In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.Smooth muscle contraction is initiated by the phosphorylation of the regulatory myosin light chain (MLC 20 ) 1 by the calcium/calmodulin-dependent activation of the myosin light chain kinase (MLCK) (1). Relaxation is effected by the dephosphorylation of MLC 20 by the smooth muscle myosin phosphatase (SMMP). Complexity is brought to this system by accessory proteins and signaling pathways that regulate the smooth muscle contractile state (reviewed in Refs. 2 and 3). The SMMP is a target of signals that are positive and negative modulators of smooth muscle tone. SMMP is a heterotrimeric protein composed of the 37-kDa catalytic subunit (PP1c␦), the 130/133-kDa myosin targeting subunit (MYPT1, also referred to as MBS), and the 21-kDa M21 subunit (4 -6). MYPT1 targets the catalytic subunit to MLC 20 (7,8) and in this way confers substrate specificity to the phosphatase, whereas the function of the M21 subunit is unknown. Activation of the Rho kinase signaling pathway leads to phosphorylation of the MYPT1 subunit, resulting in inhibition of myosin phosphatase activity and an increase in smooth muscle tone (9 -13). This signaling pathway is thought to determine the calcium-sensitizing effect of ␣-adrenergic stimulation, for example, in which greater force is produced at a given calcium concentration th...

The control of force production in vascular smooth muscle is critical to the normal regulation of blood flow and pressure, and altered regulation is common to diseases such as hypertension, heart failure, and ischemia. A great deal has been learned about imbalances in vasoconstrictor and vasodilator signals, e.g., angiotensin, endothelin, norepinephrine, and nitric oxide, that regulate vascular tone in normal and disease contexts. In contrast there has been limited study of how the phenotypic state of the vascular smooth muscle cell may influence the contractile response to these signaling pathways dependent upon the developmental, tissue-specific (vascular bed) or disease context. Smooth, skeletal, and cardiac muscle lineages are traditionally classified into fast or slow sublineages based on rates of contraction and relaxation, recognizing that this simple dichotomy vastly underrepresents muscle phenotypic diversity. A great deal has been learned about developmental specification of the striated muscle sublineages and their phenotypic interconversions in the mature animal under the control of mechanical load, neural input, and hormones. In contrast there has been relatively limited study of smooth muscle contractile phenotypic diversity. This is surprising given the number of diseases in which smooth muscle contractile dysfunction plays a key role. This review focuses on smooth muscle contractile phenotypic diversity in the vascular system, how it is generated, and how it may determine vascular function in developmental and disease contexts.

Abundant protein expression is a characteristic feature of corneal keratocytes that is lost when cells are phenotypically modulated in culture. Greater light-scattering by myofibroblasts also provides support for a link between cellular transparency and haze after injury that is possibly related to loss of protein expression or development of prominent actin filament bundles.

Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general has resulted in the development of new techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs.

Abstract-Morphogenesis and developmental remodeling of cardiovascular tissues involve coordinated regulation of cell proliferation and apoptosis. In the heart, clear evidence points toward focal apoptosis as a contributor to development of the embryonic outflow tract, cardiac valves, conducting system, and the developing coronary vasculature. Apoptosis in the heart is likely regulated by survival and death signals that are also present in many other tissues. Cell type-specific regulation may be superimposed on general cell death/survival machinery through tissue-specific transcriptional pathways. In the vasculature, apoptosis almost certainly contributes to developmental vessel regression, and it is of proven importance in remodeling of arterial structure in response to local changes in hemodynamics. Physical forces, growth factors, and extracellular matrix drive vascular cell survival pathways, and considerable evidence points to local nitric oxide production as an important but complex regulator of vascular cell death. In both the heart and vasculature, progress has been impeded by inadequate information concerning the incidence of apoptosis, its relative importance compared with the diverse cell behaviors that remodel developing tissues, and by our primitive knowledge concerning regulation of cell death in these tissues. However, tools are now available to better understand apoptosis in normal and abnormal development of cardiovascular structures, and a framework has been established that should lead to considerable progress in the coming years. (Circ Res. 2000;87:856-864.)

Smooth muscle myosin phosphatase dephosphorylates the regulatory myosin light chain and thus mediates smooth muscle relaxation. The activity of this myosin phosphatase is dependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1 have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle section of chicken and rat MYPT1 genes revealed that each gene gave rise to isoforms by cassette-type alternative splicing of exons. In chicken, a 123-nucleotide exon was included or excluded from the mature mRNA, whereas in rat two exons immediately downstream were alternative. MYPT1 isoforms lacking the alternative exon were only detected in mature chicken smooth muscle tissues that display phasic contractile properties, but the isoform ratios were variable. The patterns of expression of rat MYPT1 mRNA isoforms were more complex, with three major and two minor isoforms present in all smooth muscle tissues at varying stoichiometries. Isoform switching was identified in the developing chicken gizzard, in which the exon-skipped isoform replaced the exon-included isoform around the time of hatching. This isoform switch occurred after transitions in myosin heavy chain and myosin light chain (MLC(17)) isoforms and correlated with a severalfold increase in the rate of relaxation. The developmental switch of MYPT1 isoforms is a good model for determining the mechanisms and significance of alternative splicing in smooth muscle.

MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.Force regulation in smooth muscle is dependent on the activities of myosin light chain (MLC) 1 kinase and MLC phosphatase (3, 4). The activity of MLC kinase is regulated by Ca 2ϩ -calmodulin (3), whereas MLC phosphatase was originally thought to be constitutively active and unregulated (5). However, there is abundant evidence that the activity of MLC phosphatase can be both inhibited to produce Ca 2ϩ sensitization (reviewed in Refs. 5-7) or an increase in force at a constant [Ca 2ϩ ] and enhanced to produce Ca 2ϩ desensitization (8) or a decrease in force at a constant [Ca 2ϩ ]. NO is the classical agent to produce Ca 2ϩ desensitization (1, 9, 10). Recent evidence (11) suggests that NO produces vasodilatation by activating the soluble pool of guanylate cyclase, which in turn produces cGMP and leads to the activation of type I cGMP-dependent protein kinase (PKGI). PKGI mediates smooth muscle cell relaxation by several mechanisms. It has been demonstrated that PKGI acts on the maxi K ϩ channel to produce hyperpolarization of the smooth muscle (12), decreases Ca 2ϩ flux (13,14), and also activates MLC phosphatase (1, 15) to decrease the level of MLC 20 phosphorylation and to produce smooth muscle relaxation. In addition, PKGI-dependent pathways for vasodilatation may also include phosphorylation of telokin (16, 17) and HSP20 (18).MLC phosphatase is a holoenzyme consisting of a catalytic subunit (PP1c␦); a myosin-binding subunit (MBS), which is also referred to as the myosin-targeting subunit (MYPT1); and a 20-kDa subunit of unknown function (5). The activation of MLC phosphatase by PKGI is hypothesized to be due to a leucine zipper-leucine zipper (LZ-LZ) interaction of the N-terminal LZ of PKGI␣ and the C-terminal LZ of the MBS of MLC phosphatase (1, 2). The MBS has four major isoforms, which are produced by alternative RNA splicing of two different exons (5). Tissue-specific and developmentally regulated alternative splicing of a 123-bp central exon produces a 41-amino acid central insert (19). Alternative splicing of the 31-bp 3Ј-exon is responsible for the expression of LZ ϩ or LZ Ϫ MBS isoforms (5). Specifically, exclusion of the 3Ј-exon shifts the reading frame of the MBS transcript to encode a C-terminal LZ (2).We previously demonstrated that sensitivity to cGMP-mediated relaxation correlates with the relative expression of LZ ϩ / LZ Ϫ MBS isoforms (2), which is consistent with the activation of MLC phosphatase activity resulting from a LZ-LZ interaction of PKGI␣ with the MBS (1). In this study, we tested the hypothesis that cGMP-dependent activation of MLC phosphatase activity and smooth muscle vasodilatation are due to a LZ-LZ interaction of PKGI␣ and MBS by changing the expression of the MBS isoform, in isolation, and determining the effect on cGMP-mediated MLC 20 dephosphorylation in primary cultured smooth muscle cells (SMCs). MATERIALS AND METHODSCloning of the Chicken MBS of the MLC Phosphatase cDNA Fragment-...

Myosin phosphatase is a target for signaling pathways that modulate calcium sensitivity of force production in smooth muscle. Myosin phosphatase targeting subunit 1 (MYPT1) isoforms are generated by cassette-type alternative splicing of exons in the central and 3' portion of the transcript. Exclusion of the 3' alternative exon, coding for the leucine zipper (LZ)-positive MYPT1 isoform, is associated with the ability to desensitize to calcium (relax) in response to NO/cGMP-dependent signaling. We examined expression of MYPT1 isoforms and smooth muscle phenotype in normal rat vessels and in a prehepatic model of portal hypertension characterized by arteriolar dilation. The large capacitance vessels, aorta, pulmonary artery, and inferior vena cava expressed predominantly the 3' exon-out/LZ-positive MYPT1 isoform. The first-order mesenteric resistance artery (MA1) and portal vein (PV) expressed severalfold higher levels of MYPT1 with predominance of the 3' exon-included/LZ-negative isoform. There was minor variation in the presence of the MYPT1 central alternative exons. Myosin heavy and light chain splice variants in part cosegregated with MYPT1 isoforms. In response to portal hypertension induced by PV ligature, abundance of MYPT1 in PV and MA1 was significantly reduced and switched to the LZ-positive isoform. These changes were evident within 1 day of PV ligature and were maintained for up to 10 days before reverting to control values at day 14. Alteration of MYPT1 expression was part of a complex change in protein expression that can be generalized as a modulation from a phasic (fast) to a tonic (slow) contractile phenotype. Implications of vascular smooth muscle phenotypic diversity and reversible phenotypic modulation in portal hypertension with regards to regulation of blood flow are discussed.