M.E. Fultz

Remodeling of the actin cytoskeleton in the contracting A7r5 smooth muscle cell.

It has been proposed that the reorganization of components of the actin cytomatrix could contribute to force development and the low energy cost of sustained contraction in contractile cells which lack a structured sarcomere (A.S. Battistella-Patterson, S. Wang and G.L. Wright (1997) Can J Physiol Pharmacol75: 1287–1299). However, there has been no direct evidence of an apropos actin reorganization specifically linked to the contractile response in cells of this type. Remodeling of the α- and β-actin domains was studied in A7r5 smooth muscle cells during phorbol 12,13 dibutyrate (PDB)-induced contraction using immunohistologic staining and β-actin-green fluorescent protein (β-actin-GFP) fusion protein expression. Cell stained with phalloidin as well as cells expressing β-actin-GFP showed densely packed actin stress cables, arranged in parallel and extending across the cell body. PDB caused approximately 85% of cells to contract with evidence of forcible detachment from peripheral adhesion sites seen in many cells. The contraction of the cell body was not uniform but occurred along a principal axis parallel to the system of densely packed β-actin cables. During the interval of contraction, the β-actin cables shortened without evidence of disassembly or new cable formation. The use of cytochalasin to inhibit actin polymerization resulted in the dissolution of the actin cables at the central region of the cell and caused the elongation of precontracted cells. In unstimulated cells, α-actin formed cables similar in arrangement to the cell spanning β-actin cables. Within a short interval after PDB addition; however, the majority of α-actin cables disassembled and reformed into intensely fluorescing column-like structures extending vertically from the cell base at the center of clusters of α-actin filaments. The α-actin columns of contracting cells showed strong colocalization of α-actinin suggesting they could be structurally analogous to the dense bodies of highly differentiated smooth muscle cells. The results indicate that the α- and β-actin domains of A7r5 cells undergo a highly structured reorganization during PDB-induced contraction. The extent and nature of this restructuring suggest that remodeling could play a role in contractile function.

Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells

The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR‐108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC‐α (11.6‐fold), nPKC‐δ (5.3‐fold), and nPKC‐ϵ (22.0‐fold) but not aPKC‐ζ. Northern blot analysis indicated a significant (2.3‐fold) increase in the 10 kb transcript of cPKC‐α, a slight (1.3‐fold) increase in that of nPKC‐ϵ but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC‐α, PKC‐δ, and PKC‐ϵ and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR‐108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme‐selective increases in the protein levels of PKC isozymes of osteoblastic UMR‐108 cells upon growth to confluence which may be regulated at the transcriptional or the post‐transcriptional level. The results from UMR‐108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994 . J Bone Miner Res 9(7):999–1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton.

Ca2+-dependent actin remodeling in the contracting A7r5 cell

Previous work has shown that stimulation of contraction in A7r5 smooth muscle cells with phorbol ester (PDBu) results in the disassembly and remodeling of the α-actin component of the cytoskeleton (Fultz et al., 2000, J Mus Res Cell Motil 21: 775–781). In the present study, we evaluated the effect of increasing intracellular calcium ion concentration [Ca2+]i by A23187 and thapsigargin on α- and β-actin remodeling. The effects of A23187 and thapsigargin on cell contraction and actin remodeling were effectively identical. The two compounds caused contraction of A7r5 cells that was earlier in onset and more quickly completed than PDBu-induced contractions. Both the α- and β-actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, β-actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca2+]i resulted in partial or complete dissolution of α-actin cables without further remodeling. In addition, PDBu-mediated α-actin remodeling was blocked in the presence of A23187. Increased [Ca2+]i also caused dispersal of α-actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for α-actin cytoskeletal structure. The incubation of cells in calcium-free media prevented α-actin dissolution by A23187/thapsigargin and also blocked PDBu-mediated remodeling. Finally, of six kinase inhibitors investigated, only ML-7 partially blocked the dissolution of α-actin cables by increased [Ca2+]i. The results suggest that the sustained elevation of [Ca2+]i beyond a threshold level initiates depolymerization of α-actin but not β-actin. It further appears that PDBu-induced α-actin remodeling requires Ca2+ but increases of [Ca2+]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits α-actin dissolution in the presence of A23187/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing α-actin structure in the activated cell.

Concentration-dependent phorbol stimulation of PKCα localization at the nucleus or subplasmalemma in A7r5 cells

Abstract. The subcellular translocation of PKCα was studied in A7r5 cells by confocal microscopy through use of standard immunohistologic staining and PKCα-enhanced green fluorescent protein (PKCα–EGFP) fusion protein expression. The results from both methods were consistent in indicating that PKCα, observed to be diffusely distributed in the unstimulated cell, was translocated primarily to either the perinuclear region of the cell or to subplasmalemmal sites depending on the concentration of phorbol 12, 13 dibutyrate (PDBu) used to activate the response. Translocation of PKCα to the perinucleus but not the plasmalemma was blocked by the use of colchicine to disrupt cell microtubules. However, there was little evidence of significant colocalization of PKCα with the microtubular cytoskeleton during the interval of translocation. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on PKCα translocation to either the plasmalemma or the perinucleus. The results indicate that the target site of PKCα translocation may vary with activating stimulus strength in A7r5 cells and that the translocation of the isoform to perinuclear target loci depends on an intact microtubular cytoskeleton. This suggests that multiple pathways are available for the redistribution of PKCα that may employ different mechanisms to regulate the movement and/or docking of the isoform at specific target sites.

Effect of fasting on vascular contractility in lean and obese Zucker rats

We compared the effects of fasting (36 h) on blood pressure and aortic contractile responsiveness in lean and obese Zucker rats. Fasting of lean animals resulted in a significant loss in body weight (-9.1 +/- 0.1%) and reduction in systolic blood pressure (-11.4 +/- 1.9 mmHg). Fasting plasma triacylglycerols (-76%) and beta-hydroxybutryic acid (beta-HBA) (+ 218%) were significantly decreased and increased, respectively. The fasting plasma concentrations of insulin (-72%) were significantly decreased, whereas glucose and epinephrine (Epi) were not changed in lean rats. The fasting of obese rats also resulted in weight loss (-5.6 +/- 1.3%) but did not cause a significant reduction of blood pressure. The plasma total cholesterol (+18%) was increased, triacylglycerols (-42%) were decreased and beta-HBA levels were unchanged in fasted obese rats. Similar to lean animals, the insulin levels of fasted obese rats were significantly decreased (-85%), whereas glucose and Epi concentrations were not significantly changed. Fasting of lean animals had no effect on the maximal contractile response of aortae to high K(+) and phorbol 12, 13 dibutyrate (PDBu) but significantly reduced the response to norepinephrine (NE) (% reference: fed, 61.1 +/- 11.0; fasted, 45.6 +/- 4.5). In addition, the concentration for half-maximal response (ED(50)) to NE was increased in fasted lean rats (fed, 1.8+/-0.2 x 10(-8)M; fasted, 3.0+/-0.3 x 10(-8)M). By comparison, fasting of obese rats had no significant effect on the contractile response to K(+), NE, or PDBu. The results show that short-term food withdrawal induces significant changes in vascular contractile properties of lean but not obese rats. Because fasting-induced changes were variable depending on the agonist, the results further suggest that the mechanism did not involve a general loss or enhancement in functional status.