The Endothelium Ca2 and Ca2 Sensitization

Our studies in rat small pulmonary arteries have shown that the relationship between [Ca2+]: and tension development during sustained HPV (2060 min) is not straightforward. Whereas [Ca2+]j (measured using Fura-2 simultaneously with tension) shows an initial transient rise followed by a raised but stable plateau, after an initial transient constriction, tension continues to rise progressively without any further change in [Ca2+]; (Fig. 1) (20, 26, 28). Moreover, removal ofthe endothelium does not alter the hypoxia-induced rise in [Ca2+]i( even though the sustained component of HPV is abolished (39). This strongly suggests, as we originally proposed in 1995 (26), that the endothelium is releasing a factor that increases the Ca2+ sensitivity of the VSM contractile apparatus, and the concept of modulation of sensitivity as an important component of HPV is now gaining acceptance (1, 7, 38). The reader is also directed to the chapters in this volume on the critical role of Ca2+ sensitization in HPV, by Robertson and McMurtry (Chapters 7 and 24).

Hpv Biphasic Response

Figure I: A typical experiment on a small intrapulmonary artery (~350 nm i.d.) from rat, showing simultaneous recordings of Po2, tension, and [Ca2+]( measured with Fura PE-3. The solid lines are from an experimental trace. PGF2o was used as a priming agent, and the level of pretone is shown as a dashed line; on reoxygenation both tension and [Caz+], fall back to the level of pretone. Hypoxia elicited a biphasic response, with a transient constriction and rise in [Ca2+]| superimposed on a slowly developing but sustained vasoconstriction and a stable rise in [Ca2+]j respectively. The transient component is absent in the present of 1 |iM La3+ or with use of 20 mM K+ as a priming agent; it is shown shaded in the figure. The effect of removal of the endothelium is shown as the dotted line; although the sustained component of tension is abolished, there is no effect on [Ca2+]j (line offset for clarity).

Regulation of Ca2+ sensitivity is a recognized mechanism that contributes to the vasoconstrictor response to a variety of agonists in smooth muscle (34). Ca2+ sensitivity is determined by the balance between Ca2+-calmodulin dependent phosphorylation of the myosin light chain (MLC) by myosin light chain kinase (MLCK), and its dephosphorylation by myosin phosphatase (SMPP-1M) (Fig. 2B) (34). Several protein kinases have been implicated in pathways leading to Ca2+ sensitization, including protein kinase C (PKC), Rho-associated kinases (ROK 1 and 2), and possibly MAP kinases.

4.1. Protein Kinase C

Activation of PKC isoforms has been long been recognized as leading to Ca2+-independent vasoconstriction in smooth muscle (including pulmonary vascular smooth muscle), although it is now believed that conventional and novel PKC isoforms play little role in at least G-protein mediated regulation of Ca2+ sensitivity (34). Several studies in isolated perfused lungs have suggested that inhibition of PKC suppresses HPV and hypoxia-mediated pulmonary hypertension (3, 41). However, in isolated small pulmonary arteries, the wide spectrum PKC inhibitor, Ro31-8220, had no effect on endothelium-dependent HPV, although it completely abolished phorbol ester induced pulmonary vasoconstriction (26). Note that PKC inhibition may affect the action of endogenous priming agents (Fig. 2).

4.2. PM MAP Kinase

Comparatively little work has been performed on the role of MAP kinases in HPV, though a recent study by Karamsetty et al. (16) shows that SB202190, an inhibitor of p38 MAP kinase, abolishes the sustained phase of HPV in isolated pulmonary arteries. Even though this effect appears to be relatively specific to the sustained component of HPV, it is not yet clear whether it relates to inhibition of the signal transduction pathways leading to Ca2+ sensitization, those linking the hypoxic sensor to Ca2+ mobilization in the vascular smooth muscle cells, or production of an endothelium-derived mediator in pulmonary arteries.

4.3. RhoA and Rho Associated Kinase

There has been considerable interest in the role of the monomeric G-protein RhoA and the inhibition of myosin phosphatase by its associated kinase, Rho kinase. It is now believed that this pathway is the major intermediate for regulation of Ca2+ sensitivity in smooth muscle (34). We have shown that an inhibitor of Rho kinase, Y-27632, selectively suppresses the sustained, but not transient, component of HPV in small pulmonary arteries, and abolishes the hypoxic pressor response in isolated perfused lungs at very low concentrations (27). Another study has also shown that Rho A and Rho kinase are both activated and are important for myosin light chain (MLC) phosphorylation during acute hypoxia-mediated pulmonary vasoconstriction (38). These results strongly suggest that RhoA and Rho kinase are a key component of the mechanisms underlying sustained and endothelium-dependent pulmonary vasoconstriction induced by acute hypoxia (Fig. 2B).

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