To observe morphological changes in living cells as a response to the two isomers of compound 7, chinese hamster ovary cells (CHO K1) were adherently grown to confluence in 96-well plates with coplanar gold-film electrodes deposited on the bottom of each well. When non-invasive AC currents are applied, morphological changes are sensitively detectable at an AC frequency of 12 kHz. Altering intracellular cAMP-levels and the associated downstream signaling is assessed as changes in the time course of the impedance magnitude |Z|. Prior to substance addition, E-7 was irradiated at 400 nm and Z-7 at 505 nm to reach the PSDs with the maximum ratios for E and Z. Time courses of the impedance were zeroed to the data point preceding the addition of the modulating substances or corresponding controls for better comparison. The resulting time-resolved impedance data are shown in Figure . Forskolin, a membrane permeable activator of the adenylyl cyclase (AC), served as positive control (green curve). When AC activity is enhanced by forskolin inducing a rise of cAMP levels within the cells, the impedance drops by more than 2000 Ω within 10 min, followed by stabilization and slow recovery. The latter might be due to a loss of forskolin activity by degradation or the cells simply run out of ATP as the biosynthetic precursor of cAMP. The isomers of 7 were expected to create similar effects as forskolin as they represent direct mimics of cAMP. When comparing the two isomers of 7 at a final concentration of 500 µM, the impedance traces show differences of approximately 1200 Ω. The E-isomer levels to about -1500 Ω (red curve) no more than 10 min after start of exposure. Considering an average signal of 300 Ω as obtained by incubation with the Z-isomer (blue curve), the deviation from the control was minimal, revealing the E-isomer to be more biologically active than its counterpart when equal concentrations are applied. Following up on these initial experiments (cf. Fig. ) indicating cAMP-dependent intracellular signaling cascades as the source for a decrease in impedance when the E-isomer of 7 was applied, we performed additional experiments with a collection of well-established pharmacological modulators of intracellular cAMP levels. The aim of these experiments was to demonstrate by several independent molecular mechanisms that impedance changes indeed mirror intracellular cAMP levels. Figure illustrates four distinct ways of modulating intracellular cAMP levels and the associated signaling cascade. In resting state, the second messenger cAMP is continuously formed and degraded by constitutively active AC and PDE, resulting in baseline steady-state levels that the cells can readily control. Forskolin elevates the cAMP levels by activating the membrane-bound adenylyl cyclase (AC, blue arrows), fostering the conversion of adenosine 5'-triphosphate (ATP) to cAMP and pyrophosphate. 3-Isobutyl-1-methylxanthine (IBMX), on the other hand, is known to inhibit the phosphodiesterase (PDE, green arrows), an enzyme catalyzing the hydrolysis of the phosphodiester bond in cAMP. Changes of AC or PDE activities resulting from their interaction with forskolin or IBMX are indicated by arrow length (Fig. ). Intracellular cAMP levels are encoded by the partial filling of the cells (red). As a third and most direct experimental means, a membrane permeable analog of cAMP -8-(4-chlorophenylthio)adenosine-3', 5'-cyclic monophosphate (8-CPT-cAMP, pink arrow) -has been used to directly induce a cAMP-mediated cell response and the associated change of the impedance profile. Due to its high membrane permeability compared to endogenous cAMP, it is often used as a tool for studying the cAMP cascade in vitro. Incubating CHO cells, overexpressing the neuropeptide receptor 4 (Y4R), with its endogenous ligand, the human pancreatic polypeptide (hPP), reduces cAMP levels. Y4R is a G-protein coupled receptor (GPCR) that affects intracellular signaling by reducing AC activity, thus leading to a reduction of intracellular cAMP. We applied all the above mentioned modulators of intracellular cAMP and recorded the cell response by ECIS readings. The corresponding time-dependent impedance profiles for the various modulators are summarized in the Supporting Information. In short: A concentration-dependent decrease of the impedance was observed for forskolin, IBMX, and 8-CPT-cAMP. In contrast, a dose-dependent increase of impedance was observed after stimulating the cells with hPP (SI, Fig. ). Since forskolin, IBMX, and 8-CPT-cAMP are compounds known to accumulate intracellular cAMP, while hPP does the opposite, there is compelling evidence that impedance readings indeed mirror the changes in intracellular cAMP -mediated by effector proteins further downstream in the signaling cascade. Downstream of cAMP there are several effector proteins that are controlled in their activity by cAMP levels, like for instance, effector proteins activated by cAMP (EPACs) or protein kinase A (PKA). Modulation of any of these proteins is directly or indirectly responsible for the observed changes in cell morphology as they are picked up by ECIS readings. Thus, impedance measurements provide direct and real-time access to the cAMP levels in CHO-K1 cells.