Transfectants

Another member of the FGF family, FGF-4, protects against radiation-iaduced ceU killing and enhanced the G2 arrest when overexpressed ia a human adrenal cortical carcinoma ceU line (196). This effect is again manifested as the appearance of a shoulder on the survival curve, although neither the control nor the transfected cells undergo radiation-iaduced apoptosis. No differences ia the yield or repair of either SSBs or DSBs have been observed. 

The different furanones 104 were tested for their potency as inhibitors of PGE2 production both in transfected Chinese hamster ovarian (CHO) cells expressing human COX-2 and in human whole blood. Compound 104r proved to be an orally active and selective COX-2 inhibitor that is devoid of the ulcerogenic effect at >100 times the dose for antiinflammatory, analgesic, and antipyretic effects (99BMC3187). 

FIGURE 2.4 Occupancy-response curves for P-adrenoceptor agonists in transfected CHO cells. Occupancy (abscissae) calculated from binding affinity measured by displacement of [125I]iodocyanopindolol. Response measured as increases in cyclic AMP. Drawn from [3],... 

FIGURE 2.11 Receptor-occupancy curves for activation of human calcitonin type 2 receptors by the agonist human calcitonin. Ordinates (response as a fraction of the maximal response to human calcitonin). Abscissae (fractional receptor occupancy by human calcitonin). Curves shown for receptors transfected into three cell types human embryonic kidney cells (HEK), Chinese hamster ovary cells (CHO), and Xenopus laevis melanophores. It can be seen that the different cell types lead to differing amplification factors for the conversion from agonist receptor occupancy to tissue response. 

FIGURE 3.11 Constitutive activity in melanophores expressing hCTR2 receptor, (a) Basal melanophore activity, (b) Effect of transfection with human cDNA for human calcitonin receptors (16 j-ig/ml). (c) Concentration response curve for cDNA for human calcitonin receptors (abscissae as log scale) and constitutive activity. Data redrawn from [27]. 

FIGURE 3.12 Dependence of constitutive receptor activity as ordinates (expressed as a percent of the maximal response to a full agonist for each receptor) versus magnitude of receptor expression (expressed as the amount of human cDNA used for transient transfection, logarithmic scale) in Xenopus laevis melanophores. Data shown for human chemokine CCR5 receptors (open circles), chemokine CXCR receptors (filled triangles), neuropeptide Y type 1 receptors (filled diamonds), neuropeptide Y type 2 receptors (open squares), and neuropeptide Y type 4 receptors (open inverted triangles). Data recalculated and redrawn from [27],... 

FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor, (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors, (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and lOOnM. Dose-response curve for the effects seen in panel B. 

FIGURE 5.6 Calcitonin receptor responses, (a) Real-time melanin dispersion (reduced light transmittance) caused by agonist activation (with human calcitonin) of transfected human calcitonin receptors type II in melanophores. Responses to 0.1 nM (filled circles) and lOnM (open circles) human calcitonin, (c) Dose-response curves to calcitonin in melanophores (open circles) and HEK 293 cells, indicating calcium transient responses (filled circles). 

With the advent of molecular biology and the ability to express transfected genes (through transfection with cDNA) into surrogate cells to create functional recombinant systems has come a revolution in pharmacology. 

The first idea to consider is the effect of receptor density on sensitivity of a functional system to agonists. Clearly, if quanta of stimulus are delivered to the stimulus-response mechanism of a cell per activated receptor the amount of the total stimulus will be directly proportional to the number of receptors activated. Figure 5.8 shows Gi-protein-mediated responses of melanophores transiently transfected with cDNA for human neuropeptide Y-l receptors. As can be seen from this figure, increasing receptor expression (transfection with increasing concentrations of receptor cDNA) causes an increased potency and maximal response to the neuropeptide Y agonist PYY. 

FIGURE 5.10 Effects of co-expressed G-protein (G ) on neuropeptide NPY4 receptor responses (NPY-4). (a) Dose-response curves for NPY-4. Ordinates Xenopus laevis melanophore responses (increases light transmission). Ordinates logarithms of molar concentrations of neuropeptide Y peptide agonist PYY. Curves obtained after no co-transfection (labeled 0 jig) and co-transfection with cDNA for Gai6. Numbers next to the curves indicate jig of cDNA of Ga]g used for co-transfection, (b) Maximal response to neuropeptide Y (filled circles) and constitutive activity (open circles) as a function of pg cDNA of co-transfected G g. 

FIGURE 5.11 Microphysiometry responses to 1 nM human calcitonin, (a) Responses obtained from HEK 293 cells stably transfected with low levels of human calcitonin receptor (68 pM/mg protein). Response is sustained, (b) Response from HEK 293 cells stably transfected with high levels of receptor (30,000 pM/mg protein). Data redrawn from [8],... 

Recombinant assays have revolutionized pharmacology and now functional systems can be constructed with engineered levels of responsiveness (i.e., through difference in receptor levels or co-transfection of other proteins). 

FIGURE 11.2 Paired experimental data. Values of constitutive calcitonin receptor activity [1 -(Tr/Tj) units] in transiently transfected melanophores. Five separate experiments are shown. Points to the left indicate the basal level of constitutive activity before (filled circles) and after (open circles) addition of 100 nM AC512 (calcitonin receptor inverse agonist). Lines join values for each individual experiment. Points to the right are the mean values for constitutive activity in control (filled circles) and after AC512 (open circles) for all five experiments (bars represent standard errors of the mean). Data shown in Table 11.3. 

Values are levels of constitutive activity [1 — (Tr/Tj)] for four individual transfection experiments (denoted X ) x2 are the constitutive receptor activity values after exposure to AC512 in the same experiment. 

Differences in constitutive calcitonin receptor activity in four separate receptor transfection experiments (xj to x ). Four readings of activity taken for each transfection. 

FIGURE 11.3 One-way ANOVA (analysis of variance). One-way analysis of variance of basal rates of metabolism in melanophores (as measured by spontaneous dispersion of pigment due to G,.-protein activation) for four experiments. Cells were transiently transfected with cDNA for human calcitonin receptor (8 j-ig/ml) on four separate occasions to induce constitutive receptor activity. The means of the four basal readings for the cells for each experiment (see Table 11.4) are shown in the histogram (with standard errors). The one-way analysis of variance is used to determine whether there is a significant effect of test occasion (any one of the four experiments is different with respect to level of constitutive activity). 

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