To use the equilibrium established by creatine kinase (CK) to determine hepatic free ADP levels, the transcriptional control elements of the transthyretin gene were used to direct expression of the CK B isozyme to the livers of transgenic mice. Activities of CK ranging from 80-250 pmol per min per g (wet weight) were detected in liver extracts from five founder mice. The CK activity was stably transmitted to subsequent generations. Isozyme gels and immunoblots confirmed that the activity detected in extracts was due to the B isozyme of CK. Immunohistology indicated that the protein was expressed uniformly throughout the liver and was localized primarily to the cytoplasm. 31P NMR spectroscopy was used to detect the metabolic product of the CK reaction, phosphocreatine, demonstrating that the enzyme was active in vivo. The phosphocreatine level fell rapidly during anoxia (tQ12 = 1 min), indicating that the CK reaction was integrated into hepatic energy metabolism. The equilibrium established by CK was used to calculate a hepatic free ADP level of 0.059 ± 0.004 ,umol/g (wet weight). In vivo NMR studies of these mice will be valuable for studying the role of free ADP in regulating liver metabolism.Adenosine diphosphate (ADP) is an important regulator of many metabolic pathways in vitro, including oxidative phosphorylation (1), glycolysis (2), gluconeogenesis (2), and ion transport (3). Due to difficulties in measuring free ADP levels in intact liver it has been impossible to assess the role of this key metabolite in regulating hepatic metabolism in vivo (4). To study changes that may occur during perturbations of liver metabolism, ADP is usually assayed from whole cell extracts or from liver cells after subcellular fractionation into cytoplasmic and mitochondrial compartments (4-6). However, it is generally acknowledged that for metabolites such as ADP, the concentrations of which are low and comparable to the number of intracellular binding sites, measurements from extracts lead to an overestimation of free metabolite levels (4).Hans Krebs introduced the idea of using cellular reactions near equilibrium to determine the levels of free metabolites at low concentrations (7). This technique relies on measuring compounds present in relatively high concentrations that are in chemical equilibrium with the metabolite of interest. Ideally, a single equilibrium using metabolites that can be monitored in vivo should be used. The reaction catalyzed by creatine kinase (CK), phosphocreatine (PCr) + MgADP + H+ = MgATP + creatine, is the method of choice for the determination of ADP (4). The value of the equilibrium constant is known for a variety of conditions, and free ADP levels can be calculated from in vivo nuclear magnetic resonance (NMR) measurements of PCr, ATP, creatine, Mg2+, and pH. This approach has been successfully used to study the role of ADP in metabolic regulation in heart (8), muscle (9), and brain metabolism (10). However, it has not been useful for liver due to the lack of significant levels of CK.In...

When primary mouse mammary epithelial cells (PMME) are cultured on a basement membrane type matrix, they undergo extensive morphogenesis leading to the formation of 3-dimensional alveoli-like spherical structures surrounding a closed lumen. We show for the first time that cells cultured on basement membrane-type matrix express high levels of whey acidic protein (WAP) mRNA and secrete the protein into the lumen. The expression of WAP appears to be dependent upon the formation of the alveoli-like spheres: prevention of sphere formation by fixation or drying of the matrix abolishes the expression of WAP. Co-culturing PMME on native and fixed basement membrane matrix indicates that the suppression of WAP expression is dominant, thereby revealing the existence of a diffusible inhibitor(s). The inhibitory activity is present in the conditioned medium of PMME cultured on plastic surface and floating collagen gels, substrata that do not form alveoli and do not allow WAP expression. These findings are consistent with the model that the synthesis, or the action, of the WAP inhibitory factor is regulated by the tissue-like multicellular organization of mammary cells. When PMME do not have correct 3-dimensional structures, one (or more) inhibitor is secreted into the medium which suppresses WAP expression by an autocrine or paracrine mechanism. Nuclear run-on experiments suggest that the suppression of WAP expression is posttranscriptional. These results have obvious bearings on the understanding of the mechanisms by which cell-cell and cell-extracellular matrix interaction regulate tissue specific gene expressions.