Muscarinic Acetylcholine Receptors: Calcium Imaging With Fluo-4 AM

Muscarinic acetylcholine (mAch) receptors are instrumental in signal transmission across neurons, glandular secretion and muscle contraction. Furthermore, acetylcholine produced in the nervous system regulates cell growth, survival and apoptosis. However, in some disorders, the functioning of this system is compromised, thus necessitating the development of drugs modulating these functions (Matera and Tata, 2014). These ligands are categorized by which receptors they target. So far, interventions targeting muscarinic receptors have lacked selectivity. Therefore, the search for possible selective ligands continues.

The binding of an agonist to the muscarinic acetylcholine receptor triggers a cascade of reactions which leads to the movement of Calcium ions into the intracellular space. Activation of the receptor, which is a G-protein coupled receptor, leads to conversion of GDP to GTP which travels along the cell membrane and activates enzyme phospholipase C. Phospholipase C lyses phosphatidylinositol-4,5-bisphospate into diacylglycerol and inositol-1,4,5-triphosphate. Inositol-1,4,5-triphosphate interacts with the IP₃ receptor on the sarcoplasmic reticulum which is a storage for calcium ions. The binding of inositol triphosphate to its receptors induces a conformational change which open the channels allowing the movement of calcium ions from the endoplasmic reticulum into the cytoplasm initiating contraction of smooth muscle cells in the case of M₃ muscarinic acetylcholine receptors. Diacylglyceriol activates protein kinase C which also activates muscle contraction and calcium ion influx (Broadley and Kelly, 2001). The response to the ligand can be quantified by the use of a technique known as calcium imaging. This is a microscopic technique which quantifies the calcium ion status of a cell. Calcium ion-sensitive fluorescent dyes are used followed by microscopy to measure the calcium concentration in the cell. These are calcium ion chelators (de Melo Reis, Freitas and de Mello, 2020). In this particular experiment, Fluo-4 acetoxymethyl ester (Fluo-4 AM), a green fluorescent dye was used.

Fluo-4 AM is unable to bind calcium ions. It enters the cell following incubation and is converted by endogenous esterases to Fluo-4 which is able to bind calcium ions. Unbound Fluo-4 is non-fluorescent, however when bound to calcium ions, it gains the ability to fluoresce and is excited at an absorption maximum of 488nm.  Fluo-4 AM is used to detect calcium ions in cells by using fluorescence microplate reader, flow cytometry, fluorescence microscopy or confocal microscopy (de Melo Reis, Freitas and de Mello, 2020). In this experiment, confocal microscopy was used.

1. To increase the comprehension of confocal microscopy use and calcium ion-sensitive fluorescent dyes to examine GPCR signaling in pharmacology.
2. To assess generated data following the imaging experiments above, and further generate pharmacological variables (for instance, half maximal effective dose) in relation to ligands used.
3. To determine the type of ligands, in regards to broad pharmacological categorization (for instance, antagonist, inverse agonist, etc.).

The neuroblastoma cells were formerly grown on plates on which imaging and Drug X and Y treatment was conducted. Media was aspirated from the wells that were to be used and discarded. Imaging buffer (1ml) was utilized to wash cells. 1ml of fluorescent dye, 5µM Fluo-4AM, was added to the cells followed by incubation in the dark for 45 minutes. Fluo-4AM was aspirated from the cells followed by a washing step with imaging buffer (1ml) and discarding after washing. Imaging buffer (950µl) was added to the neuroblastoma cell line and incubated for 5 minutes. Under brightfield illumination, cells were then viewed through the eyepiece using the 10x objective lens. Cells were observed under fluorescent illumination, specifically blue light, to verify that they had loaded. By switching to ‘live view’, cells were viewed confocally (on the computer monitor). The settings were tuned to generate an image of good quality, in order to bring cells into clear visibility in the DIC (brightfield) channel and that Fluo-4AM dye was visible in the green FITC channel. Identification of a field of view to image was done. The number of images to be taken over through the experiment was set. Each experiment was ran for 2 minutes. The start key was pressed and the software began to take images every 1.5 seconds. 50µl of drug concentrations as follows were picked by a use of a pipette: Drug X, 0.1µM, Drug X 1µM, Drug X 10µM, Drug X 100µM, Drug X 300µM. The baseline was determined at the beginning of the experiment. After 10 seconds, the particular concentration of the drug was carefully added to the well. Each drug concentration was added to a separate well, in 4 separate experiments respectively

Procedure

The same procedure was redone for 50µl Drug Y (30 nM) in a separate experiment (Experiment 2). Likewise, experiment 1 was repeated in experiment 3. However, in experiment 3, after loading and washing, there was addition of imaging buffer (900µl) to the well. Also, before conducting the experiment with Drug X, 50µl Drug Y (30 nM) was added to the well.

Fold response over basal was plotted against time. Identification of peak response from the plots allowed the plotting of concentration-response curves and subsequent generation of the EC₅₀ values of Drug X both with and without Drug Y. Statistical analysis and plotting of curves was done using Graphpad Prism 9.

Figure 1. Plots of raw data showing the time-course of response to Drug X alone at varied concentrations.

Figure 2. Plots of fold response over basal (normalized fluorescence data) against time using Drug X alone at varied concentrations.

Table 1 showing the peak response to varied concentrations of Drug X as identified from the fold over basal against time plots.

0.1µM = 1 x 10^-7M, therefore log₁₀ [x] = log₁₀ (1 x 10^-7) = -7

1µM = 1 x 10^-6M, therefore log₁₀ [x] = log₁₀ (1 x 10^-6) = -6

10µM = 1 x 10^-5M, therefore log₁₀ [x] = log₁₀ (1 x 10^-5) = -5

100µM = 1 x 10^-4M, therefore log₁₀ [x] = log₁₀ (1 x 10^-4) = -4

300µM = 3 x 10^-4M, therefore log₁₀ [x] = log₁₀ (3 x 10^-4) = -3. 

Figure 3. A concentration-response curve of Drug X, with a curve of best fit. The EC₅₀ is 1.66 X 10^-6 M. The hill slope of the curve is 1.33.

Figure 4. Plots of raw data illustrating time-course of response to Drug X at varied concentrations in the presence of Drug Y.

Figure 5. Plots of normalized fluorescence data against time using Drug X at varied concentrations, in the presence of Drug Y.

Table 2 showing the peak reaction to varied concentrations of Drug X in exposure to Drug Y as identified from the fold over basal against time plots.

Figure 6. A dose-response curve of Drug X exposed to Drug Y, with a curve of best fit. The EC₅₀ value is 1.12 X 10^-5 M. The hill slope of the curve is 1.00.

The concentration response curve of Drug X alone gives an EC₅₀ value of 1.66 X 10^-6 M or 1.66 µM. On the other hand, Drug X in exposure to Drug Y gives an EC₅₀ value of 1.12 X 10^-5 M or 11.2 µM. Evidently, Drug Y reduces the potency of Drug X. Therefore, Drug X is an agonist because the response increases with increased concentration, while Drug Y is a negative allosteric modulator because it reduces the response of the receptor to Drug X. Ligands which have been reported to act similar to Drug X at the M₃ mAch receptor are acetylcholine, muscarine, carbachol (Rang et al., 2003). Drug X mimics the action of agonists which binds to and activates the G-protein coupled receptor leading to the movement of calcium ions from sarcoplasmic reticulum storage into the cytosol. Consequently initiating the process of smooth muscle cell contraction and glandular secretion (Broadley and Kelly, 2001). The binding of an agonist to the muscarinic acetylcholine receptor triggers a cascade of reactions which leads to the influx of Calcium ions into the intracellular space. Following activation of the receptor, which is a G-protein coupled receptor, leads to conversion of GDP to GTP which activates enzyme phospholipase C. Phospholipase C lyses phosphatidylinositol-4,5-bisphospate into diacylglycerol and inositol-1,4,5-triphosphate. Inositol-1,4,5-triphosphate interacts with IP₃ receptors on the sarcoplasmic reticulum which is a storage for calcium ions. The binding of inositol triphosphate to its receptors induces a conformational change which open the channels allowing the movement of calcium ions from the endoplasmic reticulum into the cytoplasm initiating contraction of smooth muscle cells in the case of M₃ muscarinic acetylcholine receptors. Diacylglyceriol activates protein kinase C which also activates muscle contraction and Calcium ion influx (Broadley and Kelly, 2001). Drug Y, on the other hand, mimics antagonistic ligands such as lidocaine. Lidocaine acts as a non-competitive inhibitor of M₃ mAch receptors which binds to a different site from the ligand binding site. Lidocaine has been reported to inhibit the m3 G-protein coupled receptor. The actual mechanism is unknown. However a possible mechanism would be that lidocaine may impede the coupling of G protein to the receptor, therefore blocking the downstream reactions following activation of the G-protein coupled receptor responsible for muscle contraction or glandular secretion (Hollmann et al., 2001).  

This experiment is however limited by the fact that a drug may have different sensitivity to different cell types. The M₃ mACh receptor is expressed in several tissues such as endocrine glands, smooth muscles, the brain, lungs and the pancreas (Matera and Tata, 2014). For this reason, making conclusions based off of one cell type may not be ideal. Therefore, more experiments should be conducted using different cells or tissues to give a more concrete conclusion.

References

Broadley, K. and Kelly, D. (2001) ‘Muscarinic receptor agonists and antagonists’, Molecules: A Journal of Synthetic Chemistry and Natural Product Chemistry, 6(3), pp. 142–193. doi: 10.3390/60300142.

Hollmann, M. et al. (2001) ‘Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics’, British Journal of Pharmacology, 133(1), pp. 207–216. doi: 10.1038/sj.bjp.0704040.

Matera, C. and Tata, A. (2014) ‘Pharmacological approaches to targeting muscarinic acetylcholine receptors’, Recent Pat CNS Drug Discov., 9(2), pp. 85–100. doi: 10.2174/1574889809666141120131238.

de Melo Reis, R., Freitas, H. and de Mello, F. (2020) ‘Cell calcium imaging as a reliable method to study neuron-glial circuits’, Frontiers in Neuroscience. Available at: https//www.frontiersin .org/articles/10.3389/fnins.

Rang, H. et al. (2003) Pharmacology. 5th edn. Elseveier Churchill Livingstone.