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Chapter 6: MOLECULAR AND CELLULAR BIOLOGY OF BLOOD VESSELS PHYSIOLOGY OF THE VASCULAR SMOOTH MUSCLE CELL

The smooth muscle cell normally responds to hormonal stimulation with contraction or relaxation. In certain disease states, however, growth and/or hypertrophy and migration to the intima are the predominant responses. Some of the biochemical signals generated by these vasoactive agonists are similar for both types of responses, with the final physiologic response dictated by the phenotype and environment of the cell, and the exact biochemical pathways activated. Thus, in normal arteries, growth factors can act as vasoconstrictors41 while, in modulated smooth muscle cells, vasoconstrictors can stimulate hypertrophy or hyperplasia.42

Second Messengers Traditionally Associatedwith Contraction

Some of the earliest signals generated within the cell following stimulation with calcium-mobilizing vasoactive agonists involve hydrolysis of a specific class of membrane lipids: the phosphoinositides.43 There are three major inositol phospholipids in the plasma membrane that serve as substrates for a class of enzymes called phospholipase Cs. Phospholipase C cleaves phospholipids to liberate the water-soluble head group and the lipophilic molecule, diacylglycerol

Fig. 6-2). The water-soluble head group that is most important for signal generation is inositol trisphosphate (IP3), which has been shown to release Ca2+ from intracellular stores.44 Ca2+, in turn, activates a cascade of enzymes leading to contraction or growth (see below). Diacylglycerol is a potent activator of protein kinase C, a Ca2+- and phospholipid-dependent enzyme that phosphorylates numerous cellular proteins.45 Diacylglycerol can be further metabolized to phosphatidic acid or to glycerol, fatty acids, and, ultimately, eicosanoids and leukotrienes that may themselves modulate tone. Additionally vasoconstrictor agents cause a sustained intracellular alkalinization46 and an influx of extracellular Ca2+,47 both of which serve to sustain and enhance vasoconstriction.

Contraction Cascade

Contractions induced by various vasoactive hormones differ not only in magnitude and time course, but also differ between vessels. In general, there is an initial, rapid component of force generation and a more sustained phase of contraction. Some agonists, such as angiotensin II, induce only a transient constriction of many vessels, whereas others, including norepinephrine and vasopressin, nearly always cause a sustained contraction. The initial phase of force development has been shown to depend on the formation of actin-myosin cross-bridges, but the mechanisms underlying the sustained phase of contraction are less clear.

A sliding-filament mechanism similar to that found in skeletal muscle is thought to regulate phasic contraction of smooth muscle. Force generation is accomplished by attachment of the myosin heads (or cross-bridges) to actin filaments. This attachment catalyzes ATP hydrolysis to generate tension and occurs in a cyclic manner for the duration of the stimulus. Smooth muscle has a relatively greater content of actin and a lower content of myosin than does skeletal muscle and, in contrast to skeletal muscle, the major site of calcium regulation of smooth muscle actomyosin is on the myosin molecule. Smooth muscle myosin consists of two large subunits, each with a molecular weight of 200 kDa, and two small subunits of 20 and 16 to 17 kDa, known as the myosin light chains. Force generation in smooth muscle is regulated by the phosphorylation/dephosphorylation of the 20-kDa protein (Fig. 6-3). Once phosphorylation occurs, actin-activated Mg2+ATPase activity is stimulated, resulting in cross-bridge cycling. Myosin light chain phosphorylation is mediated by an enzyme known as myosin light chain kinase (MLCK). This protein associates with calmodulin, a calcium-binding protein required for activation of numerous cytoplasmic enzymes. Thus, when Ca2+ increases within the cell in response to hormonal stimulation, it binds to calmodulin, which, in turn, associates with MLCK, converting it from an inactive to an active form. MLCK then phosphorylates the myosin light chain, enabling actin activation of the Mg2+-ATPase ultimately resulting in cross-bridge formation. When the intracellular Ca2+ concentration drops below about 100 nM, Ca2+ dissociates from calmodulin, calmodulin detaches from MLCK, and MLCK becomes inactive. Myosin light chain phosphatase activity then predominates, myosin is dephosphorylated, and cross-bridge cycling ceases. During sustained contraction, however, the intracellular Ca2+ concentration is low, and energy consumption is reduced, suggesting the development of a latch-bridge, or of a low cycling state.48 Alternatively, the sensitivity of the contractile apparatus to Ca2+ may be increased, a response posited to be regulated by protein kinase C.49

Healthy Coronary Artery

Figure 6-3: Contraction cascade. Activation of smooth muscle by a vasoconstrictor hormone leads to a cascade of biochemical signals, ultimately resulting in phosphorylation of actomyosin, cross-bridge formation, and force generation. The release of Ca2+ from intracellular stores is one of the major initiating events, since Ca2+ combines with calmodulin to activate myosin light chain kinase. This enzyme phosphorylates the myosin light chain, which is then able to interact with actin. Abbreviations: R = receptor; PLC = phospholipase C; DG = diacylglycerol; PIP2 = phosphatidylinositol 4,5-bisphosphate; IP3 = inositol trisphosphate; CaM = calmodulin; MLCK = myosin light chain kinase; MLC = myosin light chain; P = phosphate. (Courtesy of Bernard Lassegue, Ph.D.)

Figure 6-3: Contraction cascade. Activation of smooth muscle by a vasoconstrictor hormone leads to a cascade of biochemical signals, ultimately resulting in phosphorylation of actomyosin, cross-bridge formation, and force generation. The release of Ca2+ from intracellular stores is one of the major initiating events, since Ca2+ combines with calmodulin to activate myosin light chain kinase. This enzyme phosphorylates the myosin light chain, which is then able to interact with actin. Abbreviations: R = receptor; PLC = phospholipase C; DG = diacylglycerol; PIP2 = phosphatidylinositol 4,5-bisphosphate; IP3 = inositol trisphosphate; CaM = calmodulin; MLCK = myosin light chain kinase; MLC = myosin light chain; P = phosphate. (Courtesy of Bernard Lassegue, Ph.D.)

Biochemical Signals Traditionally Associatedwith Proliferation

Classic growth factors, such as platelet-derived growth factor (PDGF), activate many of the same signaling pathways as do vasoconstrictors: phosphoinositide hydrolysis, Ca2+ mobilization and influx, Na+/H+ exchange and intracellular alkalinization. Receptors for these growth factors are intrinsic tyrosine kinases, leading to the tyrosine phosphorylation of numerous proteins that are essential for growth. The importance of tyrosine phosphorylation in mediating the growth response is shown by the observation that mutant PDGF receptors, which lack the normal, intrinsic tyrosine kinase domain, are incapable of mediating proliferation in response to PDGF.50 In addition, tyrosine kinase inhibitors have been shown to inhibit growth.5! There is also increasing evidence that tyrosine phosphatases can counteract the mitogenic effects of growth factors by inhibiting tyrosine phosphorylation of specific substrates.52

A complex of substrates becomes associated with activated growth factor receptors and subsequently activates multiple signaling cascades leading to the final cellular response.53 Some proteins, such as phospholipase C-g, the tyrosine kinase c-Src, and phosphatidylinositol-3-kinase, bind directly to receptor tyrosine kinases, whereas others, including the tyrosine kinase Pyk-2 and the cytoskeletal protein paxillin, associate with the receptor via linker proteins such as Grb and Shc. Upon addition of growth factors, the receptors dimerize and auto-tyrosine phosphorylate, and each of the aforementioned proteins is phosphorylated on tyrosine, presumably leading, either directly or indirectly via association with the activated receptor, to their activation. In addition, Shc and Grb2 link these receptors to Ras, a ubiquitous GTPase that initiates a kinase cascade that includes mitogen-activated protein kinase (MAP kinase) and ultimately leads to growth. Recent evidence suggests that many of these proteins are also activated by seven-transmembrane-spanning G-protein-coupled receptors,54 an observation that may partially explain the growth-promoting properties of some vasoconstrictor hormones.

An additional pathway that is activated under some conditions by growth factors and vasoactive agonists is phospholipase D-mediated hydrolysis of plasma membrane phosphatidylcholine.55 In this reaction, phosphatidic acid and choline are released. This pathway is receiving increasing attention because phosphatidic acid may have a role in mediating the growth response56 and because phospholipase D activation seems to be required for the proliferative response.57

Growth

Vascular smooth muscle cell growth occurs via two processes: hypertrophy and hyperplasia. In general, hypertrophy occurs in response to long-term stimulation with vasoconstrictor-type agents, whereas hyperplasia occurs in response to the classic growth factors. Hypertrophy is characterized by an increase in smooth muscle cell mass due to increased protein synthesis and has been shown to occur in response to angiotensin II58 and thrombin59 and in large vessels during hypertension. Hyperplasia is characterized by cell replication and is stimulated by growth factors such as PDGF and fibroblast growth factor (FGF)60-62 following vascular injury. The biochemical processes leading to hypertrophy and hyperplasia are currently under investigation. It is clear that the aforementioned tyrosine kinase pathways are important in both types of growth.53,54 In addition, generation of reactive oxygen species, including superoxide and hydrogen peroxide, serves to transduce the growth signal by activating specific proteins such as p38 mitogen-activated protein kinase and Akt/protein kinase B.6364

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Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

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