Isolated tissue assays

Commonly overlooked when performing these assays is the possibility of metabolism to an active form that can be assessed in this in vitro model. The test material should be tested in both original and metabolized forms. The metabolized form is prepared by incubating a 5% solution (in aerated Tyrodes) or other appropriate physiological salt solution with strips of suitably prepared test species liver for 30 min. A filtered supernatant is then collected from this incubation and tested for activity. Suitable metabolic blanks should also be tested. This is a classic nervous system pharmacology approach. 

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This example emphasizes the need for data upon the intraventricular potency of narcotic analgesics whereby transport factors would largely be eliminated and it is to be hoped that procedures for the direct administration of drugs to the brain (technically difficult and often unreliable in their present state [281]) will be improved in the near future. Alternatively, the development of a meaningful isolated tissue assay for analgesia would do much to improve our interpretation of structure-activity relationships in this field (see page 262). 

Figure 6.10b shows a pattern of antagonism often observed in isolated tissue studies but not so often in cell-based assays. Saturation of uptake systems for the agonist or saturation of an adsorption site for the agonist can account for this effect. The linear portion of the regression can be used to estimate the pKB or the pA2. If there is a loss of concentration dependence of antagonism, as seen in... 

Functional assays (continued) dissimulation, 87-88 group I, 81-82 group II, 81, 83 group III, 83-84 group IV, 84 isolated tissues for, 81 membrane, 81 output from, 79... 

Cytokine profiling has also been measured as a function of changes in cytokine mRNA expression using either reverse transcription polymerase chain reaction (RT-PCR) [87, 91-93] or ribonuclease protection assay (RPA) [94-97], Measurement of cytokine transcripts by RT-PCR revealed that prolonged exposure to TMA induced increased levels of IL-4 mRNA expression compared with treatment with DNCB [87,92-93]. However, expression of the type 1 cytokine IFN-y by DNCB-activated LNC was variable and failed to discriminate between contact and respiratory allergens [87,91,93). A similar profile was observed for freshly isolated tissue analyzed by RPA. This somewhat less... 

It should be noted that much of the early SAR data on opioids was generated using whole-animal analgesic assays rather than with isolated tissue preparations rich in receptor subtypes. 

The results suggest that the affinities of cyprodime and its 3-hydroxy analogues (34)-(38), measured in binding assays and by bioassay, agree for the H receptor site but not for k and 8 receptor sites. A possible explanation is that the receptors in guinea-pig brain and the isolated tissues may be different and are distinguishable by this series of compounds. 

Efforts to predict the risk of potential QT prolongation over the last decade have resulted in the number of preclinical in vitro and in vivo methodologies. In vitro methods to predict QT liabilities range from simple recombinant cellular assays to disaggregated cells, isolated tissues and perfused heart preparations." ... 

ECE inhibitors can be evaluated in enzyme assays, isolated tissues and whole animals. A range of tissues (for example, vascular endothelium, vascular smooth muscle, lung) demonstrate phosphoramidon-sensitive ECE activity and provide a choice of starting points for an enzyme assay for ECE inhibitors. Partially purified ECE, such as that from rabbit lung, is... 

Figure 8.3). Thiorphan is less effective than phosphoramidon and enalapril is inactive. Thus, there is an apparent correlation between the ECE studied in the enzyme assay and that in the isolated tissue.

Isolated tissue / whole organ assays Isolated rabbit heart Purklnje fiber assay Isolated ventricular tissue... 

More rapid responses to a stressor are observed in assays based on isolated tissues or cells, or microorganisms where contact with a pollutant is identified through an interaction with a molecular target and the expression of biomarkers. 

Adenyi cyclase - The synthesis of cyclic AMP from ATP is catalyzed by the enzyme adenyi cyclase, which itself is responsive to hormone stimulation in various tissues . Catecholamines have been shown to stimulate the formation of cyclic AMP in a variety of tlssues (Table 1 ), and adenyi cyclase has been shown to be wide-spread in various tissues . Since procedures for the isolation and assay of this enzyme and measurement of the products of its reaction are available - , direct effects of analogues of ATP and/or cyclic AMP (or hormones) on the enzyme can be determined in an in vitro system. 

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