Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs.

BACKGROUND AND PURPOSE: Prolongation of the cardiac QRS complex is linked to increased mortality and may result from drug-induced inhibition of cardiac sodium channels (hNaV 1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling. EXPERIMENTAL APPROACH: Using the human cardiac gap junction connexin 43 (hCx43), a dye transfer 'parachute' assay to determine IC50 values for compound ranking was validated with compounds known to uncouple gap junctions. Uncoupling activity (and hNaV 1.5 inhibition by automated patch clamp) was determined in a set of marketed drugs and preclinical candidate drugs, each with information regarding propensity to prolong QRS. KEY RESULTS: The potency of known gap junction uncouplers to uncouple hCx43 was ranked (according to IC50 ) as phorbol ester>digoxin>meclofenamic acid>carbenoxolone>heptanol. Among the drugs associated with QRS prolongation, 29% were found to uncouple hCx43 (IC50 < 50 µM), whereas no uncoupling activity was observed in drugs not associated with QRS prolongation. In preclinical candidate drugs, hCx43 and hNaV 1.5 IC50 values were similar (within threefold). No consistent margin over preclinical Cmax (free) was apparent for QRS prolongation associated with Cx43 inhibition. However, instances were found of QRS prolonging compounds that uncoupled hCx43 with significantly less activity at hNaV 1.5. CONCLUSION AND IMPLICATIONS: These results demonstrate that off-target uncoupling activity is apparent in drug and drug-like molecules. Although the full ramifications of Cx inhibition remain to be established, screening for hCx43 off-target activity could reduce the likelihood of developing candidate drugs with a risk of causing QRS prolongation.

Inhibition of TGF-beta 3 (but not TGF-beta 1 or TGF-beta 2) activity prevents normal mouse embryonic palate fusion.

The critical stage of palatal development is the fusion of apposing individual palatal shelves. Palatal shelves, from the day 13 murine embryo, cultured in vitro fuse normally in the absence of exogenous factors. Therefore, some endogenous factor(s) is responsible for the normal fusion process. Prevention of mature TGF-beta 3 activity during a specific time window of development in palate organ cultures, either by antisense oligodeoxynucleotides or neutralizing antibody, resulted in failure of palate fusion. Northern analysis was used to demonstrate that the antisense treatment down-regulated TGF-beta 3 mRNA. Inhibition of TGF-beta 1 or -beta 2 activity (by either antibodies or antisense oligodeoxynucleotides) had no such effect on palate development and palate fusion was normal. These data indicate an isoform specific role for TGF-beta 3 in palatal fusion.

The distribution of epidermal growth factor binding sites in the developing mouse palate.

The distribution of epidermal growth factor (EGF) receptors in the developing mouse palate was mapped using 125I-EGF labeling of paired palate organ cultures. 125I-EGF binding sites were localized throughout the palate mesenchyme except in a region immediately adjacent to the midline seam. The EGF receptor was detected in all palatal epithelia at the beginning of culture, but as seam formation and subsequent degeneration took place it was down-regulated in the medial edge epithelia. Using submerged culture the mechanism of this down-regulation was investigated by treating with various growth factors such as EGF, basic fibroblast growth factor (bFGF), insulin-like growth factor-II (IGF-II) and transforming growth factors alpha and beta (TGF-alpha, TGF-beta). Conventional Trowell organ culture was not used because it was observed that the Millipore filter blocked growth factor uptake. All three TGF-beta isoforms accelerated palate fusion and TGF-beta 1 reduced 125I-EGF binding throughout the palate, suggesting a potential level of regulation during palatogenesis. Contrary to previous reports, EGF treatment in the absence of serum prevented palatal shelf fusion, and also down-regulated subsequent 125I-EGF binding.

Localisation of acidic and basic fibroblast growth factors during mouse palate development and their effects on mouse palate mesenchyme cells in vitro.

The distribution of acidic and basic fibroblast growth factors (aFGF, bFGF) was mapped during mouse embryonic palate development. Generally, they localised most intensely in the basement membrane and epithelia rather than the mesenchyme. Localisation was predominantly restricted to the palatal nasal, and medial edge epithelia. Staining was particularly intense in the medial edge epithelia at the time of mid-line epithelial seam formation. Intense staining persisted in the epithelia of the degenerating seam and later in the oral and nasal epithelial triangles. Mouse embryonic palate mesenchyme (MEPM) cells cultured in vitro on a variety of substrata (on plastic, on the surface of a collagen gel and within a collagen gel) responded to treatment with aFGF or bFGF. These responses were modulated by the culture substratum. The FGFs stimulated MEPM cell proliferation on plastic and on collagen, but inhibited cell growth in collagen. The FGFs had little effect on protein production when cells were cultured on plastic, but caused a large reduction in on-collagen and incollagen cultures. This reduction was greater in collagenous than non-collagenous proteins. Generally, treatment with FGFs stimulated the production of glycosaminoglycans (GAGs), particularly hyaluronan (HA) and dermatan sulphate (DS). In addition, the size class of HA was shifted to a higher molecular weight form. These data indicate that aFGF and bFGF may play a role in modulating mesenchymal cell matrix biosynthesis, so facilitating palatal epithelial seam degeneration.

Differential expression of insulin-like growth factors I and II (IGF I and II), mRNA, peptide and binding protein 1 during mouse palate development: comparison with TGF beta peptide distribution.

Development of the mammalian secondary palate involves a series of epithelial mesenchymal interactions: during one of these, a mesenchymal signal specifies regionally distinct palatal epithelial differentiation. Extracellular matrix molecules and soluble growth factors may be involved in this signalling process. In this study, we have mapped the expression of the genes for insulin-like growth factors (IGF I and II), the peptides they encode, and the IGF binding protein 1 (IGF BP-1) during murine palatogenesis (embryonic days (E) 12-15). IGF-I gene expression was below detectable levels in the craniofacial region at all ages. IGF-I peptide was at the threshold of immunocytochemical detection and widely distributed in the palatal mesenchyme, decreasing in staining intensity from E12 to E14. By contrast, IGF-II mRNA was intensely localised in several tissues. IGF-II gene expression within the forming palate was developmentally regulated. In the vertical palatal shelves (E12 to E13) IGF-II gene expression was absent. On early E14, in the horizontal prefusion palate, significant expression was present in the palatal mesenchyme, but not the epithelium. Once palatal fusion had occurred, mesenchymal expression fell rapidly to undetectable levels. IGF-II mRNA was next detectable in the secondary palate on late E15 at sites of membranous bone formation. By contrast to the mRNA distribution, IGF-II peptide was localised predominantly in the palatal epithelia (particularly the nasal and medial edge epithelia) but also in the mesenchyme of the E14 prefusion palate. Significantly, the IGF binding protein had a similar distribution pattern to the IGF-II peptide. At all ages, the developing tongue myotubes labelled heavily for IGF-II mRNA, protein and binding protein. These data suggest that IGF-II may play a localised paracrine role during murine palatogenesis, perhaps in the mesenchymal signalling of epithelial differentiation. IGF-II may also serve to coordinate the development of the tongue and palate. The distribution of IGF-II peptide was very similar to that of TGF-beta, suggesting a possible interactive role of these growth factors during palate development. Finally, evidence that the IGF-II gene is imprinted (Ferguson-Smith et al. 1991) and may be the target for uniparental disomy in the human Beckwith Wiedemann syndrome (Henry et al. 1991), which is characterised by the overgrowth of tissues (especially the tongue) expressing IGF II in the embryo, indicates the necessity of reanalysing human cleft palate families for disruption (including uniparental disomy) of the genes encoding IGFs, their receptors and binding proteins.

Modulation of the epidermal growth factor receptor of mouse embryonic palatal mesenchyme cells in vitro by growth factors.

A single class of high-affinity receptors for EGF were detected on mouse embryonic palatal mesenchyme (MEPM) cells cultured in vitro. The degree of confluence of the cultured cells did not affect the number or affinity of the binding sites. Culture of MEPM cells in the presence of bFGF, IGF-II or TGF-beta 1 induced changes in 125I-EGF binding. TGF-beta 1 caused a marked reduction in binding to 40% of control levels. This reduction was achieved after 2 h and persisted for 24 h after addition of the growth factor. IGF-II induced a similar reduction but this effect was transitory; after a 12 h pretreatment with IGF-II, binding was restored to control levels. The effects of bFGF were biphasic. Initially, a short pre-treatment period (3-5 h) with bFGF caused a small reduction in 125I-EGF binding; longer periods of pre-incubation (24 h) resulted in a large increase in receptor number. Pre-incubation in medium containing both bFGF and TGF-beta 1 resulted in a decrease in EGF binding. Thus, TGF-beta 1 negated the large increase in receptor number induced by bFGF alone. Changes in receptor number were usually, but not always, directly related to changes in the biological activity of EGF, as assessed by a thymidine incorporation assay. This study highlights the possible interactive role of growth factors known to be present in the developing palate.

The effects of transforming growth factor-beta 1 on protein production by mouse embryonic palate mesenchymal cells in the presence or absence of serum.

Mouse embryonic palatal mesenchyme cells were cultured on a variety of substrata (plastic, on a collagen gel or within a collagen gel). On each substratum TGF-beta 1 (1 ng/ml) inhibited cell proliferation. Cells cultured within a collagen gel had the lowest rate of proliferation, but were metabolically the most active in terms of incorporation of [3H]-proline into both collagenous and non-collagenous proteins. TGF-beta 1, in the presence of 2.5% donor calf serum stimulated the production of fibronectin and the major collagen types I, III and V. However, in serum-free medium, TGF-beta 1 induced a large reduction in total collagen production, mainly due to an effect on type I collagen, whilst stimulating production of some non-collagenous proteins. Experiments involving combinations of TGF-beta 1 with other growth factors suggested that the different effects of TGF-beta 1 on collagen production, in the presence and absence of serum, may be due to an interaction with platelet-derived growth factor.

Comparative biochemistry of mouse and chick secondary-palate development in vivo and in vitro with particular emphasis on extracellular matrix molecules and the effects of growth factors on their synthesis.

A biochemical study analysing the wet weight, dry weight, water, protein and DNA content, collagen and GAG composition of all stages of the developing secondary palate in vivo and in vitro was undertaken to investigate differences between a species in which the palatal shelves elevate (mouse) and one in which they do not (chick). The effects of EGF, bFGF, PDGF and TFG-beta 1 on collagen and GAG synthesis by cultured mouse and chick palatal shelves of different embryonic stages were also studied. The total GAG content of developing mouse palatal shelves decreased with developmental time; heparan sulphate proteoglycan formed the major species in early palates but hyaluronan was the major species in mid-late palates. There was a peak of hyaluronan synthesis in embryonic palatal shelves in vitro at day 13 (T.21), i.e. immediately before shelf elevation. By contrast the total GAG content of chick palates increased with development; chondroitin-6-sulphate formed the major GAG species and there was no peak in hyaluronan synthesis. The water content of developing murine palates rose rapidly at day 14 (T.22), i.e. the time of shelf elevation. No such peak was seen in the chick, where the water content rose exponentially with developmental time. Mouse palates synthesized chondroitin-4-sulphate and novel proteins around the time of shelf elevation; chick palates synthesized chondroitin-6-sulphate and no novel proteins at any developmental stage. Collagen synthesis also peaked in vitro in T.21 murine palates. EGF markedly stimulated murine palatal collagens and GAG synthesis between stages T.20-T.22, but had no effect thereafter. Basic FGF had similar but smaller stage-related effects. PDGF had no effect on mouse palatal collagen and GAG synthesis whilst TGF-beta 1 inhibited GAG synthesis at T.21. The ratios of collagens I, III and V produced by mouse palates were unaltered by the growth factors. All the growth factors had no effect on chick palatal collagen synthesis at any stage and minimal effect on GAG synthesis; TGF-beta 1 stimulated it in early but inhibited it in mid- to late-stage chick palates. These data indicate that extracellular matrix molecule metabolism within the palate is markedly different in the two species studied and suggest that the differing profiles of such molecules may be regulated at certain developmental stages by specific growth factors.

Mesenchymal influences on epithelial differentiation in developing systems.

Mesenchyme tissue: cells, matrix and soluble factors, influence the morphogenesis, proliferation and differentiation of a variety of embryonic epithelia, e.g. in the tooth, skin, mammary and salivary glands. Mesenchyme derivatives also 'maintain' adult epithelia, e.g. the local proliferation rate and cytokeratin composition of oral mucosa. Abnormalities in such epithelial-mesenchymal interactions lead to a variety of pathologies such as premalignant lesions, e.g. leukoplakia, tumours and psoriasis, whilst therapeutic manipulation of such interactions can prevent the exfoliation of dental implants. In all of these systems it is critical to understand, at the cellular and molecular levels, how the mesenchyme signals to the epithelium and how the latter processes and responds to such signals. We have investigated such questions using the developing embryonic palate both as a model system and as an important organ: failure of mesenchymal signalling leads to the common and distressing birth defect of cleft palate. Bilateral palatal shelves arise from the maxillary processes of embryonic day 11 (E11) mice, grow initially vertically down the sides of the tongue, elevate on E13.8 to a horizontal position above the dorsum of the tongue and fuse with each other in the midline on E14. The medial edge epithelia of each shelf fuse with each other to form a midline epithelial seam, suprabasal cells die, and the basal (stem) cells synthesize extracellular matrix molecules and turn into mesenchymal cells. Simultaneously the oral epithelia differentiate into stratified squamous cells and the nasal epithelia into pseudostratified ciliated columnar cells. Oral, medial and nasal epithelial differentiation is specified by the underlying mesenchyme in vivo and in vitro. Signalling involves a bifurcating action of a combination of soluble growth factors e.g. TGF-alpha, TGF-beta, PDGF and FGF on palatal epithelia and mesenchyme. These factors stimulate the synthesis of specific extracellular matrix molecules by palate mesenchyme cells, and the appearance of receptors for such molecules on epithelial cells. In this way, a combination of mesenchymal soluble factors and extracellular matrix molecules direct palatal epithelial differentiation. These signals act on epithelial basal (stem) cells, causing them to synthesize unique proteins, which may direct subsequent differentiation of daughter cells. In the most extreme example, namely the medial edge epithelia, these signals result in the basal epithelial cells transforming into mesenchymal cells, thus demonstrating that they are indeed multipotential stem cells.

The effect of manipulating growth in sheep by diet or anabolic agents on plasma cortisol and muscle glucocorticoid receptors.

1. The cortisol status (total plasma cortisol concentration, free cortisol concentration, transcortin capacity) and the characteristics of skeletal muscle binding for cortisol and dexamethasone were examined in female lambs either implanted with Zeranol or trenbolone acetate or whose dietary intake was restricted. 2. The skeletal muscle glucocorticoid receptor had a high affinity for the glucocorticoid triamcinolone (relative binding affinity 0.85) and cortisol (relative binding affinity 0.51) with virtually no affinity for trenbolone. 3. Trenbolone acetate treatment reduced the binding capacity of sheep skeletal muscle for cortisol within 2 d of implantation. The other treatments had little effect except a small reduction in the animals where food intake was restricted. Similarly, binding capacity for dexamethasone was reduced by trenbolone acetate treatment but was not affected by the other treatments. This reduction in trenbolone acetate-treated animals is, at least in part, due to a reduction in glucocorticoid receptors. 4. Transcortin capacity was elevated by Zeranol treatment but reduced with diet restriction or trenbolone treatment. 5. No support for the suggestion of free cortisol concentration being important in the growth-promoting mechanism of trenbolone or Zeranol was obtained. 6. Although insulin concentrations were not significantly altered by treatment (P greater than 0.05), when combining all the animals there was evidence of a negative correlation between total cortisol/insulin value (P less than 0.05) or free cortisol:insulin value and growth rate (P less than 0.001). Free cortisol was negatively correlated to growth rate (P less than 0.05) and transcortin capacity positively correlated (P less than 0.01).