M. S. Pampusch

IGF-I mRNA levels in bovine satellite cell cultures: Effects of fusion and anabolic steroid treatment

Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known. Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens. The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17β‐estradiol (E2) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)‐α, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin‐like growth factor (IGF)‐I, IGF binding protein (IGFBP)‐3, and myostatin). BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 h with various concentrations of E2 or trenbolone ranging from 0.001 to 10 nM. IGF‐I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9‐times control values, P < 0.02) and at 0.1, 1, and 10 nM E2 (2.9‐, 3.5‐, and 3.5‐times control values, respectively, P < 0.0001). Additionally both 1 and 10 nM trenbolone increased IGF‐I mRNA levels to 1.7‐times control values (P < 0.02). ER‐α mRNA was detectable in BSC cultures, and levels were increased (2.3‐times control levels, P < 0.001) in cultures treated with 0.001 nM E2 but not in cultures treated with higher concentrations of E2. Androgen receptor mRNA levels also were increased (1.5‐times control levels, P < 0.02) in cultures treated with 0.001 nM trenbolone but not by treatment with higher concentrations of trenbolone. Levels of IGFBP‐3 were increased (1.4‐times control values, P < 0.02) by treatment with 0.001 nM E2 but not by treatment with high concentrations of E2. Myostatin mRNA levels were not affected by any concentration of either of the steroids. Although, levels of IGF‐I mRNA were 10‐times greater (P < 0.02) in fused BSC cultures than in proliferating cultures, treatment of fused cultures for 48 h with 10 nM E2 increased IGF‐I mRNA levels (2.5‐times control levels, P < 0.02). Both E2 and trenbolone increased 3H‐thymidine incorporation rate (1.5‐times control levels, P < 0.001) in BSC cultures in media containing serum from which IGFBP‐3 had been removed by anti‐IGFBP‐3 affinity chromatography. In summary, treatment of BSC cultures with either E2 or trenbolone increased IGF‐I mRNA level and proliferation rate, thus, establishing that these steroids have direct anabolic effects on cells present in the BSC culture.

Cloning, expression and functional role of a nociceptin/orphanin FQ receptor in the porcine gastrointestinal tract

The heptadecapeptide nociceptin/orphanin FQ is the cognate ligand for the opioid receptor-like orphanin FQ (OFQ) receptor, a member of the G protein-coupled receptor superfamily. The gastrointestinal tract is a major site of opioid action, and preliminary evidence suggests that an OFQ receptor may be expressed in rat small intestine. We addressed the hypothesis that this receptor is expressed in the gastrointestinal tract of the pig, a model for the human digestive system. A 1205-bp cDNA was isolated from porcine forebrain which contained the 370 amino acid open reading frame encoding the OFQ receptor. The receptor mRNA is likely to arise from a single gene, as determined by Southern blotting of porcine genomic DNA restriction digests using a porcine OFQ receptor cDNA probe. A semi-nested reverse transcriptase-polymerase chain reaction survey of receptor mRNA indicates that it is expressed in the porcine cerebral cortex and kidney, and along the length of the gastrointestinal tract. OFQ decreased initial contractile responses of porcine ileal smooth muscle strips to trains of electrical field stimulation with an IC50 value of 1.3 nM; its effects were resistant to the opioid antagonist, naloxone. The peptide, at concentrations > or =3 nM, also attenuated Isc elevations evoked by electrical transmural stimulation of mucosa-submucosa sheets from porcine ileum. The actions of OFQ appeared to differ from those previously reported for opioid receptor agonists in these tissue preparations. These results indicate that an OFQ receptor is expressed in the porcine intestine which modulates the neural control of intestinal smooth muscle contractility and mucosal transport.

Inducible nitric oxide synthase expression in porcine immune cells

Porcine immune cells were examined for the ability to produce inducible nitric oxide synthase following in vitro or in vivo stimulation. Enzyme activity and product formation were not detected following stimulation of porcine peripheral blood mononuclear cells (PBMC), splenocytes, or alveolar macrophages with a combination of ConA and lipopolysaccharide (LPS) or recombinant porcine interferon gamma and LPS. In vitro engulfment of Haemophilus parasuis by macrophages also failed to induce inducible nitric oxide synthase (iNOS) activity or nitrite formation. Swine Herpes Virus infection led to a small but significant increase in level of nitrite detected in lung lavage fluid, whereas the infection of pigs with Porcine Respiratory and Reproductive Syndrome Virus did not alter the lavage fluid nitrite levels. iNOS mRNA was detected in both stimulated and unstimulated porcine immune cells and in macrophages from both control and infected animals suggesting that it is constitutively expressed with little or no upregulation following cellular stimulation. The results presented in this paper indicate that the reactive nitrogen intermediate pathway is not an vital innate immune response in the pig.

Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-β- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures

Both transforming growth factor (TGF‐β) and growth and development factor (GDF)‐8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin‐like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high‐affinity insulin‐like growth factor binding proteins (IGFBP 1–6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF‐β1 or GDF‐8 suppressed proliferation and increased production of IGFBP‐3 protein and mRNA (P < 0.005). An anti‐IGFBP‐3 antibody that neutralizes the biological activity of IGFBP‐3 reduced the ability of either TGF‐β1 or GDF‐8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF‐β1 and GDF‐8. These data show that IGFBP‐3 plays a role in mediating the activity of either TGF‐β1 or GDF‐8 alone but not when both TGF‐β1 and GDF‐8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP‐3 did not result in increased levels of phosphosmad‐2. Since TGF‐β and GDF‐8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP‐3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP‐3 also may be involved in regulating these processes in myogenic cells. J. Cell. Physiol. 197: 225–231, 2003© 2003 Wiley‐Liss, Inc.

Monoclonal antibodies putatively identifying porcine B cells

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.

Expression of nociceptin/OFQ receptor and prepro-nociceptin/OFQ in lymphoid tissues

This study was undertaken to examine the presence of functional nociceptin/orphanin FQ (N/OFQ) receptors in the immune system. Receptor mRNA signals were detected by RT-PCR in porcine thymus, lymph nodes, spleen and freshly-isolated splenocytes; the distribution of prepro-nociceptin/-orphanin FQ (PP-N/-OFQ) mRNA was similar, with the exception of lymph nodes. However, specific [(3)H]nociceptin binding sites were not detected in rat or porcine lymphoid tissues, and 0.1-100 nM nociceptin had no effect on forskolin-stimulated cyclic AMP concentrations in porcine splenocytes. Thus, it appears that nociceptin/orphanin FQ receptor mRNA, but not a functional receptor protein is expressed in the immune system.

Effects of implants of trenbolone acetate, estradiol, or both, on muscle insulin-like growth factor-I, insulin-like growth factor-I receptor, estrogen receptor-α, and androgen receptor messenger ribonucleic acid levels in feedlot steers

We previously showed that a combined trenbolone acetate (TBA)/estradiol-17beta (E2) implant significantly increases IGF-I mRNA levels in the LM of feedlot steers by 28 d after implantation. Here we compare the effects of E2 (25.7 mg), TBA (120 mg), and combined TBA (120 mg)/E2 (24 mg) implants on IGF-I, IGF-I receptor (IGFR-1), estrogen receptor (ER)-alpha and androgen receptor (AR) mRNA levels in the LM of steers. Twenty yearling crossbred steers with an average initial BW of 421.1 +/- 3.6 kg were stratified by BW and randomly assigned to 1 of 4 treatments: 1) nonimplanted, control; 2) implanted with TBA and E2; 3) implanted with E2; or 4) implanted with TBA. Steers were weighed weekly starting on d 0, and muscle biopsy samples were taken from each steer on d 0 (before implantation), 7, 14, and 28. Ribonucleic acid was prepared from each sample and real-time reverse transcription-PCR was used to determine the levels of IGF-I, IGFR-1, ER-alpha, and AR mRNA. Body weight of implanted steers, adjusted by using d-0 BW as a covariant, tended (P = 0.09) to be greater than that of control steers. On d 7 and 28, IGF-I mRNA levels were greater (58 and 78%, respectively; P < 0.009) in E2-implanted animals than in control steers. Similarly, on d 28 the LM IGF-I mRNA level was 65% greater (P = 0.017) in TBA/E2-implanted steers than in control animals. In contrast, the TBA implant did not increase (P = 0.99) LM IGF-I mRNA levels after 28 d of implantation. Muscle IGFR-1, AR, and ER-alpha mRNA levels were not different (P > 0.47) in any of the treated groups compared with the control group. These data suggest that E2 is responsible for the increased muscle IGF-I mRNA level observed in steers implanted with a combined TBA/E2 implant.

Porcine myelomonocytic markers: summary of the second international swine CD workshop

Forty five mAbs submitted to the Second International Swine CD workshop were analyzed by six different laboratories for their possible reactivity with porcine myelomonocytic cells using flow cytometry and immunohistochemistry. As a result of these analyses, a new swine workshop cluster, SWC9, composed of two mAbs that recognize an antigen selectively expressed on mature macrophages, was defined. In addition, several mAbs were identified, allowing the differentiation of granulocytes from monocytes/macrophages, or monocytes from macrophages. Further work is required to identify the antigen recognized by these mAbs. Nevertheless, they should already prove useful for the identification of different stages in the macrophage maturation/differentiation, and will certainly aid analyses on the complexity of the mononuclear phagocyte system in the pig. Finally, the cross-reactivity of three anti-human CD14 mAbs with porcine myelomonocytic cells was established in this workshop.

Delta-Opioid Receptor mRNA Expression and Immunohistochemical Localization in Porcine Ileum

Opiates have potent antidiarrheal actions thatare mediated in part by delta-opioid receptors (DOR). Weexamined DOR localization within subregions of porcineileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR wasobtained after reverse transcription-polymerase chainreaction cloning of forebrain RNA; it encoded the end oftransmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93%nucleotide identity with human DOR. Positive signals forDOR mRNA were found in all subregions of the porcineileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-likeimmunoreactivity was localized in neurons withinmyenteric and submucous ganglia, longitudinal andcircular smooth muscle, and villous lamina propria. TheDOR agonist [D-Ser2, Leu5, Thr6]enkephalin(DSLET) attenuated circular smooth muscle contractionsin porcine ileum that were evoked by electricalstimulation of myenteric cholinergic neurons. Theseresults are consistent with previous reports of the DOR-mediatedneuromodulation that underlies the antipropulsive andantisecretory effects of opioids in the intestinaltract.

Effect of recombinant porcine IGFBP-3 on IGF-I and long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells

Insulin‐like growth factor (IGF)‐I stimulates both proliferation and differentiation of myogenic precursor cells. In vivo, IGFs are bound to one of the members of a family of six high‐affinity IGF binding proteins (IGFBP 1–6) that regulate their biological activity. One of these binding proteins, IGFBP‐3, affects cell proliferation via both IGF‐dependent and IGF‐independent mechanisms and it has generally been shown to suppress proliferation of cultured cells; however, it also may stimulate proliferation depending upon the cell type and the assay conditions. Cultured porcine embryonic myogenic cells (PEMCs) produce IGFBP‐3 and its level drops significantly immediately prior to differentiation. Additionally, IGFBP‐3 suppresses both IGF‐I and Long‐R3‐IGF‐I‐stimulated proliferation of embryonic porcine myogenic cells. In this study, we have examined the effects of recombinant porcine IGFBP‐3 (rpIGFBP‐3) on IGF‐I‐ and Long‐R3‐IGF‐I‐stimulated proliferation and differentiation of the L6 myogenic cell line. L6 cells potentially provide a good model for studying the actions of IGFBP‐3 on muscle because they contain no non‐muscle cells and they do not produce detectable levels of IGFBP‐3. RpIGFBP‐3 suppresses both IGF‐I and Long‐R3‐IGF‐I‐stimualted proliferation of L6 cells, indicating that it suppresses proliferation via both IGF‐dependent and IGF‐independent mechanisms. Our data also show that rpIGFBP‐3 causes IGF‐independent suppression of proliferation without increasing the level of phosphosmad‐2 in L6 cultures. Additionally, rpIGFBP‐3 suppresses IGF‐I‐stimulated differentiation of L6 cells. In contrast, however, rpIGFBP‐3 does not suppress Long‐R3‐IGF‐I‐stimulated differentiation. This suggests that rpIGFBP‐3 does not have IGF‐independent effects on L6 cell differentiation.