Vascular Smooth Muscle Contraction Nancy L. Kanagy The University of New Mexico, Albuquerque, USA Stephanie W. Watts Michigan State University, East Lansing, USA ã 2007 Elsevier Inc. All rights reserved.
In general, all types of muscle depend on the calcium-dependent interaction of actin and myosin filaments to generate in contraction. However, the regulation of smooth muscle contraction differs from cardiac or skeletal muscle contraction. In addition, intermediate filaments are uniquely present in smooth muscle cells to provide cytoskeletal structure for the cell. The actual shortening of smooth muscle occurs in a manner similar to that of skeletal and cardiac muscle, with the movement of thin actin filaments along myosin thick filaments. This is mediated by the formation of cross-bridges between myosin head groups and actin groups in the thin filaments. When the two are attached, the myosin head moves from a ‘‘cocked’’ to a relaxed position, pulling the thin filament along. The head then disengages and moves back to the cocked position by an ATP-dependent mechanism. The energy for the head movement and actin binding (‘‘cross-bridge cycling’’) is generated by the hydrolysis of ATP via an ATPase intrinsic to the myosin head (Fig. 1). In smooth muscle, the rate of ATP hydrolysis, and hence the movement of the filaments, is regulated primarily by the concentration of intracellular calcium. In smooth muscle, the enzyme myosin light chain kinase (MLCK) is activated by calcium-bound calmodulin to phosphorylate myosin light chain 20 (MLC20) and initiate contraction. Therefore, regulation is through phosphorylation of the heavy chain myosin. This is in contrast to skeletal and cardiac muscle where intracellular calcium regulates Myosin Light Chain Kinase C binding to the light chain to regulate contraction. The myosin isoform in smooth muscle differs from that in skeletal muscle and only binds actin if the myosin light chain is phosphorylated. In fact, smooth muscle does not express troponin C.
Fig. 1. Model depicting smooth muscle cell contractile filaments.
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Filaments/structure of smooth muscle cell Calcium plays a central role in regulating arterial and venous smooth muscle contraction. A rise in cellular calcium levels can be elicited by either the release of intracellular calcium from the sarcoplasmic reticulum or by calcium moving into the cell from the extracellular environment through the L-type voltage-gated calcium channels. The increase in intracellular calcium activates both MLCK and other intracellular signaling pathways. In addition to increased intracellular calcium, many contractile agents regulate myosin light chain phosphorylation through the regulation of myosin light chain phosphatase (MLCP). This enzyme removes the phosphate from myosin light chain ending contraction. Contraction is therefore initiated by intracellular signaling pathways that increase intracellular calcium levels to activate MLCK and by pathways that inhibit MLCP.
Electromechanical modulation of cytosolic calcium concentrations Calcium channels, regulated by voltage, serve as a primary source for allowing calcium to enter the cytosol from the extracellular space down its concentration gradient. Extrusion mechanisms in the plasma membrane and storage sites (sarcoplasmic reticulum, mitochondria) keep free cytosolic calcium levels below 100 nM in the resting cell to maintain a large concentration gradient across the cell membrane. In smooth muscle cells, two calcium channel subtypes are most responsible for allowing extracellular calcium to enter in the cell.
The L-type voltage gated calcium channel The open probability of L-type voltage gated calcium channels (also known as Cav1.1Cav 1.4) is increased with membrane depolarization. The activation threshold is relatively high at about-35mV, well above the normal resting membrane potential of vascular smooth muscle cells (about-45 to-60 mV) Caterall (2000). Therefore, few L-channels are open in resting cells. Upon membrane depolarization, channels open and calcium flows into the cytosol. The channel remains open for a relatively long time (hence the name of L-type, or long-type). The channel closes with a moderate rate of inactivation and a fast rate of deactivation. A direct agonist of the L-type voltage gated calcium channel is BayK8644. Antagonists of this channel are numerous and are represented by three different chemical classes: the phenylalkylamines, e.g., verapamil; the dihydropyridines, e.g., nifedipine, nitrendipine, and nisoldipine; and the benzothiazapines, e.g., diltiazemTriggle (1999). Therapeutically, L-type voltage gated calcium channel antagonists are used in the treatment of hypertension, a condition in which arterial smooth muscle tone is inappropriately high.
The T type calcium channel While L-type voltage gated calcium channels are clearly the primary channel type, the Ttype calcium channels (known as Cav3.1-Cav3.3) represent between 10-15% of the calcium channels in smooth muscle cells. In contrast to L-channels, T-channels are low voltage activated (activation threshold of-45 mV), have fast inactivation and slow deactivation. While there are no selective T-type channel agonists, mibefradil, nickel, and flunarizine inhibit these sites. Calcium can also be increased in cells by exposing them to the ionophore A23187. This compound inserts pores in the plasma membrane allowing calcium to enter the cell down its concentration and electrical gradient. This compound
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is useful for increasing intracellular calcium levels independent of channel or receptor stimulation. Pharmacomechanical modulation of cytosolic calcium concentrations When stimulated by agonists, both heptahelical and growth factor type receptors in the plasma membrane can activate L-type voltage-gated calcium channels and allow calcium to enter the cell. In addition, heptahelical receptors coupled to G proteins, such as Gq, G11, Gz, and Gi, activate phospholipase C, resulting in the hydrolysis of phosphotidylinositol to diacylglycerol and inositol 1,4,5 trisphosphate (InsP3). InsP3 interacts with InsP3 receptors in the membrane of the sarcoplasmic reticulum to release stored calcium. Sarcoplasmic reticulum calcium stores are also liberated by activation of cyclic ADP ribose (ryanodine-sensitive) receptors. These stores are rapidly depleted, however, and sustained contraction requires calcium influx from the extracellular space. Sarcoplasmic reticulum stores are repleted by ATP-driven calcium pumps, which sequester calcium that is released along with calcium that enters the cell through membrane channels. Calcium can also flow into the cell by way of receptor-operated calcium channels (ROCC) and store-operated calcium channels (SOCC).
Currently, there are 4 accepted mechanisms of vascular smooth muscle contraction
1. Myosin Light Chain Kinase (MLCK) dependent contraction This process was the first described and is considered the basic mechanism for smooth muscle contraction. When intracellular levels of calcium increase, calmodulin binds calcium with high affinity, is activated and, in turn, activates MLCK, the kinase responsible for phosphorylating one of the light chains attached to the myosin head, myosin light chain 20 (MLC20). When MLC20 is phosphorylated, the rate of ATP hydrolysis by myosin is elevated 100-fold, increasing the rate of interaction between the myosin head and the
Fig. 2 Myosin Light Chain Kinase (MLCK) dependent contraction.
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actin thin filaments. Myosin light chain is dephosphorylated by myosin light chain phosphatase (Fig. 2).
2. Protein kinase C (PKC)-dependent (MLCK dependent and independent) contraction Several different forms of PKC are expressed in vascular smooth muscle Andrea and Walsh (1992)Rassmussen et al (1987). Several studies suggest that activation of smooth muscle contraction through a calcium and MLCK dependent process may not be the only way for arterial contraction to occur. Specifically, it has been hypothesized that PKC may be able to phosphorylate myosin light chain to regulate cross-bridge cycling independent of intracellular calcium concentrations. PKC may also inhibit the phosphatase activity of myosin light chain phosphatase and activate raf, a protein in the extracellular signal regulation kinase (Erk) mitogen activated protein kinase (MAPK) pathway, thereby promoting contractility Khalil and Morgan (1993).
3. Rho kinase-dependent contraction The phosphorylation of MLC20 regulates actin-myosin cross-bridge cycling. Rho kinase causes phosphorylation and inhibition of myosin light chain phosphatase (MLCP) to promote contraction Solaro (2000)Gohla et al (2000). MLCP contains three subunits, one of which is called myosin binding site (MBS) peptide. When MBS is serine and threonine phosphorylated, the activity of the MLCP holoenzyme is inhibited. The phosphoryation of MBS is stimulated by Rho-kinase, inactivating MLC P. Rho kinase activity can therefore increase contraction at any given calcium concentration. Rho kinase can be inhibited by Y27623 with resulting increased MLCP activity Ishizaki et al (2000), Fu et al (1998). This, in turn, increases the rate of myosin light chain dephosphorylation and promotes smooth muscle relaxation.
4. Tyrosine kinase dependent contraction In the past 5-10 years it has become clear that activation of pathways dependent on tyrosine kinases, including mitogen activated protein kinase (MAPK) pathways, participate in modulating arterial tone. This includes both the extracellular regulated signal kinase (Erk) MAPK and p38 MAPKHollenberg (1994)Yamboliev et al (2000). Studies by Adam and Hathaway have revealed that caldesmon, a thin filament protein is a substrate for the Erk MAPK proteins Adam et al (1989), Adam et al (1992), Katsuyama et al (1992). Caldesmon inhibits the actinomyosin ATPase that regulates actin/myosin cross-bridge cycling and contraction. Unphosphorylated caldesmon binds tightly to the actin filament exerting this inhibition. When caldesmon is phosphorylated, the binding is diminished and actinomyosin ATPase activity increases, augmenting contraction. Studies are underway to determine whether MAPK alters contractility independently of caldesmon.
Relaxation of smooth muscle Generally, smooth muscle can be relaxed by blocking calcium entry through plasma membrane channels, by blocking release of intracellular calcium, by modulating the level of myosin light chain phosphorylation, or by promoting the production of cyclic AMP and cyclic GMP Ignarro and Kadowitz (1985).
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Journal Citations Adam, L.P., Haeberle, J.R., Hathaway, D.R., 1989. Phosphorylation of caldesmon in arterial smooth muscle. J. Biol. Chem., 264, 7698–7703. Adam, L.P., Gapinski, C.J., Hathaway, C.R., 1992. Phosphorylation sequences in h-caldesmon from phorbol ester stimulated canine aortas. FEBS Lett., 302, 223–226. Andrea, J.E., Walsh, M.P., 1992. Protein kinase C of smooth muscle. Hypertension, 20, 585–595. Caterall, W.A., 2000. Structure and regulation of voltage-gated Ca2+ channels. Ann. Rev. Cell Dev. Biol., 16, 521–555. Fu, X., Gong, M.C., Jia, T., Somlyo, A.V., Somlyo, A.P., 1998. The effects of the rho-kinase inhibitor Y-27632 on arachidonic acid-, GTPgs and phorbol ester-induced Ca2+ sensitization of smooth muscle. FEBS Lett., 440, 1843–1847. Gohla, A., Schultz, G., Offermanns, S., 2000. Role for G12/G13 in agonist-induced vascular smooth muscle contraction. Circ. Res., 87, 221–227. Hai, C.-M., Murphy, R.A., 1989. Ca2+, crossbridge phosphorylation and contraction. Ann. Rev. Physiol., 51, 285–298. Hollenberg, M.D., 1994. Tyrosine kinase pathways and the regulation of smooth muscle contractility. Trends Pharmacol. Exi., 15, 108–114. Ignarro, L.J., Kadowitz, P.J., 1985. The pharmacological and physiological role of cycli GMP in vascular smooth muscle relaxation. Ann. Rev. Pharmacol. Toxicol., 25, 171–191. Ishizaki, T., Uehata, M., Tamechika, I., Keel, J., Nonomura, K., Maekawa, M., Narumiya, S., 2000. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol. Pharmacol., 57, 97–983. Katsuyama, H., Wang, C.-L.A., Morgan, K.G., 1992. Regulation of vascular smooth muscle tone by caldesmon. J. Biol. Chem., 267, 14555–14558. Khalil, R.A., Morgan, K.G., 1993. PKC-mediated redistribution of mitogen activated protein kinase during smooth muscle cell activaton. Am. J. Physiol., 265, 401–C411. Rassmussen, H., Takuwa, Y., Park, S., 1987. Protein kinase C in the regulation of smooth muscle contraction. FASEB J., 1, 177–185. Solaro, R.J., 2000. Myosin light chain phosphatase: a cinderella of cellular signaling. Circ. Res., 87, 173–175. Triggle, D.J., 1999. The pharmacology of ion channels; with particular reference to voltage gated calcium channels. Eur. J. Pharmacol., 375, 311–325. Yamboliev, I.A., Hedges, J.C., Mutnick, J.L.-M., Adam, L.P., Gerthoffer, W.T., 2000. Evience for modulation of smooth muscle force by the p38 MAPK kinase/HSP27 pathway. Am. J. Physiol., 278, 1899–H1907.
Further Reading Berne and Levy, Cardiovascular Pharmacology, Mosby-Year Book, Inc., Edition 8, 1997. Garland, C. J. and Angus, J. A., Pharmacology of Vascular smooth Muscle, Oxford University Press, Oxford, 1996. Barany, Michael. Biochemistry of smooth muscle contraction. Academic Press, Inc., San Diego, C A. 1996.