CDNA cloning and sequencing of phospholamban from pig stomach smooth muscle.
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ABSTRACT:
SUBMITTER: Verboomen H
PROVIDER: S-EPMC1133268 | biostudies-other | 1989 Aug
REPOSITORIES: biostudies-other
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Project description:The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.
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Project description:The stomach serves as food reservoir, mixing organ and absorption area for certain substances, while continually varying its position and size. Large dimensional changes during ingestion and gastric emptying of the stomach are associated with large changes in smooth muscle length. These length changes might induce history-effects, namely force depression (FD) following active muscle shortening and force enhancement (FE) following active muscle stretch. Both effects have impact on the force generating capacity of the stomach, and thus functional relevance. However, less is known about history-effects and active smooth muscle properties of stomach smooth muscle. Thus, the aim of this study was to investigate biomechanical muscle properties as force-length and force-velocity relations (FVR) of porcine stomach smooth muscle strips, extended by the analysis of history-effects on smooth muscle force. Therefore, in total n = 54 tissue strips were dissected in longitudinal direction from the ventral fundus of porcine stomachs. Different isometric, isotonic, and isokinetic contraction protocols were performed during electrical muscle stimulation. Cross-sectional areas (CSA) of smooth muscles were determined from cryo-histological sections stained with Picrosirius Red. Results revealed that maximum smooth muscle tension was 10.4 ± 2.6 N/cm2. Maximum shortening velocity (Vmax ) and curvature factor (curv) of the FVR were 0.04 ± 0.01 [optimum muscle length/s] and 0.36 ± 0.15, respectively. The findings of the present study demonstrated significant (P < 0.05) FD [up to 32% maximum muscle force (Fim )] and FE (up to 16% Fim ) of gastric muscle tissue, respectively. The FE- and FD-values increased with increasing ramp amplitude. This outstanding muscle behavior is not accounted for in existing models so far and strongly supports the idea of a holistic reflection of distinct stomach structure and function. For the first time this study provides a comprehensive set of stomach smooth muscle parameters including classic biomechanical muscle properties and history-dependent effects, offering the possibility for the development and validation of computational stomach models. Furthermore, this data set facilitates novel insights in gastric motility and contraction behavior based on the re-evaluation of existing contractile mechanisms. That will likely help to understand physiological functions or dysfunctions in terms of gastric accommodation and emptying.
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Project description:Smooth muscle cells (SMCs) play an important role in physiology and production in farm animals such as pigs. Here, we report the generation of a pig SMC line. Our original objective was to establish an enteroendocrine cell line from the pig ileum epithelium through lentiviral transduction of the Simian Virus (SV) 40 large T antigen. However, an initial expression analysis of marker genes in nine cell clones revealed that none of them were enteroendocrine cells or absorptive enterocytes, goblet cells, or Paneth cells, some of the major cell types existing in the ileum epithelium. A more detailed characterization of one clone named PIC7 by RNA-seq showed that these cells expressed many of the known smooth muscle-specific or -enriched genes, including smooth muscle actin alpha 2, calponin 1, calponin 3, myosin heavy chain 11, myosin light chain kinase, smoothelin, tenascin C, transgelin, tropomyosin 1, and tropomyosin 2. Both quantitative PCR and RNA-seq analyses showed that the PIC7 cells had a high expression of mRNA for smooth muscle actin gamma 2, also known as enteric smooth muscle actin. A Western blot analysis confirmed the expression of SV40 T antigen in the PIC7 cells. An immunohistochemical analysis demonstrated the expression of smooth muscle actin alpha 2 filaments in the PIC7 cells. A collagen gel contraction assay showed that the PIC7 cells were capable of both spontaneous contraction and contraction in response to serotonin stimulation. We conclude that the PIC7 cells are derived from an enteric SMC from the pig ileum. These cells may be a useful model for studying the cellular and molecular physiology of pig enteric SMCs. Because pigs are similar to humans in anatomy and physiology, the PIC7 cells may be also used as a model for human intestinal SMCs.
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Project description:A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.
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