Ca2 Sensitization and Vascular Smooth Muscle Contraction

Vascular smooth muscle tone is determined primarily by the phosphorylation/ dephosphorylation ratio of MLC20, which in turn is regulated by the relative activities of MLCK and myosin phosphatase (MLCP, also known as SMPP-1M) (49). Increases in the activity of MLCK or decreases in MLCP activity will therefore increase MLC20 phosphorylation and contraction. Ca2+-

sensitization can therefore be due to Ca2+-independent activation of MLCK, or Ca2+-independent inhibition ofMLCP. Although the former has been observed in isolated kinase studies (26), there is no evidence that Ca2+-independent activation of MLCK occurs in intact arteries. However, the agonist-induced inhibition ofMLCP leading to increased Ca2+-sensitivity and contraction was first proposed over a decade ago (48), and it has become apparent that this is a key mechanism of Ca2+-sensitization in vascular smooth muscle (50). MLCP is comprised of regulatory and catalytic (PP-1C) domains coupled to a third subunit ofyet identified function, and can be regulated by several intracellular enzymes (11). The pathways to be discussed in this chapter that may lead to inhibition of MLCP during HPV are detailed in Figure 1.

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Figure 1. Putative pathways mediating Ca2+-sensitization during HPV. Ca2+sensitization in vascular smooth muscle is thought to be primarily mediated by inhibition of MLCP. Hypoxia, possibly via an endothelium-derived constricting factor (EDCF), elicits activation of Rho-kinase by RhoA. In its cytosolic (inactive) form, GDP-bound RhoA is complexed with RhoGDI (Rho-associated guanine nucleotide dissociation inhibitor). Upon agonist stimulation, RhoA is activated by Rho guanine nucleotide exchange factors that cause the replacement of GDP by GTP, in turn causing dissociation of RhoA from RhoGDI. Activated RhoA then translocates to the plasma membrane whereupon it activates Rho-kinase. Rho-kinase inhibits MLCP (myosin light chain phosphatase) by phosphorylating the regulatory subunit (MBS). However, it is unclear whether a direct interaction occurs in intact arteries; it is possible that Rho-kinase inhibits MLCP indirectly via phosphorylation of CPI-17. When phosphorylated, CPI-17 is a potent inhibitor of the catalytic subunit (PP-1C) ofMLCP. PKC has also been shown to phosphorylate CPI-17. PKC may be an upstream regulator of Rho-kinase as PKC has been shown to phosphorylate RhoGDI, in turn, resulting in activation of RhoA. Inhibition of MLCP results in a net increase of MLC20 phosphorylation and contraction. An increase in Ca2+-sensitivity results in a leftward shift in the tension/[Ca2+]| curve (inset). The pathways that may mediate PTK and p38 MAP kinase associated Ca2+-sensitization are yet to be determined. M20, small non-catalytic subunit.

3. Ca2+ Sensitization in Acute HPY

3.1. Acute HPV: A Transient or Sustained Constrictor Response?

HPV is a homeostatic mechanism within the lung. Since HPV occurs in isolated arteries, it is apparent that the sensor and effector mechanisms reside in the pulmonary artery smooth muscle and/or endothelium. In vivo and in isolated blood-perfused lungs, the pressor response to acute hypoxia is usually immediate in onset and sustained. In contrast, HPV in isolated arteries is often biphasic, consisting of an immediate transient constrictor response superimposed on a more slowly developing, sustained contraction (22). It has been proposed that the latter response is the more physiologically relevant process, since it is sustained. Indeed, when considering the role of HPV in the normal lung, it would appear important that HPV be a sustained response. It is apparent that HPV responds to localized alveolar hypoxia, thereby diverting blood flow to better oxygenated areas of the lung and maintaining ventilation/perfusion matching. If HPV were transient, then the re-direction of blood flow would also be transient, and ventilation/perfusion matching would not be maintained, presumably promoting hypoxemia, which does not seem to occur in vivo. Conversely if HPV is sustained, the re-direction of blood flow in response to localized hypoxia is maintained for the duration of the hypoxic episode and optimal ventilation/perfusion matching is maintained.

3.2. Ca2+ Sensitization During Acute HPV

The first studies designed to determine the relationship between [Ca2+]j and tension development during acute HPV were performed in Dr. Jeremy Ward's laboratory in the early 1990's (34, 40). In these experiments, the Ca2+-sensitive fluorophore Fura-2 was used to monitor levels during HPV in rat isolated small intrapulmonary arteries mounted in small vessel myographs to simultaneously measure tension development. The experimental record from the first ofthese experiments in shown in Figure 2. The contractile response to acute hypoxia in rat intrapulmonary arteries is biphasic. During the initial transient constriction there is a simultaneous transient increase in [Ca2+]j. However, the pivotal result from these experiments was the demonstration of a dissociation between tension and [Ca2+]j during the sustained constriction. Specifically, subsequent to the transient increases in [Ca2+]j and tension, [Ca2+]j remained constant (at a level higher than that prior to the induction of hypoxia) whereas tension increased for the remainder of the hypoxic challenge. Upon re-oxygenation, both vascular smooth muscle tension and [Ca2+]j returned to their pre-hypoxic levels.

Hypoxia

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Hypoxia

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Figure 2. Sustained HPV involves Ca2+ sensitization. Record of [Caa+]( changes (F34(/F380) and tension in a Fura-2 loaded rat intrapulmonary artery during acute hypoxia. In the presence of a small degree of agonist-induced tone (3 |xM prostaglandin F2a, PGF2a), hypoxia elicited a transient increase in both [Ca2+]j and tension. However, whereas tension progressively increased for the remainder of the hypoxic challenge, [Ca2+]; remained at a constant level.

Figure 2. Sustained HPV involves Ca2+ sensitization. Record of [Caa+]( changes (F34(/F380) and tension in a Fura-2 loaded rat intrapulmonary artery during acute hypoxia. In the presence of a small degree of agonist-induced tone (3 |xM prostaglandin F2a, PGF2a), hypoxia elicited a transient increase in both [Ca2+]j and tension. However, whereas tension progressively increased for the remainder of the hypoxic challenge, [Ca2+]; remained at a constant level.

In subsequent work, it became apparent that the use of Fura-2 (and the related compound Fura-PE3) to measure [Ca2+]j during hypoxia might overestimate the rise in [Ca2+]j (21). Hypoxia increases NAD(P)H levels in pulmonary arteries, as determined by monitoring the ratio of autofluorescence in isolated pulmonary arteries when excited at 340 and 380 nm, respectively (21, 43). Coincidentally, these are the same excitation wavelengths employed when using Fura-2 or Fura-PE3 as Ca2+-sensitive fluorophores. Therefore, the increase in fluorescence ratio observed in isolated arteries during HPV when using these probes will be due partly to the concomitant increase in NAD(P)H levels. Leach and co-workers (2001) calculated that this amounted to -25% of the increase in fluorescence ratio during HPV, indicating that the rise in [Ca2+]| during sustained HPV may be less and, in turn, that the dissociation between tension and [Ca2+]( during sustained HPV greater than that reported previously using similar techniques (34). The dissociation between [Ca2+]j and tension during sustained HPV can be readily explained by Ca2+ sensitization.

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