Shortening Of Smooth Muscle Cells Research Articles

Smooth muscle: a stiff sculptor of epithelial shapes.

Smooth muscle is increasingly recognized as a key mechanical sculptor of epithelia during embryonic development. Smooth muscle is a mesenchymal tissue that surrounds the epithelia of organs including the gut, blood vessels, lungs, bladder, ureter, uterus, oviduct and epididymis. Smooth muscle is stiffer than its adjacent epithelium and often serves its morphogenetic function by physically constraining the growth of a proliferating epithelial layer. This constraint leads to mechanical instabilities and epithelial morphogenesis through buckling. Smooth muscle stiffness alone, without smooth muscle cell shortening, seems to be sufficient to drive epithelial morphogenesis. Fully understanding the development of organs that use smooth muscle stiffness as a driver of morphogenesis requires investigating how smooth muscle develops, a key aspect of which is distinguishing smooth muscle-like tissues from one another in vivo and in culture. This necessitates a comprehensive appreciation of the genetic, anatomical and functional markers that are used to distinguish the different subtypes of smooth muscle (for example, vascular versus visceral) from similar cell types (including myofibroblasts and myoepithelial cells). Here, we review how smooth muscle acts as a mechanical driver of morphogenesis and discuss ways of identifying smooth muscle, which is critical for understanding these morphogenetic events.This article is part of the Theo Murphy meeting issue 'Mechanics of Development'.

Observation of Cell Shortening and Dynamic Changes of Actin Filaments during Cell Detachment from Thermoresponsive-Gelatin-Coated Substrate

We observed cell shortening and dynamic changes of actin filaments during detachment from the substrate by using a thermoresponsive-gelatin. The thermoresponsive-gelatin, a mixture of the poly(N-isopropylacrylamide) grafted gelatin (PNIPAAm-grafted-gelatin) and PNIPAAm, was coated on the glass bottom culture dishes. Rat aortic smooth muscle cells (SMC) expressed GFP-actin were cultured on the thermoresponsive-gelatin-coated dishes filled with serum free Dulbecco’s Modified Eagle’s medium (DMEM) or Ca2 + -Mg2 + -free Hank’s balanced salt solution (HBSS(-)). They adhered normally at 37°C, and became shortened and detached from the dishes when the ambient temperature was dropped below 34°C due to melting of the gelatin substrate. The shortening of SMCs was larger in DMEM (51.0 ±3.0%, mean ± SEM, n=14) than in HBSS(-) (35.8 ±3.6%, n=14). Actin filaments remained straight during detachment in HBSS(-), while in DMEM, they locally concentrated and disappeared at cell periphery. The shortening of SMCs upon detachment from ordinary plastic culture dishes by trypsinization was more than for both media. No significant difference was observed between the two, indicating detrimental effects of the trypsin. The present method was useful to study the cell contraction and the actin filament behavior during cell detachment for this causes minimal damage to cells.

Modulation of smooth muscle phenotype in vitro by homologous cell substrate.

We have developed a novel cell culture system that supports the shortening of smooth muscle cells. Primary rat airway smooth muscle cells were plated on an ethanol-fixed, confluent monolayer of homologous smooth muscle cells (homologous cell substrate, HCS). Cells grown on HCS exhibited morphological and functional characteristics consistent with a differentiated phenotype. Cells on HCS were spindle shaped with a well-defined long axis, whereas cells grown on glass were larger and irregularly shaped. Smooth muscle-specific alpha-actin immunostained diffusely in cells on HCS, whereas it appeared as stress fibers in cells on glass. Agonists recruited a greater fraction of HCS cells to contract, resulting in greater changes in cell area or length on average, but the maximal capacity of shortening of individual cells was similar between the groups. Unlike cells on glass, cells on HCS shortened to methacholine. HCS was reversible and persisted over several passages. Agonists stimulated intracellular Ca(2+) oscillations in cells on HCS, whereas they elicited biphasic peak and plateau transients in cells on glass. HCS modulates smooth muscle cell phenotype in vitro.

Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells.

Smooth muscle cells (SMC) were isolated from cat cerebral arteries and three sizes of pulmonary artery (less than 200-, 200- to 600-, and greater than 800-microns diameter) and used within 72-96 h. Change in cell length in response to hypoxia and other vasoactive agents was measured in a specially constructed cell chamber on an inverted microscope. Pulmonary artery SMC responded to hypoxia differently according to artery size. SMC from less than 200- and 200- to 600-microns-diameter pulmonary arteries shortened 18.6 +/- 4.85 and 24.2 +/- 2.70%, respectively. However, SMC from greater than 800-microns-diameter pulmonary arteries shortened 0.81 +/- 0.44%. Cerebral artery SMC plated on a flexible polydimethyl siloxane membrane showed loss of tension during exposure to hypoxia. The shortening of SMC from the 200- to 600-microns pulmonary arteries was accompanied by myosin phosphorylation. SMC from greater than 800-microns-diameter pulmonary arteries and cerebral arteries contained myosin that did not phosphorylate during hypoxia. The SMC from both artery types responded to norepinephrine, serotonin, prostaglandin F2 alpha and indomethacin and exhibited alpha-adrenergic receptor population patterns similar to those of intact arteries. The pattern of hypoxic responses exhibited by these nondedifferentiated pulmonary and cerebral artery SMC supports the idea that, at least in the cat, the hypoxic sensor is located within the SMC.

Unusual [Ca2+] dependence of vascular smooth muscle cell shortening velocity

The relationship between unloaded shortening velocity and Ca2+ concentration was determined for hog carotid arterial smooth muscle cells, freshly isolated by digestion with papain. Cells were exposed to various [Ca2+] for 60 s at 37 degrees C and then stimulated with 10 microM histamine. Cell length was measured by a video analysis system. Shortening velocity was expressed as an exponential rate constant by fitting the cell lengths to the following equation: length = Lmin + (Lmax-Lmin)exp[-v (time-latency)], where Lmax is length before contraction, Lmin is shortest length reached, time is time elapsed after addition of agonist, latency is time from addition of agonist until contraction starts, and v is the exponential rate constant (s-1). Cells shortened substantially, usually reaching one-fourth to one-third of their initial length within 1 min. Shortening velocities of the cells were much faster than published values of maximum shortening velocity in muscle strips from this same tissue. At 1.6 mM Ca2+, v was 0.173 +/- 0.015 s-1. When Ca2+ was increased to 5 or 10 mM, v was not significantly different. However, when Ca2+ was decreased to 0.5 and 0.16 mM, v increased to 0.288 and 0.258 s-1, respectively. The difference between 0.5 and 1.6 mM was significant. The unexpected increase in shortening velocity at low Ca2+ was also seen when 10 mM caffeine was used as a stimulus: v at 1.6 mM Ca2+ was 0.156 s-1, whereas v at 0.16 mM Ca2+ was 0.272 s-1. The high shortening velocities we measured suggest that measurements made on multicellular tissues seriously underestimate the potential shortening velocity of isolated individual cells.

Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria

In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, including the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.

Alterations of excitation-contraction coupling by platelet-derived growth factor in enzymatically isolated and cultured vascular smooth muscle cells

We studied stimulus-specific alterations of the excitation-contraction coupling pathway in freshly isolated contractile and subcultured non-contractile vascular smooth muscle cells. Using the calcium indicator aequorin, we detected physiological increases in cytoplasmic free calcium [( Ca2+]i) in subcultured smooth muscle cells subjected to angiotensin or 33 mM potassium depolarization. These increases were qualitatively identical to those previously measured in intact vascular strips. Platelet-derived growth factor (PDGF) induced a slow, sustained [Ca2+]i increase when applied to the subcultured smooth muscle cells at low picomolar concentrations. Freshly isolated, contractile vascular smooth muscle cells, prepared by a novel technique, exhibited a slow shortening of 20% of resting length in response to PDGF. PDGF also markedly potentiated smooth muscle cell shortening in response to an ED50 dose of phenylephrine. This effect was PDGF concentration dependent. The time course of shortening induced by PDGF alone was consistent with the time course of the PDGF-induced [Ca2+]i increase in the cultured smooth muscle cells. These data suggest that agonists which induce [Ca2+]i changes in contractile smooth muscle cells may retain this ability with respect to cultured smooth muscle cells. PDGF, a peptide mitogen for proliferative smooth muscle cells, may also serve to modulate vascular tone by modestly raising [Ca2+]i in contractile smooth muscle cell and, therefore, sensitizing the cells to alpha adrenergic agonists.

Occurrence of a contractile tissue in the theca externa of atretic follicles in the mouse ovary.

In mature mouse ovaries, smooth muscle cells (SMC) are as a rule concentrated in the perifollicular stroma of developing follicles but are also present in different ovarian regions. In growing antral follicles SMC form a conspicuous investment ('capsule') of concentric layers of stretched and elongated elements located in the zones of the theca externa. On the contrary, in antral follicles reaching approximately the same growth phase but with clear signs of atresia, the contractile capsule of the follicles has an irregular outline due to an apparent dislocation and shortening of SMC. The ultrastructural findings show that in atretic follicles the majority of the cells forming this capsule possess the cytological characteristics of SMC fixed in contraction. The occurrence of compressed SMC in the wall of atretic follicles suggests that some relationship exists between follicle contraction and the atretic process. In the case of the follicle at ovulation, it may be suggested that the activation of such perifollicular contractile tissue, even if incapable of producing alone the dehiscence of the follicle, may nevertheless be capable of squeezing the follicular wall. Then, if other factors do not simultaneously occur to cause a normal ovulatory process, the contraction of this tissue, instead of assisting in the rupture of the follicle, may favor its collapse and subsequent involution during atresia.