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It is well established that heat stress (HS) negatively impacts growth rate in swine. Although reduced feed intake undoubtedly plays a significant role in this reduction, studies in laboratory animals and other non-swine species indicate muscle growth also is affected by HS-related alterations in muscle physiology. There is now emerging evidence that heat shock proteins (Hsp), produced in response to HS and other types of cellular stress, may play an important role in regulating rate and efficiency of muscle growth. Because muscle satellite cells play a crucial role in postnatal muscle growth, the effects of HS on rates of satellite cell proliferation, protein synthesis, and protein degradation play an important role in determining the rate and extent of muscle growth. Consequently, in the current study we have examined the effects of mild HS (40.5°C for 48 h) on the rates of proliferation, protein synthesis, and protein degradation and on levels of Hsp90, Hsp70, and Hsp25/27 mRNA and protein in cultured porcine muscle satellite cells (PSC). Mild HS of PSC cultures resulted in 2.5-, 1.4-, and 6.5-fold increases (P < 0.05) in the levels of Hsp90, Hsp70, and Hsp25/27 mRNA, respectively, relative to the levels in control cultures. Levels of Hsp 90, 70, and 25/27 proteins were also increased in HS PSC cultures compared to those in control cultures. Proliferation rates in HS PSC cultures were 35% less (P < 0.05) than those in control cultures. Protein synthesis rates in HS fused PSC cultures were 85% greater (P < 0.05) than in control cultures, and protein degradation rates in HS fused PSC were 23% less (P < 0.05) than in control cultures. In light of the crucial role satellite cells play in postnatal muscle growth, the HS-induced changes we have observed in rates of proliferation, protein turnover, and in levels of Hsp mRNA and protein in PSC cultures indicate that mild HS affects the physiology of PSC in ways that could affect muscle growth in swine. [ABSTRACT FROM AUTHOR]

We previously showed that a combined trenbolone acetate (TBA)/estradiol-17β (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, ICF-I receptor (IGFR-1), estrogen receptor (ER)-α 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-α, 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-α 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 ICF-I mRNA level observed in steers implanted with a combined TBA/E2 implant. [ABSTRACT FROM AUTHOR]

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 TGFβ1 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. [ABSTRACT FROM AUTHOR]

Opiates have potent antidiarrheal actions that are mediated in part by delta-opioid receptors (DOR). We examined DOR localization within subregions of porcine ileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR was obtained after reverse transcription-polymerase chain reaction cloning of forebrain RNA; it encoded the end of transmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93% nucleotide identity with human DOR. Positive signals for DOR mRNA were found in all subregions of the porcine ileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-like immunoreactivity was localized in neurons within myenteric and submucous ganglia, longitudinal and circular smooth muscle, and villous lamina propria. The DOR agonist [D-Ser2, Leu5, Thr6]enkephalin (DSLET) attenuated circular smooth muscle contractions in porcine ileum that were evoked by electrical stimulation of myenteric cholinergic neurons. These results are consistent with previous reports of the DOR-mediated neuromodulation that underlies the antipropulsive and antisecretory effects of opioids in the intestinal tract. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor binding proteins (IGFBP)-3 and -5 play a significant role in the mechanism by which TGF-beta and myostatin suppress proliferation of porcine embryonic myogenic cells (PEMC) and porcine muscle satellite cells (PMSC). RNA interference (RNAi) utilizing small inhibitory RNA (siRNA) is currently extensively used to silence specific genes in mammalian cells. Consequently, we have cloned a small hairpin (sh) IGFBP-3 RNA sequence (complementary 19mer siRNA sequences separated by a hairpin loop) into the pSilencer 2.1 (Ambion) siRNA expression vector. Electroporation was used to transiently transfect this construct into PEMC cells (IGFBP-3-silenced PEMC). As a control, the same electroporation procedure was used to transfect the vector containing a nonsense sequence supplied by the manufacturer into PEMC (mock-silenced cells). As compared to mock-silenced or non-transfected control cells, IGFBP-3-silenced PEMC showed a 90% reduction (p<0.01) in IGFBP-3 protein and mRNA levels. Neither IGFBP-2 nor IGFBP-5 mRNA or protein levels were significantly affected in the IGFBP-3-silenced cell population, indicating that the suppression of IGFBP-3 production was specific. Suppression of IGFBP-3 production in IGFBP-3-silenced PEMC persisted for at least 120 h after transfection, providing ample time to accomplish experiments assessing the effects of IGFBP-3 knock-down on proliferation and cell signaling. Additionally, immunohistochemical studies show that intracellular IGFBP-3 levels were dramatically reduced in IGFBP-3-silenced cells as compared to mock-silenced cells. We also have identified an shRNA that reduces IGFBP-5 mRNA in PEMC by 95% (as compared to mock-silenced control PEMC) (p<0.01) without significantly altering the levels of IGFBP-2 or -3 mRNA or protein. Silencing IGFBP-3 and IGFBP-5 production in PEMC cells will provide a valuable research tool for use in assessing the role of these IGFBPs in mediating the anti-proliferative effects of myostatin and TGF-beta on these cells. [ABSTRACT FROM AUTHOR]

The proliferation-suppressing actions of myostatin and TGF-beta in porcine embryonic myogenic cell (PEMC) cultures are mediated, at least in part, by insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5. Consequently, understanding the mechanism of action of these IGFBPs in myogenic cells is important to understanding how TGF-beta and myostatin regulate growth of muscle. We have used anti-rpIGFBP-3 (anti-BP3), anti-rpIGFBP-5 (anti-BP5) and anti-desmin antibodies to localize IGFBP-3, IGFBP-5 and desmin, respectively, in PEMC. IGFBP-3 was detected in the cytoplasm and nuclei of desmin-positive, mononucleated cells in proliferating PEMC cultures; thereby, establishing that IGFBP-3 is present in PEMC (controls using non-specific IgG show no staining). Similarly, IGFBP-3 was detected in cultured PEMC myotubes. In proliferating PEMC cultures, treatment for 24 h with 20 ng TGF-beta/ml medium resulted in an 80% increase (p<0.01) in the number of nuclei containing IGFBP-3. Myogenic cells pre-treated with anti-BP3 for 24 h prior to immunohistochemical localization showed dramatically reduced intracellular levels of IGFBP-3 as compared to control cells that received no pre-treatment. This confirms reports in other cell types that a significant portion, if not all, of the intracellular IGFBP-3 represents uptake of secreted IGFBP-3. Additionally, these results establish that anti-BP-3 interferes with the transport of IGFBP-3 into myogenic cells. IGFBP-5 was detected in the cytoplasm and nuclei in proliferating and fused PEMC cultures (controls with non-specific IgY show no staining). Localization of IGFBP-3 and IGFBP-5 in PEMC at different stages of differentiation or after treatment with specific growth factors should lead to a greater understanding of the roles of these IGFBPs in muscle growth and differentiation. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Insulin-like growth factor binding protein-3 (IGFBP-3) suppresses proliferation of numerous cell types, including myogenic cells, via both insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms; however, the mechanism of IGF-independent suppression of proliferation is not clearly defined. In nonmuscle cells, binding of IGFBP-3 to the low-density lipoprotein receptor-related protein-1 (LRP-1)/activated α(2)M receptor is reportedly required for IGFBP-3 to inhibit proliferation. These findings suggest that binding to this receptor also may be required for IGFBP-3 to suppress proliferation of cultured myogenic cells. To investigate the role of the LRP-1 receptor in suppression of myogenic cell proliferation by IGFBP-3, we have examined the effect of receptor-associated protein, an LRP-1 receptor antagonist, on recombinant porcine (rp)IGFBP-3 inhibition of L6 myogenic cell proliferation. Treatment with receptor-associated protein results in a 37% decrease (P < 0.05) in the ability of rpIGFBP-3 to inhibit L6-cell proliferation. In L6 cells subjected to LRP-1 small interfering RNA treatment for 48 h (LRP-1 silenced), LRP-1 mRNA levels were reduced by greater than 80% compared with control cultures treated with nonsense small interfering RNA (mock silenced). In addition, the 85-kDa transmembrane subunit of LRP-1 was undetectable in Western immunoblots of total protein lysates from LRP-1-silenced cells. Even though LRP-1 mRNA and protein levels were dramatically reduced in LRP-1-silenced L6 cells compared with mock-silenced controls, rpIGFPB-3 suppressed proliferation rate to the same extent in both LRP-1-silenced and mock-silenced cultures. Our results strongly suggest that, in contrast to data obtained for nonmuscle cell lines, the LRP-1 receptor is not required for IGFBP-3 to suppress proliferation of L6 myogenic cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-beta superfamily members myostatin and TGF-beta1 have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-beta1 or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-beta1 and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-beta1 or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-beta1 or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-beta1 or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-beta and myostatin to suppress proliferation of PEMC.

IGF-binding protein-5 (IGFBP-5) is produced by porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of this study was to produce recombinant porcine IGFBP-5 (rpIGFBP-5) and assess its IGF-I-dependent and IGF-I-independent actions on the proliferation of PEMCs. To accomplish this, we have expressed porcine IGFBP-5 in the baculovirus system, purified and characterized the expressed rpIGFBP-5 and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography, suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P>0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P>0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5, demonstrating the IGF-independent activity that rpIGFBP-5 possesses in PEMCs, since Long-R3-IGF-I is an IGF analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby can stimulate proliferation. The anti-rpIGFBP-5 IgY produced against rpIGFBP-5 specifically recognized native porcine IGFBP-5 in PEMC media that also contained porcine IGFBP-2, -3, and -4. This antibody is capable of neutralizing the effects of both rpIGFBP-5 and endogenously produced porcine IGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots. The production of rpIGFBP-5 and a neutralizing antibody to porcine IGFBP-5 provides a powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitro muscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo.

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 17beta-estradiol (E(2)) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)-alpha, 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 E(2) 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 E(2) (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-alpha 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 E(2) but not in cultures treated with higher concentrations of E(2). 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 E(2) but not by treatment with high concentrations of E(2). 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 E(2) increased IGF-I mRNA levels (2.5-times control levels, P < 0.02). Both E(2) and trenbolone increased (3)H-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 E(2) 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., (Copyright 2004 Wiley-Liss, Inc.)

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-stimulated 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., (Copyright 2004 Wiley-Liss, Inc.)

We used a muscle biopsy technique in conjunction with real-time PCR analysis to examine the time course of changes in muscle IGF-I, IGFBP-3, myostatin, and hepatocyte growth factor (HGF) mRNA in the longissimus muscles of Revalor-S-implanted and nonimplanted steers on d 0, 7, 12, and 26 after implantation (nine steers/treatment group). Administration of a Revalor-S implant increased (P < 0.01) ADG and improved (P < 0.05) feed efficiency, 36 and 34%, respectively, compared with steers that received no implant during the 26-d trial. Daily dry matter intake did not differ (P > 0.15) between nonimplanted and implanted steers. Steers receiving the Revalor-S implant had increased (P < 0.001) circulating IGF-I concentrations compared with nonimplanted steers. The longissimus muscles of steers receiving the Revalor-S implant contained increased (P < 0.001) IGF-I mRNA levels compared with longissimus muscles of nonimplanted steers over the 26-d duration of the study. Longissimus muscle IGF-I mRNA levels in implanted steers were increased (P < 0.003) relative to d-0 concentrations on d 7 and 12 (101% and 128%, respectively), and byd 26, longissimus muscle mRNA levels were more than three times (P < 0.0001) those in the longissimus muscles of the same steers on d 0. There was no treatment effect on the level of IGFBP-3, myostatin, or HGF mRNA in the longissimus muscle at any time point; however, levels of IGFBP-3, myostatin, and HGF mRNA increased with time on feed. Based on current and previous studies, we hypothesize that the increased IGF-I level in muscle of implanted steers by d 7 of implantation stimulates satellite cell proliferation and maintains a high number of proliferating satellite cells at a point in the growth curve where satellite cell numbers and activity are normally dropping off. This would prolong the period of rapid muscle growth, resulting in the observed increased rate and efficiency of muscle deposition in implanted steers.

The effects of subtherapeutic antimicrobial supplementation and weaning on serum levels of IGF-I and insulin-like growth factor binding proteins (IGFBP)-2, -3 and -4 were determined in crossbred weanling pigs. At weaning, pigs were allotted to a diet containing 21.8% crude protein and 1.15% lysine with or without Aureozol (110 mg/kg of Aureomycin chlortetracycline, 110 mg/kg of sulfathiazole, and 55 mg/kg of penicillin) for 4 wk. Insulin-like growth factor-binding proteins and IGF-I analyses were performed on blood samples that were drawn weekly. Weaning decreased serum IGFBP-3 levels in both control and Aureozol-treated groups on d 6 and d 14 (P < 0.05) relative to preweaning levels. The IGFBP-3 values returned to preweaning levels by d 21. Although the circulating levels of both the 43-kDa and the 39-kDa glycosylation variants of IGFBP-3 were affected by weaning, the level of the 39-kDa IGFBP-3 was affected relatively more than that of the 43-kDa IGFBP-3 (P < 0.05). Compared with circulating IGFBP-3 levels in control pigs, Aureozol-treated pigs had higher circulating IGFBP-3 levels on d 21 (43%, P < 0.05) and d 27 (46%, P < 0.05). In direct contrast to the effect of weaning on serum IGFBP-3 level, serum IGFBP-2 levels increased on d 6 and d 14 after weaning (P < 0.05) and decreased to preweaning levels by d 21. The IGFBP-2 levels continued to decline and were less than preweaning levels by d 27 (P < 0.05). Aureozol treatment had no effect on serum IGFBP-2 levels at any time. Serum levels of nonglycosylated IGFBP-4 were not affected by either weaning or Aureozol supplementation. Weaning decreased circulating IGF-I concentration on d 6 in both control and Aureozol-treated pigs (76 and 73%, respectively, P < 0.05) and on d 14 (62%, P < 0.05) and d 21 (32%, P < 0.05) in control pigs. Aureozol-supplemented pigs had higher serum IGF-I concentrations than control pigs on d 14 (82%, P < 0.05), d 21 (55%, P < 0.05), and d 27 (36%, P < 0.05). The Aureozol-fed pigs had a 14.2% increase in BW gain (P < 0.05) and a 59.6% increase in ADG (P < 0.05) compared with pigs fed the control diet. Both Aureozol-supplementation and weaning cause changes in serum IGFBP levels and IGF-I concentrations that might be involved in regulating rate and efficiency of growth.

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.

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.

The porcine mu opioid receptor (pMOR), was cloned from cerebral cortex RNA using PCR methodologies. Porcine MOR is 96% identical with human MOR in amino acid sequence. An RT-PCR survey for pMOR mRNA indicated that pMOR is widely distributed in the gut, and is present in thymus and Peyer's patches but absent in other immune tissues and in isolated immune cells. Based on these findings, it appears that opioids do not exert an immunosuppressive effect through direct interaction with the mu-opioid receptor on immune cells. In certain tissues, however, opioids may modulate immune function indirectly through neuronal MOR.

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.

We report comparative linkage mapping of eleven genes in the swine genome by RFLP analysis. These genes include: Acid phosphatase type 5 (ACP5), Cholecystokinin Type B Receptor (CCKBR), Antibiotic Peptide (FALL39), Insulin-like Growth Factor 1 Receptor (IGF1R), Integrin Alpha M (ITGAM), Integrin Beta 2 (ITGbeta2), Opioid Receptor Mu-1 (OPRM1), Pro-hormone Converter (PC1/3), Retinol Binding Protein 3 (RBP3), Ribosomal DNA (RNR1), and Zona Pellucida Glycoprotein 1 (ZP1). The CCKBR and ITGbeta2 loci define the ends of the linkage groups on Chromosomes (Chro) (SSC) 9p and 13qter, respectively.

Insulin-like growth factor binding proteins (IGFBP) may act locally as autocrine or paracrine regulators of insulin-like growth factor activity in specific tissues such as muscle. Although secretion of IGFBP by cultured myogenic cell lines has been examined, little is known about secretion of IGFBP by primary myogenic cell cultures. This may be because primary myogenic cultures contain non-muscle cells (fibroblasts) that complicate interpretation of IGFBP determinations. We have circumvented this problem by subculturing nonfusing cells from extensively fused porcine myogenic cultures and comparing the IGFBP production of these nonfusing, porcine muscle-derived cells with that of primary porcine myogenic cell cultures. Immunoprecipitation with specific antibodies and 125I-IGF-I ligand blot analysis showed that myogenic cultures secreted IGFBP-3 (doublet band, 43 kDa and 39 kDa), IGFBP-2 (34 kDa), IGFBP-4 (30 and 24 kDa), and IGFBP-5 (30 and 28 kDa). Muscle-derived fibroblasts secreted no detectable IGFBP-3 but approximately 10 times more IGFBP-2 than did myogenic cell cultures. Treatment of myogenic cultures for 24 h with transforming growth factor (TGF) beta-1 caused a concentration-dependent increase in IGFBP-3 secretion with a maximum 1.5-fold increase occurring at .5 ng of TGF beta-1/mL. In contrast, TGF beta-1 treatment did not stimulate detectable IGFBP-3 secretion by muscle-derived fibroblast cultures. Northern analysis of total RNA using a porcine IGFBP-3 probe revealed that TGF beta-1 treatment resulted in a fourfold increase in the steady-state level of IGFBP-3 mRNA in myogenic cultures. Insulin-like growth factor binding protein-3 mRNA was not detectable in fibroblast cultures either before or after TGF beta-1 treatment. This is the first report of IGFBP-3 secretion by cultured myogenic cells.

Myogenic cells isolated from lamb fetuses (approximately mid-gestation) exhibited a concentration-dependent decrease in myogenic cell proliferation in response to transforming growth factor (TGF) beta-1 (P < .001). Half-maximal inhibition of proliferation occurred at approximately .05 ng of TGF beta-1/mL and maximal inhibition of proliferation occurred at approximately .1 ng of TGF beta-1/mL. The specificity of this inhibition was confirmed by neutralization of the activity following exposure to a TGF beta antibody. The TGF beta-1 also suppressed proliferation of ovine satellite cells isolated from 5-d-old lambs (P < .0035), but to a lesser extent than observed for embryonic cells. In contrast, TGF beta-1 did not significantly suppress serum-stimulated proliferation of ovine satellite cells isolated from 30- or 150-d-old lambs. Similarly, TGF beta-1 did not suppress proliferation of skeletal muscle fibroblast-like cells isolated from either fetal lambs or 150-d-old lambs. In fact, proliferation of fibroblast-like cells derived from embryonic ovine muscle was enhanced by exposure to TGF beta-1 at all levels tested; however, a concentration-dependent response was not observed. Media transfer experiments showed that conditioning of culture media by postnatally derived cells did not render TGF beta-1 inactive. The studies described in this manuscript suggest that sensitivity of ovine myogenic cells to the antiproliferative effect of TGF-beta may vary with the stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

We have evaluated the effect of transforming growth factor beta-1 (TGF beta-1) on proliferation and fusion of cultured ovine satellite cells isolated from 5-month-old wether lambs. The isolation and culture protocols were validated by clonal analysis of the original cell preparation and assessment of proliferation and fusion of control cultures. Approximately 85% of the original cells isolated were myogenic as assessed by clonal analysis. The ovine cells doubled approximately every 18 hours during their exponential growth period and achieved a maximum percent fusion of 39.5% after 144 hours in culture. TGF beta-1 inhibited fusion of these cells in a dose-dependent manner with half-maximal inhibition occurring at .08 ng/ml. Maximal inhibition (95% suppression) occurred between .1 and .5 ng/ml. TGF Beta-1 (.05-3.0 ng/ml) did not inhibit proliferation of cultured ovine satellite cells in serum-containing medium or in serum-free defined medium. In contrast, TGF beta-1 did significantly suppress serum-stimulated proliferation of either porcine or bovine satellite cells that were isolated by using a procedure identical to that used to isolate the ovine satellite cells. Thus, proliferation of ovine satellite cells appears to respond differently to TGF beta-1 than does proliferation of either porcine or bovine satellite cells.

Sera obtained from pigs before and after subtherapeutic levels of ASP250 supplementation (pre and post serum pools) have been subjected to comparative fractionation by using gel filtration and affinity chromatography on immobilized Cibacron Blue F3G-A. Comparable serum fractions obtained from pre- and post-ASP250 blood sera were assayed in muscle cell culture bioassays designed to measure their effect on proliferation. Pre- and post-ASP250 sera were subjected to gel filtration and divided into the following fractions: fraction 1, Kav less than .17; fraction 2, Kav = .17 to .41; fraction 3, Kav = .41 to .59. Post-ASP250 fractions 2 and 3 increased proliferation rate in cultured muscle cells to a greater extent than comparable pre-ASP250 fractions (P less than .001). Chromatography of fraction 3 on immobilized Cibacron Blue F3G-A showed that both pre- and post-ASP250 fraction 3 contained a putative inhibitor of myogenic cell proliferation as well as mitogenic factors. However, negative growth factor activity was greater in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). Additionally, positive growth factor activity was lower in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). These data suggest that levels and(or) activities of both positive and negative muscle growth factors in serum may be altered by the addition of antimicrobials to the diets of growing pigs.

We have examined the effect of Transforming Growth Factor (TGF) beta on proliferation of L6 and embryonic porcine myogenic cells. Proliferation of L6 cells was suppressed by both TGF beta-1 and TGF beta-2 in a dose-dependent manner. Half-maximal suppression of proliferation occurred at .036 ng TGF beta-1/ml and .06 ng TGF beta-2/ml. Maximal inhibition (60% suppression of proliferation for TGF beta-1 and 52% for TGF beta-2) occurred between .1 and .3 ng/ml for each growth factor. Suppression of proliferation was completely abolished in the presence of an anti-TGF beta antibody that inhibited the biological activity of TGF beta-1 and TGF beta-2. When we evaluated the effect of TGF beta-1 on proliferation of embryonic porcine myogenic cells we obtained results which were very similar to those obtained for L6 cells. Insulin-like growth factor (IGF)-I stimulated proliferation of L6 cells in a dose-dependent manner in serum-free, defined medium. However as little as .02 ng TGF beta-1/ml detectably suppressed this stimulation and .3 ng TGF beta-1/ml caused a 60% reduction in cell number in cultures treated with 30 ng IGF-l/ml. Thus TGF beta-1 significantly suppressed IGF-I-stimulated proliferation of L6 cells.