Embryonic Chicken Gizzard Research Articles

Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (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). Alternative splicing of a 3'-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p<0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.

Determinants of the contractile properties in the embryonic chicken gizzard and aorta.

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening (V(max)) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V(max) and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC(17a)). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103-110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V(max) in intact tissues. In the current study, differences in V(max) did not correlate with previously published differences in MHC or MLC(17a) isoforms. Rather, V(max) was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)) in the gizzard, suggesting that a significant internal load exists. Furthermore, V(max) in the gizzard increased during postnatal development without changes in MHC or MLC(17) isoforms. Although the rate of MLC(20) phosphorylation was similar at ED 20, the rate of MLC(20) dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC(17) isoform expression marks the phasic contractile phenotype.

Differentiation of smooth muscle cells from undifferentiated cells of chicken gizzard occurs on the layer of fibroblast-like cells.

We examined the in vitro differentiation of smooth muscle cells from undifferentiated cells of embryonic chicken gizzard. We used the gizzard from 7-day-old embryos (Hamburger and Hamilton's stage 26-28) as a source of culture, since the expression of myosin is extremely low, and the gizzard consists of round cells, which are not stained by anti-smooth muscle myosin antiserum. When the dissections of the gizzard were cultured, they attached to the dish, and fibroblast-like cells migrated within 4 days after the culture. Then round cells migrated from the transplants over the layer of fibroblast-like cells. At 12-14 days after the culture, smooth muscle cells, which were ribbon-shaped and stained by anti-smooth muscle myosin antiserum, appeared in the layer of round cells. The dissected transplant itself was not stained by anti-smooth muscle myosin antiserum even after being cultured for 15 days. We concluded then that the smooth muscle cells were differentiated from the round cells, which spread on the layer of the fibroblast-like cells. We also observed the differentiation of smooth muscle cells when the cells separated from 7-day-old embryo cultured on the layer of cloned fibroblast cells. We suggest that the fibroblast-like cell may play an important role in the differentiation of smooth muscle cells.

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, alpha-actin-in and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryo-specific isoform with a different molecular mass was detected.

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M(r) of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.

Embryonic chicken gizzard: immunolocalization of collagen and smooth muscle myosin.

Antibodies to chicken gizzard myosin and to chicken skin collagen type I allow the myofibrillar and connective tissue development in the embryonic chicken gizzard to be followed. Fibroblasts are assumed to synthesize collagen prior to the onset of smooth muscle cell development in the muscle primordium (day 5); they are presumably also responsible for collagen synthesis close to the presumptive lamina propria and in the developing tubular glands (day 14 to 17). From day 6 to 8, myosin and collagen are colocalized intracellularly, and from day 9 onward collagen fibers start to appear extracellularly, eventually forming the trellis-like connective tissue septa that give the rhomboid profile found in the adult muscle. The close association of collagen and myosin in early development suggests that the muscle cells themselves produce and export collagen.

Phosphorylatioh of vertebrate smooth muscle and nonmuscle myosin heavy chains in vitro and in intact cells

In this article we summarize our recent experiments studying the phosphorylation of vertebrate myosin heavy chains by protein kinase C and casein kinase II. Protein kinase C phosphorylates vertebrate non-muscle myosin heavy chains both in vitro and in intact cells. A single serine residue near the end of the helical portion of the myosin rod is the only site phosphorylated in a variety of vertebrate nonmuscle myosin heavy chains. There does not appear to be a site for protein kinase C phosphorylation in vertebrate smooth muscle myosin heavy chains. Casein kinase II phosphorylates a single serine residue located near the carboxyl terminus of the 204 x 10(3) Mr smooth muscle myosin heavy chain in vitro as well as in cultured smooth muscle cells. It does not phosphorylate the 200 x 10(3) Mr smooth muscle myosin heavy chain. However, the site is present in vertebrate nonmuscle myosin heavy chains. The 204 x 10(3) Mr myosin heavy chain of embryonic chicken gizzard smooth muscle is exceptional in not containing a site for casein kinase II phosphorylation.

Two isoforms of smooth muscle myosin regulatory light chain in chicken gizzard.

We isolated a cDNA clone for a new isoform of chicken smooth muscle myosin regulatory light chain (MRLC) from a cDNA library of embryonic chicken gizzard. The deduced amino acid sequence was different in 10 amino acid residues from the previously reported polypeptide sequences of chicken gizzard MRLC. The in vitro transcription/translation product from the cDNA comigrated with a minor isoform of chicken gizzard MRLC (L20-B) in a two-dimensional gel electrophoresis. This isoform was detected only in the embryonic gizzard and was slightly more acidic than the predominant isoform (L20-A). The partial polypeptide sequence of L20-A was confirmed to be identical to the previously reported MRLC sequence. Nevertheless, Northern blot analysis showed that L20-B-related mRNAs were present in both the embryonic and adult gizzard. Non-denaturing pyrophosphate polyacrylamide gel electrophoresis showed that the in vitro transcription/translation product could be associated with native myosin when mixed and coprecipitated in a low-ionic-strength buffer with adult chicken gizzard myosin. Moreover, the coprecipitated translation product was phosphorylated in vitro by chicken gizzard myosin light chain kinase apparently more rapidly than L20-A on the native myosin heavy chain. From these findings, we concluded that at least two isoforms of smooth muscle MRLC exist in chicken gizzard and that their expression may be regulated translationally depending on the developmental stage.

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence: Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) hase been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated M r of 229 × 10 3, and can be divided into a long α-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two “hinges” for the 6S/10S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg 2+ ATP is bound to a pocket between two opposing α β domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.

Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

Differentiation of contractile proteins of smooth muscle

Tropomyosin was prepared from different developmental stages of embryonic chicken gizzard, and its differentiation was examined by one-and two-dimentional gel electrophoresis. There existed different four tropomyosin chains: two were the same as those of adult gizzard, f-chain and s-chain; another one was close to the f-chain; the last smallest one was very like to be non-muscle tropomyosin. Examination of the gels showed that the last two chains were gradually decreased in their amounts and finally disappeared just before hatching. These 4 chains, especially the last two abnormal chains were identified from their special behavior on IEF-SDS and urea-SDS-gel electrophoresis. The Mg2+-paracrystals prepared from 19 day tropomyosin showed two typical types of gizzard tropomyosin from the negatively stained image on electron microscopy, but that of 12 day tropomyosin gave another view like the non-muscle tropomyosin tactoid in addition to the two types of adult tropomyosin. Sectioned images of the fixed embryonic gizzard in the different stages showed relatively many non-muscle cells like fibroblasts in 12 day gizzard except for smooth muscle cells, and only smooth muscle cells were observed in 19 day gizzard. In conclusion, one chain of embryonic tropomyosin came from non-muscle cells in the early embryonic gizzard. Another chain exists which closely resembles the f-chain, and this might be the real embryonic tropomyosin.

Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery

Rhodamine-labeled actin microinjected into living embryonic chicken gizzard cells became associated with its characteristic cytoskeletal structures. In these domains the translational diffusion coefficients (D) of rh-actin were determined in vivo by fluorescence photobleaching recovery (FPR) measurements. Two classes of actin molecules with respect to its mobilities were detected: rh-actin with a half-time of recovery of 5–10 min in stress fibers and focal contacts (immobile on the time-scale of FPR measurements) and rh-actin with D = 2–3 × 10 −9 cm 2/sec in the cytoplasm and leading lamellae. The slow recovery on stress fibers exhibited similar kinetics whether a short segment or the entire structure were photobleached, indicating that recovery occurs predominantly by exchange with the surrounding diffusable actin. We propose that a steady-state equilibrium between the soluble and cytoskeletal pool of actin exists in living cells.