-activated myosinATPase activity.
2. Correlation between MLC phosphorylation and contraction.
3. Correlation between MLC dephosphorylation and relaxation
4. Inhibition of MLC phosphorylation and contraction by calmodulin antagonists
and MLCK inhibitors
5. Contraction induced by phosphatase inhibitors.
MLC
MLC
MLCK
MLCPase
MLC-p
Contraction
MLCK
MLCPase
MLC-p
Relaxation
Figure 5. The ratio of activities of myosin light chain kinase (MLCK) and
myosin light chain phosphatase (MLCPase) effects contraction and relaxation in
smooth muscle. Left: Increased cytosolic calcium activates MLCK resulting in increased
MLC phosphorylation (MLC-p) and contraction. Inhibition of MLC phosphatase also
increases MLC phosphorylation and contraction. Right: Decreased cytosolic calcium
inhibits MLC kinase, thereby decreasing MLC phosphorylation and leading muscle
relaxation. Stimulation of MLC phosphatase also decreases MLC phosphorylation
resulting muscle relaxation.
MLCK: myosin light chain kinase, MLCPase: myosin light chain phosphatase,
MLC-p: phosphorylated myosin light chain.
C. Source of Calcium
An essential step in smooth muscle contraction is phosphorylation of the 20-kDa
regulatory myosin light chain (MLC20) by a Ca2+/calmodulin-dependent MLC kinase. An
increase in cytosolic calcium is a prerequisite for stimulation of MLC kinase activity.
Two mechanisms lead to an increase in [Ca2+]i. In the first, interaction of a contractile
agonist with its receptor on the plasma membrane generates a messenger that causes the
release of Ca2+ from intracellular stores. In the second, interaction of the contractile
agonist with its receptor generates a messenger that induces depolarization of the plasma
membrane, which opens voltage-gated Ca2+ channels and causes Ca2+ influx, followed by
Ca2+-induced Ca2+ release from intracellular stores.
Smooth muscle cells, like other cells, possess efficient mechanisms to dispose of
the increase in calcium that occur during contraction. In the resting state, the cells
maintain low concentrations of Ca2+ in the cytosol despite large chemical (e.g., 2 mM
Ca2+ outside versus 100 nM Ca2+ inside the cell) and electrical (e.g., membrane potential
of -40 to -80 mV) gradients favoring the movement of Ca2+ into the cell. The disposal
mechanism in the plasma membrane include a Ca2+/Mg2+-ATPase, which acts as a highaffinity Ca2+ pump sustained by ATP hydrolysis, and a low-affinity, high-capacity Na+Ca2+ exchanger. In the sarcoplasmaic reticulum, a high-affinity sarco-endoplasmic
reticulum Ca2+-ATPase pump (SERCA) participates in dissipating the increase in
cytosolic calcium.
D. Calcium-dependent contraction
The interaction between actin and myosin provides molecular basis for muscle
contraction. This interaction is regulated by calcium in all muscle types, but mechanisms
of regulation are fundamentally different for smooth muscle compared with skeletal and
cardiac muscle. Smooth muscle differs from skeletal muscle in having two mechanisms
for initiation of processes leading to contraction. Increases in cytosolic calcium may
occur by influx of calcium through calcium channels in the plasma membrane or by
release of calcium from sarcoplasmic reticulum.
Electro-mechanical coupling: Depolarization of the membrane electrical
potential leads to opening of voltage-gated calcium channels followed by elevation in
cytosolic calcium, which in turn activates the contractile proteins to initiate contraction.
This is called electro-mechanical coupling.
The resting membrane potential of smooth muscle is, like that of other cells,
negative (-40 to –70 mv, depending on the cell type). More positive potentials
(depolarization) can open voltage-gated calcium channels, causing calcium influx to
increase cytosolic calcium and trigger contraction.
The plasma membrane of smooth muscle contains a great variety of ion channels.
The distribution and properties of these channels vary among different (for example,
intestine, large and small vessels, uterus) tissues, contributing to the diversity of smooth
muscle. Voltage-dependent calcium channels and calcium-activated K+ channels are the
main channels involved in the regulation of cytosolic calcium. The activity of these
channels can be modulated by excitatory and inhibitory neurotransmitters.
The depolarization-induced rise in cytosolic calcium is due, in part, to the influx
of extracellular calcium through voltage-gated calcium channels, but this calcium also
releases additional calcium from sarcoplasmic reticulum, a process known as calciuminduced calcium release.
Depolarization
VO channel
Ca2+ Influx
[Ca2+]i
MLC
+
RyR
SR
[Ca2+]i
Ca2+/CaM
MLCK
MLCPase
MLC-p
+
Contraction
Figure 6. The electro-mechanical pathway of excitation-contraction coupling
in smooth muscle. Depolarization of the membrane opens voltage-gated calcium
channels (VO channel) to induce calcium influx. The increase in cytosolic calcium
([Ca2+]i) binds calmodulin and activates myosin light chain kinase and increases myosin
light chain phosphorylation leading muscle contraction. The initial increase in [Ca2+]i
also releases additional Ca2+ via ryanodine receptors (RyR) (calcium-induced calcium
release).
VO channel: voltage-gated calcium channel, MLCK: myosin light chain kinase,
MLCPase: myosin light chain phosphatase, MLC-p: phosphorylated myosin light chain,
RyR: ryanodine receptors, SR: sarcoplasmic reticulum, Ca2+/CaM: calcium-calmodulin
complex, + denotes stimulation of activity.
Pharmaco-mechanical coupling: Smooth muscles have an additional
mechanism in which binding of contractile agonist to its receptor on the muscle
membrane leads to elevation in cytosolic calcium without any change in the membrane
electrical potential. This is called pharmaco-mechanical coupling.
Activation of receptors coupled to contractile agonists result in G proteindependent stimulation of phospholipase C-β isozymes, which hydrolyze membrane
phosphoinositides, mainly phosphatidylinositol 4,5-bisphosphate to generate two
messengers: 1-2-diacylglycerol, an activator of protein kinase C and inositol 1,4,5trisphosphate trisphosphate (IP3), the main calcium mobilizing messenger in smooth
muscle. Inositol 1,4,5-trisphosphate diffuses through the sarcoplasm and binds to the IP3
receptor located in the sarcoplasmic reticulum membrane. This receptor is also a Ca2+
release channel, allowing Ca2+ to diffuse down its concentration gradient from
sarcoplasmic reticulum lumen into sarcoplasm, thereby triggering contraction.
Agonist
Gαq
PLC-β1
IP3
IP3R
DAG
SR
[Ca2+]i
Ca2+/CaM
MLC
MLCK
MLCPase
MLC-p
+
Contraction
Figure 7. The pharmaco-mechanical pathway of excitation-contraction
coupling in smooth muscle. Binding of contractile agonist to its receptors on the cell
membrane activates a heterotrimeric G protein (Gαq), which in turn stimulates
phosphoinositide-specific phospholipase-C β1 (PLC-β1).
PLC-β1 hydrolyzes
phophotidylinositol bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG). IP3 binds to its receptors (IP3R) on the sarcoplasmic reticulum
(SR) membrane to release calcium. The increase in cytosolic calcium ([Ca2+]i) binds
calmodulin and activates myosin light chain kinase and increases myosin light chain
phosphorylation leading muscle contraction.
MLCK: myosin light chain kinase, MLCPase: myosin light chain phosphatase,
MLC-p: phosphorylated myosin light chain, PLC-β1:phospholipase C-β1, IP3R: inositol
1,4,5-trisphosphate receptor, SR: sarcoplasmic reticulum, Ca2+/CaM: calciumcalmodulin complex, + denotes stimulation of activity.
In addition to inositol 1,4,5-trisphosphate receptors, the sarcoplasmic reticulum
also contains ryanodine receptors. It has been shown that the ryanodine receptors
respond, as in cardiac muscle, to elevations in cytosolic calcium by calcium-induced
calcium release. The sarcoplasmic reticulum is the physiological intracellular source of
activator calcium and inositol 1,4,5-trisphosphate is the physiological calcium mobilizing
second messenger.
The sarcoplasmic reticulum in smooth muscle also contains phospholamban, a
phosphoprotein that regulates calcium uptake by the sarcoplasmic reticulum. The
calcium-storage capacity of the sarcoplasmic reticulum is enhanced by the intraluminal
calcium-binding proteins calsequestrin and calreticulin.
E. Calcium-independent contraction (calcium sensitization)
There is little doubt that calcium-dependent myosin light chain phosphorylation
represents a major pathway by which muscle tone is regulated. The question is whether
this is the only pathway involved. One of the recent developments in smooth muscle
contraction has been the identification of secondary mechanisms of regulation that can
modify, independently of cytosolic calcium, the levels MLC phosphorylation. The
mechanism(s) through which contractile agonists can increase force (i.e. MLC
phosphorylation) without necessarily increasing calcium is referred to as calciumindependent contraction or calcium sensitization.
Increased MLC phosphorylation of smooth muscle can be affected not only by
increasing cytosolic calcium and, thereby, the activity of MLC kinase, but also by
inhibiting MLC phosphatase. MLC phosphatase is trimer containing a regulatory subunit,
a catalytic subunit and a subunit with unknown function. The inhibitory signal for
calcium sensitization is communicated by a small monomeric G protein RhoA to Rhokinase that phosphorylates MLC phosphatase regulatory subunit and inhibits the catalytic
activity of MLC phosphatase, resulting in increased MLC phosphorylation and
contraction. The RhoA/Rho kinase pathway plays an important physiological role in
smooth muscle contraction. Protein kinase C (PKC) activated by diacylglycerol can also
inhibit MLC phosphatase by phosphorylating and thereby activating CPI-17, an
endogenous inhibitor of MLC phosphatase.
Agonist
RhoA
Rho Kinase
PKC
MLC
MLCPase
MLCK
CPI-17
MLC-p
Contraction
Figure 8. Calcium-independent (calcium-sensitization) contraction in smooth
muscle. The major calcium-independent pathway that increases myosin light chain
(MLC) phosphorylation and induces contraction is through activation of Rho-kinase by
RhoA. Activated Rho-kinase inhibits myosin light chain phosphatase (MLCPase) activity
resulting in increased levels of MLC phosphorylation leading to muscle contraction. The
other pathway that enhances MLC phosphorylation is through protein kinase C (PKC)mediated phosphorylation of CPI-17, an inhibitor of MLC phosphatase.
MLCK: myosin light chain kinase, MLCPase: myosin light chain phosphatase,
MLC-p: phosphorylated myosin light chain, - denotes inhibition of activity.
E. Regulation of contraction by thin-filament-associated proteins
Contractile regulation by thin-filament-associated proteins (for example,
caldesmon and calponin), and possibly through their phosphorylation, has been
intensively studied.
Caldesmon and calponin regulate smooth muscle contractility through their ability
to inhibit actin-activated myosin-ATPase activity and to inhibit contraction.
Phosphorylation of caldesmon and calponin reverses the inhibitory effect on myosinATPase thus facilitating contraction. Biochemical studies of caldesmon and calponin
phosphorylation in intact muscle suggest important regulatory functions for these
proteins, but the details of signal transduction pathways and the molecular effects of
caldesmon and calponin on the crossbridge cycle are still unclear.
V. Molecular Mechanism of Smooth Muscle Relaxation
Smooth muscle relaxation has classically been described as a default process
resulting from the reduction in cytosolic calcium. Cytosolic calcium can be reduced,
causing relaxation, through several mechanisms. The rate and extent of calcium pumping
into the sarcoplasmic reticulum is sufficient to cause relaxation. Relaxation can also be
achieved by the active extrusion of calcium (via sarcolemmal Ca2+ ATPase) and by a
reduction in the sensitivity of the contractile system to calcium. Alternatively, inhibition
of Ca2+ influx through hyperpolarization can also reduce cytosolic calcium and cause
relaxation.
The intracellular second messengers involved in smooth muscle relaxation are the
cyclic nucleotides, cAMP and cGMP, generated by the activity of adenylyl cyclase and
guanylyl cyclase, respectively. They exert their intracellular effects by activating cAMPand cGMP-dependent protein kinases. cAMP is the dominant mediator of smooth muscle
relaxation stimulated by β-adrenergic drugs, whereas cGMP-mediated relaxation is
triggered by nitric oxide.
The nitric oxide-cGMP pathway is responsible, at least in part, for the vascular
smooth muscle relaxation produced by many agents, including nitrovasodilators and
acetylcholine. These agents stimulate the endothelial cell to produce nitric oxide and
nitric oxide-dependent cGMP.
It is generally accepted that cAMP and cGMP triggers relaxation of smooth
muscle by activating an intracellular molecular cascade, which revolves around the
activity of cAMP-dependent protein kinase (PKA) or cGMP-dependent protein kinase
(PKG). This cascade results in a reduction of cytosolic Ca2+. Two mechanisms for
smooth muscle relaxation exist: the first is the reduction of intracellular calcium, and the
second is the reduction of the sensitivity of the contractile system to the calcium.
A. Reduction of intracellular calcium concentration.
Cyclic nucleotides trigger a reduction in cytosolic calcium through the activation
of cAMP-dependent protein kinase and/or cGMP-dependent protein kinase. Activated
kinase phosphorylates several key target proteins, including ion channels, ion pumps,
receptors, and enzymes, all involved in the control of intracellular Ca2+ concentration.
Phosphorylation of these target proteins reduces intracellular Ca2+ and results in
relaxation of smooth muscle.
Activation of calcium-activated K+ channels: Calcium-activated K+ channels
are a family of channel proteins expressed in multiple cell types, including
smooth muscle cells. The efflux of K+ through these channels induces
hyperpolarization leading to decrease in calcium influx via voltage-dependent
calcium channels. Phosphorylation by PKA or PKG increases the channel
activity resulting in hyperpolarization, which in turn decreases calcium influx via
voltage-gated calcium channels.
Direct inhibition of membrane calcium channel activity: Membrane voltagedependent calcium channels, which normally open in response to a membrane
depolarization, can also be regulated by cAMP- and cGMP-dependent protein
kinases. Phosphorylation of membrane calcium channels by PKA or PKG
decreases intracellular calcium, leading to relaxation of smooth muscle
Activation of calcium-ATPase pump in the sarcoplasmic reticulum (SR):
Calcium-ATPase pump activation is responsible for the uptake of calcium into the
SR, actively decreasing intracellular calcium and thereby relaxing smooth muscle.
The activity of calcium-ATPase pump in the SR is regulated by the protein
phospholamban. Phosphorylation of phospholamban by PKG increases calciumATPase pump activity and sequestration of calcium into the SR, thereby inducing
smooth muscle relaxation.
Inhibition of inositol 1,4,5-trisphosphate (IP3) generation: Because IP3 is the
principal compound responsible for releasing calcium from sarcoplasmic
reticulum; the inhibition of IP3 formation by PKA- or PKG-dependent
phosphorylation of phospholipase C-β is a potential mechanism of action of
cyclic nucleotide’s capacity to induce smooth muscle relaxation.
Inhibition of the IP3 receptor function (inhibition of calcium release): After
its generation by phospholipase C-β activity, IP3 acts as a second messenger for
smooth muscle contraction by binding to a specific receptor. The IP3 receptor is a
channel protein located in the sarcoplasmic reticulum, which opens up when
bound to IP3. The open IP3 receptor channel permits calcium release into the
cytoplasm, resulting in contraction of smooth muscle. Phosphorylation of IP3
receptor by PKA or PKG reduces the channel activity in response to IP3.
B. Reduction of contractile system calcium sensitivity (calcium desensitization).
Desensitization to calcium is defined as a decline in myosin light chain (MLC)
phosphorylation and force in the absence of proportional, or any, decline in cytosolic
calcium. The contractile state of smooth muscle is primarily dependent on the level of
MLC kinase and MLC phosphatase activity. Inhibition of MLC phosphatase could
produce contraction by increasing MLC phosphorylation in the absence of increased
intracellular calcium (calcium sensitization).
Alternately, an increase in MLC
phosphatase activity could produce relaxation by decreasing MLC phosphorylation
without changing intracellular calcium (calcium desensitization).
K+
Ca2+
Plasma
membrane
PLC-β
+
-
-
PKA/PKG
MLC
MLCK
-
+
+
IP3R
MLCPase
MLC-p
SR
Ca2+
Ca2+
Relaxation
Figure 9. Mechanisms of action of cAMP-dependent protein kinase (PKA) and
cGMP-dependent protein kinase (PKG) to mediate smooth muscle relaxation. The
kinases decrease cytosolic calcium levels by 1) inhibiting the activity of phospholipase Cβ (PLC-β), thereby diminishing the synthesis of calcium mobilizing messenger, inositol
1,4,5-trisphosphate (IP3), 2) inhibiting calcium release from sarcoplasmic reticulum
stores (inhibiting IP3 receptor function), 3) stimulating calcium uptake by activating
sarcoplasmic reticulum Ca2+-ATPase pump, and 4) inhibiting the activity of
plasmalemmal Ca2+ channels either directly or indirectly (by activating K+ channels).
Decreased cytosolic calcium inhibits myosin light chain kinase activity, thereby
decreasing MLC phosphorylation. PKA and PKG also decrease myosin light chain
phosphorylation by stimulating myosin light chain phosphatase activity.
MLCK: myosin light chain kinase, MLCPase: myosin light chain phosphatase, MLC-p:
phosphorylated myosin light chain, PKA: cAMP-dependent protein kinase, PKG: cGMPdependent protein kinase, PLC-β: phospholipase C-β, SR: sarcoplasmic reticulum, IP3R:
inositol 1,4,5-trisphosphate receptor, + denotes stimulation, - denotes inhibition.
Table 1.
muscle
Differences between smooth (unitary and multiunit) and skeletal
Feature
Smooth Muscle
Skeletal Muscle
Unitary
Multiunit
Thick and Thin
Filaments
Yes
Yes
Yes
Striations
No
No
Yes
T-tubule
No
No
Yes
Nucleus
Single
Single
Many
Innervation
Sparse
Dense
Each cell
Type of control
Involuntary
Involuntary
Voluntary
Excitation/
inhibition
Excitation/
inhibition
Excitation
only
Gap junctions
Many
Some
None
Spontaneous activity
Yes
No
No
Source of Ca2+
Influx/
release (SR)
Influx/
release (SR)
Release (SR)
Site of
action of Ca2+
Calmodulin
Calmodulin
Troponin
Speed of
contraction
Very slow
Very slow
Fast/slow
Effect of nerve
stimulation
Sample questions
1. Which of the following statements about smooth muscle is true?
(a)
(b)
(c)
(d)
(e)
Smooth muscle is striated and involuntary
Nuclei are peripherally located in the fibers
Fibers are small and spindle shaped
Branching fibers are a characteristic
Contractions are rapid and forceful
Answer: C
2. Unitary smooth muscles have numerous
(a)
(b)
(c)
(d)
(e)
Axon terminals
Z-lines
Gap junction
T-tubules
Intercalated discs
Answer: C
3. 17-kilodalton light chains are part of
(a)
(b)
(c)
(d)
(e)
Myosin molecule
Actin filaments
Caldesmon
Calponin
Tropomyosin
Answer: A
4. The role of calcium in smooth muscle contraction is to
(a)
(b)
(c)
(d)
(e)
Answer: B
Bind troponin and activate actomyosin interaction
Bind calmodulin and activate myosin light chain kinase
Bind directly to myosin light chain kinase
Inhibit myosin light chain phosphatase
Activate cAMP-dependent protein kinase
5. Which of the following events occur before inositol 1,4,5-trisphosphate (IP3)
generation in smooth muscle in the mechanism of excitation-contraction coupling?
(a)
(b)
(c)
(d)
(e)
Answer: E
Release of calcium from ryanodine receptors
Release of calcium from IP3 stores
Activation of myosin light chain kinase
Activation of cAMP-dependent kinase
G protein-dependent stimulation of PLC-β