Assessment of a preclinical model for studying the survival and engraftment of human stem cell derived osteogenic cell populations following orthotopic implantation.

INTRODUCTION: Preclinical studies with osteoprogenitor cells derived from human embryonic stem cells (hESC) do not lead to substantial bone regeneration in vivo. The degree of survival following implantation might play a role in their long term efficiency. We investigated the initial engraftment of hESCs-derived cells during two weeks post-implantation and compared it to such response for adult bone marrow stromal cells (hBMSC)-derived osteoprogenitor cells. METHODS: hBMSC and H9-hES cells pre-treated with osteogenic factors were implanted into a calvarial defect in both adult WT and nude rats. At days 7 and 14 post-implantation, samples were analysed for persistence of implanted cells, initiation of regeneration of host bone, angiogenesis and apoptosis. RESULTS: At day 7, hESC and hBMSC were detected within defects in both rat strains. By day 14 human cells were only detected in immune-deficient rats whilst still maintaining an osteoblastic phenotype and engendered a significant increase in bone formation. In WT animals, the participation of implanted cells was very limited due to their poor survival. CONCLUSION: This study demonstrates the ability of hESC and hBMSC derived osteoprogenitor cells to survive transplantation, to engraft and to develop an osteogenic phenotype during the early stage following implantation, validating the appropriate preclinical model.

Bone tissue formation from human embryonic stem cells in vivo.

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.

Selective estrogen receptor modulator inhibits osteocyte apoptosis during abrupt estrogen withdrawal: implications for bone quality maintenance.

Estrogens exert positive effects on the quantity and quality of bone, including the maintenance of osteocytes through the inhibition of their apoptosis. Ideally, selective estrogen receptor modulators (SERMs) confer all of the positive bone-associated effects of estrogens without any adverse effects. In a similar way to estrogen, the raloxifene analog LY 117018 has been shown to prevent bone loss in ovariectomized (OVX) rats. In this study, we investigated whether the osteocyte-sparing effect of 17beta-estradiol can be mimicked by the SERM LY 117018 in a rat model of OVX. Twenty-four juvenile female rats were divided into four treatment groups: sham-operated (SHAM), OVX, OVX + 17beta-estradiol (OVX+E(2)), and OVX + LY 117018 (OVX+SERM). At 7 or 14 days following the start of treatment, the radius and ulna were removed. The percentage of apoptotic osteocytes, determined using an in situ nick-translation method, was increased (2.5-fold at 7 days and sixfold at 14 days) in the OVX group compared with SHAM in both the radius and ulna. Treatment of OVX animals with either 17beta-estradiol at a dose rate of 0.125 mg/kg/day or LY 117018 at a dose rate of 3 mg/kg/day prevented these increases in osteocyte apoptosis similarly. These observations demonstrate that LY 117018 exerts a powerful inhibitory effect upon osteocyte apoptosis directly after estrogen loss, in a similar way to the known effect of 17beta-estradiol replacement. These results point to the potential benefits of SERMs on both the quantity and quality of bone in E(2)-depleted rats.

The effect of in vivo mechanical loading on estrogen receptor alpha expression in rat ulnar osteocytes.

The presence of estrogen receptor alpha (ER alpha) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 microm distal to the midshaft of normal neonatal and adult male and female rat ulnas (n = 3 of each) and from adult male rat ulnas that had been exposed to 10 days of in vivo daily 10-minute periods of cyclic loading producing peak strains of either -3000 (n = 3) or -4000 microstrain (n = 5). Each animal ambulated normally between loading periods, and its contralateral ulna was used as a control. In animals in which limbs were subject to normal locomotor loading alone, 14 +/- 1.2% SEM of all osteocytes in each bone section were ER alpha positive. There was no influence of either gender (p = 0.725) or age (p = 0.577) and no interaction between them (p = 0.658). In bones in which normal locomotion was supplemented by short periods of artificial loading, fewer osteocytes expressed ER alpha (7.5 +/- 0.91% SEM) than in contralateral control limbs, which received locomotor loading alone (14 +/- 1.68% SEM; p = 0.01; median difference, 6.43; 95% CI, 2.60, 10.25). The distribution of osteocytes expressing ER alpha was uniform across all sections and thus did not reflect local peak strain magnitude. This suggests that osteocytes respond to strain as a population, rather than as individual strain-responsive cells. These data are consistent with the hypothesis that ER alpha is involved in bone cells' responses to mechanical strain. High strains appear to decrease ER alpha expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ER alpha levels and therefore impair the capacity for appropriate adaptive remodeling.

Osteocyte lacunar occupancy in the femoral neck cortex: an association with cortical remodeling in hip fracture cases and controls.

In adult humans, osteocytes die and disappear from their lacunae in the cortex of bones which remodel slowly, such as the proximal femur, and osteocyte death is particularly prevalent in the elderly. We have investigated the statistical determinants of osteocyte density in microscopic fields (0.71 mm2) within thin, complete femoral neck cross-sections cut from biopsies embedded in methyl methacrylate and stained with solochrome cyanine R. Lacunae were counted under phase contrast and osteocytes within lacunae were counted in the same fields under epifluorescence. The percentage of lacunae containing an osteocyte varied between 12.4% and 99.2%, according to subject and quadrantic region of the cortex examined. The microscopic determinants of field-specific osteocyte density included the porosity measured in the field itself and the regional measurement of the proportion of cortical canals bearing osteoid. There was significant variation between subjects and, within subjects, between cortical regions. Also the inferior region showed a significantly higher density of lacunae than the superior region (+8.2%; P = 0.013). However, cases of fracture were not significantly different from controls with respect to osteocyte lacunar occupancy after adjusting for osteoid-bearing canals and porosity. It is concluded that in subjects in their 7th-9th decades of age, osteocyte lacunar occupancy is statistically associated with bone turnover, implying that high turnover (locally young bone age) might favor lacunar occupancy (ln% osteoid; P = 0.021). Alternative explanations of the association are that porosity reflects a better nutritional supply via the vasculature or that porosity of the cortex is associated with osteocyte density through an effect of osteocytes on bone remodeling.

Bcl-2, tissue transglutaminase and p53 protein expression in the apoptotic cascade in ribs of premature infants.

Apoptotic cells of the human growth plate have not previously been demonstrated in situ. We have investigated the distribution of apoptotic cells in costosternal growth plates and bone of premature infants aged 4-11 d with a gestational age of approximately 26 wk. In addition, we investigated the immunolocalisation of apoptosis-related proteins within the growth plates and associated bone. A proportion of late hypertrophic chondrocytes and osteocytes within newly formed primary spongiosa showed evidence of highly fragmented DNA. The incidence of osteocyte apoptosis decreased as the distance from the chondroosseous junction increased. Tissue transglutaminase (tTG) expression was associated with apoptosis of osteocytes and hypertrophic chondrocytes. In contrast the presence of tTG was demonstrated in osteoblasts and bone lining cells but it did not colocalise with evidence of apoptosis. The anti-apoptotic gene product Bcl-2 was absent from the growth plate but was present in osteocytes. Visual assessment indicated a greater occurrence of the protein in cells occupying regions of low apoptosis. P53 was not demonstrated in the growth plate or bone. These findings would indicate that human growth plate chondrocytes appear to show little provision for ensuring cell longevity. In contrast osteocyte apoptosis appears negatively correlated with the skeletal distribution of Bcl-2 protein in the human infant, implying a potential selective vulnerability in individual cells. Lack of Bcl-2 and the high incidence of osteocyte apoptosis in the more rapidly remodelling bone of the human infant suggest a potential role of osteocyte apoptosis in the remodelling process.

Osteocyte function, osteocyte death and bone fracture resistance.

The function of the most numerous cell in bone, the osteocyte, has until recently been mysterious and at times controversial. There is now an emerging consensus that osteocytes modulate signals arising from mechanical loading and so direct the appearance and disappearance of bone tissue at the microscopic level, which allows bone as an organ both to grow and to adapt efficiently to the body's mechanical needs for strength with lightness. Osteocytes appear to use some molecular signalling pathways that are familiar from other tissues, such as the generation of nitric oxide and prostaglandins as well as directing cell-cell communication via gap junctions. They may also direct the removal of damaged or redundant bone through mechanisms linked to their own apoptosis or via the secretion of specialised cellular attachment proteins such as osteopontin. Osteocytes possess receptors for parathyroid hormone/parathyroid hormone related peptide and both oestrogen receptors alpha and beta. They also express molecules which in nerve cells are involved with glutamate neuro-transmission. At least some of these receptors and their ligands may regulate osteocyte apoptosis and modulate osteocyte signalling.

Characterization of 11beta-hydroxysteroid dehydrogenase activity and corticosteroid receptor expression in human osteosarcoma cell lines.

Studies in vitro and in vivo have shown that corticosteroids play an important role in bone physiology and pathophysiology. It is now established that corticosteroid hormone action is regulated, in part, at the pre-receptor level through the expression of isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which are responsible for the interconversion of hormonally active cortisol to cortisone. In this report we demonstrate 11beta-HSD activity in human osteoblast (OB) cells. Osteosarcoma-derived OB cell lines TE-85, MG-63 and SaOS-2 and fibrosarcoma Hs913T cells express the type 2 isoform of 11beta-HSD, as determined by reverse transcription polymerase chain reaction (RT-PCR) and specific enzyme assays. Enzyme activity was shown to be strictly NAD dependent with a Km of approximately 71 nM; 11beta-HSD type 1 mRNA expression and enzyme activity were not detected. All four cell lines expressed mRNA for the glucocorticoid receptor (GR) and mineralocorticoid receptor, but specific binding was only detectable with radiolabelled dexamethasone (Kd=10 nM) and not aldosterone. MG-63 cells had two to three times more GR than the other OB cells, which correlated with the higher levels of 11beta-HSD 2 activity in these cells. In contrast to the osteosarcoma cell studies, RT-PCR analysis of primary cultures of human OB cells revealed the presence of mRNA for 11beta-HSD 1 as well as 11beta-HSD 2. However, enzyme activity in these cells remained predominantly oxidative, i.e. inactivation of cortisol to cortisone (147 pmol/h per mg protein at 500 nM cortisol) was greater than cortisone to cortisol (10.3 pmol/h per mg protein at 250 nM cortisone). Data from normal human OB and osteosarcoma cells demonstrate the presence of an endogenous mechanism for inactivation of glucocorticoids in OB cells. We postulate that expression of the type 1 and type 2 isoforms of 11beta-HSD in human bone plays an important role in normal bone homeostasis, and may be implicated in the pathogenesis of steroid-induced osteoporosis.

Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells.

Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regulating osteoblast function is less clear and the isoforms of nitric oxide synthase (NOS) that are expressed by human osteoblasts have not been determined. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on osteoblast growth and differentiation. Low levels of NO were produced constitutively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was found in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produced only by hOB cells after stimulation with the cytokines IL-1beta, TNF-alpha, and IFN-gamma. The NOS inhibitor, L-NMMA, did not affect growth or alkaline phosphatase activity in unstimulated osteoblasts. Incubation of hOB cells with cytokines inhibited growth and stimulated alkaline phosphatase activity and these effects were abrogated by L-NMMA. Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO independent because they were not influenced by L-NMMA. Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect on osteoblast growth or differentiation under basal conditions. In contrast, IL-1beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting and differentiation-inducing effects on osteoblasts that were partly NO dependent, indicating that NO may act predominantly as a modulator of cytokine-induced effects on osteoblast function.

The role of estrogen in the control of rat osteocyte apoptosis.

We have previously shown that estrogen withdrawal by gonadotrophin-releasing hormone analogs (GnRHa) induces osteocyte death via apoptosis in human bone. Although it is likely that the increase in osteocyte death via apoptosis was related to the loss of estrogen, these experiments could not rule out a direct role for the GnRHa. Therefore, in this study, we have used a rat model of ovariectomy (OVX) to determine whether the effect of estrogen withdrawal extends to other species and to clarify the role of estrogen in the maintenance of osteocyte viability. Twelve 9-week-old rats were divided into three treatment groups: sham operated (SHAM) (n = 4), OVX (n = 4), and OVX + estrogen (E2) (25 microg/day) (n = 4). At 3 weeks following the start of treatment, tibial bones were removed. The percentage of osteocytes displaying DNA breaks, using an in situ nick-translation method, was significantly higher in the OVX group compared with the SHAM control in both cortical bone (10.04% vs. 2.31%, respectively; p < 0.0001) and trabecular bone (6.44% vs. 1.58%, respectively; p = 0.003). Addition of estrogen in the OVX animals completely abrogated the increase in osteocyte apoptosis in cortical bone (0.78%) and trabecular bone (1.17%). The percentage of apoptotic osteocytes decreased with increasing distance from the primary/secondary spongiosa interface below the growth plate in the OVX model and the OVX + E2 model. Nuclear morphology and electrophoresis of DNA confirmed the presence of apoptotic cells in the samples. In conclusion, OVX in the rat results in an increase in osteocyte apoptosis as a direct or indirect result of E2 loss. Addition of estrogen in the OVX animals prevents this increase in osteocyte apoptosis. These data confirm an important role for estrogen in the control of osteocyte apoptosis and the maintenance of osteocyte viability. Estrogen deficiency might, through compromising the viability of osteocyte networks, reduce the ability of bone to respond appropriately to loading.

Effect of estrogen suppression on the mineralization density of iliac crest biopsies in young women as assessed by backscattered electron imaging.

The effects of estrogen suppression on bone mineralization in young women were studied by quantitative backscattered electron (BSE) imaging of transiliac biopsies taken before and after treatment for endometriosis. Treatment (6 months) was with analogs of gonadotrophin releasing hormone (GnRH) given either alone (six paired biopsies), which resulted in a marked reduction in the levels of circulating estrogen, or in conjunction with tibolone, a synthetic steroid with estrogenic, progestrogenic, and androgenic properties (four paired biopsies). Estrogen withdrawal increased (p < 0.01) and concomitant tibolone treatment decreased (p < 0.05) the overall mean bone density. Estrogen withdrawal increased the fraction of bone with a high mineralization density [pretreatment: 0.236+/-0.007; GnRH: 0.279+/-0.009, mean +/- standard error of the mean (SEM); p < 0.01]. The concomitant addition of tibolone reversed these effects and increased the proportion of bone with a low mineralization density (pretreatment: 0.198 +/- 0.005; tibolone: 0.230 +/-0.008, p < 0.01). Using previously published data, the mean bone density was inversely correlated with mean wall thickness in cancellous bone (p = 0.030) and with the percentage of active osteons (p = 0.023) in cortical bone. Although treatment had similar effects on the mean bone mineralization density of cortical and cancellous bone, there were different distributions of mineralization between the two sites, with cancellous bone having more skewed and kurtotic distributions both before and after estrogen withdrawal. This study indicates that a short-term estrogen suppression results in the accumulation of bone with a higher mineralization density. As bone with a high mineral content has a decreased impact resistance, this might increase fracture risk. Understanding the cellular and biochemical mechanisms responsible for the local distribution of bone mineral when estrogen is withdrawn may allow the development of new strategies for maintaining bone quality after menopause.

The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone.

Estrogen withdrawal in women leads initially to rapid bone loss caused by increased numbers or activity of osteoclasts. We previously have noted apoptosis of lacunar osteocytes associated with conditions of high bone turnover. Therefore, in this study, we investigated whether the increased bone loss associated with GnRH analogue (GnRH-a)-induced estrogen withdrawal affects osteocyte viability in situ in a way that would be directly contrary to the effect of estrogens on osteoclast viability. Transiliac biopsies were obtained from six premenopausal women, between 30-45 yr old, diagnosed as having endometriosis. Biopsies were taken before and after 24 weeks of GnRH-a therapy. Biopsies were snap-frozen and cryostat sectioned. Osteocyte viability, determined by the presence of lactate dehydrogenase (LDH) activity, was reduced in all but one subject after treatment. Furthermore, in every subject, the proportion of osteocytes showing evidence of DNA fragmentation typical of apoptosis increased, as demonstrated using in situ DNA nick translation (P = 0.008). Gel electrophoresis of extracted DNA and morphological studies of chromatin condensation and nuclear fragmentation confirmed that changes typical of apoptosis were affecting the osteocytes. It was concluded that GnRH-a therapy caused a higher prevalence of dead osteocytes in iliac bone, probably caused by the increase in the observed proportion of osteocytes showing apoptotic changes. The capacity of bone to repair microdamage and to modulate the effects of mechanical strain is currently believed to be dependent on osteocyte viability. Our findings have therefore revealed a possible mechanism whereby estrogen deficiency could lead to increased bone fragility with or without an accompanying net bone loss.

Identification of apoptotic changes in osteocytes in normal and pathological human bone.

Previous work on bone growth and biomechanics suggests that osteocytes might sense the requirement for bone remodeling and signal to cells in the basic multicellular unit that undertake this function. The present study looked for evidence of apoptosis in human osteocytes in adult, pediatric, and pathological bone to compare these situations of differing levels of turnover and considered the possibility of a functional role for this death mechanism in bone modeling and remodeling. Apoptosis was identified in bone tissue by agarose gel electrophoresis of DNA (to demonstrate DNA ladders). In cryostat sections it was possible to visualize individual cells with fragmented DNA in situ using a modified nick translation technique (NT). In addition, visualization of apoptotic morphology was undertaken using light and electron microscopy. Adult femoral head and iliac crest bone showed no evidence of DNA ladders and very small numbers of osteocytes with DNA fragmentation using NT. In contrast, samples of pediatric calvaria, adult heterotopic bone, and osteophytes all displayed characteristic laddering of extracted DNA and showed evidence of potentially apoptotic osteocytes in situ using NT. In agreement with these findings, transmission electron microscopy showed numbers of osteocytes in infant calvaria with advanced chromatin condensation and cell shrinkage indicative of apoptosis. Since all three types of positive bone are involved in rapid matrix turnover, apoptotic changes in human osteocytes in vivo might be related in general terms to the modeling and remodeling activity level of the bone sampled. It was further found that the distribution of potentially apoptotic cells in the infant and pathological bone was anatomically nonuniform, raising the intriguing possibility of a functional relationship between bone turnover and the controlled cell death of osteocytes.

Dextran sulfate promotes the rapid aggregation of porcine bone-marrow stromal cells.

Despite the fact that cells of the mammalian stromal compartment of bone marrow have been shown to contain multipotential stem cells when studied in diffusion chambers it is notable that the same range of possible phenotypes (e.g., chondrocytic) has not been induced in freshly isolated marrow stromal cells in vitro. To investigate the possible role of glycoconjugates on phenotype expression, the effects of chondroitin sulfate, dermatan sulfate, dextran 500, and dextran sulfate on the cell morphology and differentiation of confluent porcine bone-marrow stromal-cell monolayers were studied. Of these glycosaminoglycan molecules only dextran sulfate induced confluent porcine bone-marrow stromal-cell monolayers to retract into tight, circular cell aggregates. Retraction began within 6 h, was complete after 3-5 days, and was dose dependent. Subsequent removal of dextran sulfate from the culture medium resulted in a return to a monolayer culture. Aggregated cells were essentially nonmitotic but dye exclusion indicated high cell viability. Dexamethasone, ascorbate, and beta-glycerophosphate produced no morphological change within 6 days when administered alone, but increased proliferation and aggregation in dextran sulfate-treated cultures. Immunocytochemistry of monolayer cultures revealed positive staining for type I but not type II collagen and addition of dexamethasone, ascorbate, and beta-glycerophosphate increased type I collagen deposition. In contrast, the centers of dextran sulfate-induced aggregates were positive for type II collagen, whereas type I collagen was only present at the periphery of the aggregates. Further addition of dexamethasone, ascorbate, and beta-glycerophosphate had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin gene related peptide stimulates differentiation of neonatal rat myogenic cultures.

The effect of the nerve derived peptide, calcitonin gene related peptide (CGRP) on myoblast fusion/differentiation was studied in rat myogenic cell cultures, using creatine kinase activity as an index of fusion. Addition of CGRP at 10(-7) M to myoblast cultures resulted in an enhancement of kinase activity after 11 days of treatment. This response was not accompanied by increased protein content of cultures implying an effect primarily on fusion rather than on myotube growth/size. Neither was it a direct effect on enzyme activity alone since no increase in creatine kinase activity occurred when CGRP was added to mature myotube cultures. These data suggest a role for CGRP in the myoblast fusion process itself and raise questions as to its importance in the growth and development of muscle in vivo.

Visualisation and selective elimination of a subpopulation of mitogen-responsive bone marrow stromal cells using bromodeoxyuridine and photo-induced cell killing.

Porcine bone marrow stromal cells (PBMSC) are a source of skeletogenic mesenchymal progenitor cells. Unfortunately, the heterogeneous nature of these cells in culture complicates the interpretation of their growth-factor responsiveness. We have therefore pulse-labelled mitogen-stimulated PBMSC with bromodeoxyuridine (BrdU) in order to study individual, growth-factor responsive cells in the presence of large numbers of nonresponsive PBMSC. Transfer of growing cells to low serum medium reduced BrdU labelling from 35% to 4% over a period of 24 hours. Subsequent addition of foetal calf serum (FCS) to serum-arrested cultures increased the number of BrdU positive cells to 54% by 16 hours. Addition of basic fibroblast growth factor (bFGF) to serum-arrested cultures induced DNA synthesis in 28% of cell by 16 hours after stimulation. In order to selectively eliminate mitogen responsive cells from mixed cultures, BrdU-substituted cells were photosensitised with bisbenzimide and exposed to bright light. BrdU-labelled PBMSC died within 20-40 hours of bisbenzimide treatment and subsequent illumination, whereas BrdU-labelled cells survived in the dark despite treatment with bisbenzimide. The photokilling procedure appeared to have no long term effect upon the viability of non-BrdU-labelled cells because if subconfluent cells were brought to serum arrest prior to photokilling, no change in DNA content relative to controls was observed after a subsequent 4 or 7 day incubation in Dulbecco's modified Eagle's medium (DMEM)/10% FCS.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of the beta-adrenergic agonist clenbuterol on growth and protein metabolism in rat muscle cell cultures.

Cultures were established from neonatal rat muscle cells, satellite cells, and L6 myoblasts and changes in protein metabolism were determined as development proceeded. For all three cell types, culture protein content increased with increasing myotube content. The beta-adrenergic agonist clenbuterol (added to a final concentration of 10(-7) M) significantly stimulated fusion (as indicated by creatine kinase activity) in neonatal muscle cultures and also increased culture protein content. This was associated with a stimulation in both the fractional (ks, percentage/day, +13%, P less than .05) and absolute (As, micrograms/day, +19%, P less than .05) rates of protein synthesis within 24 h after drug administration. At 48 h, As was increased by 42% above that of controls (P less than .01). In contrast, in satellite cell cultures, clenbuterol had no consistent effects on either protein accretion, creatine kinase activity, or protein synthesis (ks and As). Similarly, the drug had no stimulatory effect on protein synthesis and protein accretion in L6 myoblast or L6 myotube cultures (and no effect in neonatally derived fibroblast cultures). It is concluded that the fusion response to clenbuterol and, therefore, changes in protein metabolism and protein accretion are greatly dependent on the origin and genetic integrity of muscle cells.

Effects of the cyclo-oxygenase inhibitor, fenbufen, on clenbuterol-induced hypertrophy of cardiac and skeletal muscle of rats.

1. When rats were fed with clenbuterol for 7 days skeletal muscle mass increased by 21% in the tonic soleus and phasic plantaris muscles and a 16% hypertrophy of the heart was also induced. Fenbufen, fed to rats for the same period, blocked the hypertrophy of the heart but not that of the skeletal muscles. 2. When feeding of fenbufen commenced 3 days before the administration of clenbuterol, plasma prosta-glandin F2 alpha (PGF2 alpha) was reduced by 79%; there was again no effect of fenbufen on clenbuterol-induced increases in the RNA or protein content of plantaris, nor in the increased area of fast or slow twitch fibres in the soleus. In the heart the clenbuterol-induced increases in the RNA (+21%) and protein content (+20%) were totally inhibited. 3. The effects of clenbuterol on heart muscle appear to be mediated by a cyclo-oxygenase metabolite of arachidonic acid whilst the effects on skeletal muscle are not.