In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

Summary

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

Abstract

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.

In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

Introduction

Urinary ATP is thought to play a significant sensory role in the control of micturition¹. For example, it is thought that ATP is released from the urothelium in response to distension where it can act on receptors on bladder afferent nerves to increase their excitability, leading to sensations of fullness². Thus, it is also thought that urinary ATP could be an important player in the development of bladder pathologies. In support of this hypothesis, urinary ATP concentrations are significantly increased in patients suffering from overactive bladder (OAB)³, bladder pain syndrome/interstitial cystitis (BPS/IC)⁴, or a urinary tract infection (UTI)^5,6, all conditions characterized by increased urgency, frequency and, sometimes, pain. Conversely, patients suffering from underactive bladder (UAB), which is characterized by an inability to empty one's bladder and can sometimes include a decreased ability to sense bladder fullness, have been shown to have decreased urinary ATP concentrations⁷. Experimentally, manipulation of urinary ATP concentrations can alter bladder reflexes in the rat; increasing ATP concentrations by blocking endogenous ATPases in the bladder lumen can increase voiding frequency, while decreasing ATP concentrations by instilling exogenous ATPases into the bladder reduces voiding frequency⁸. Thus, the importance of urinary ATP to bladder function is clear.

Given the apparent importance of urinary ATP to bladder pathology, accurate measurement of urothelial ATP release is an important step in understanding the mechanisms that control release. Many studies have been completed using different experimental models to measure urothelial ATP release. Foremost among these are cell cultures, either primary cultures or cell lines. However, the use of cultured urothelial cells is complicated by the fact that urothelial cells do not take on their physiological polarized morphology unless they are grown on special permeable membranes (such as Transwell technology [well inserts])⁹. Thus, it is difficult to relate any ATP release measured to physiology. Urothelial cells grown on well inserts can polarize and form a barrier akin to what is seen in vivo; however, the growth of a fully differentiated urothelium can take days or weeks. Additionally, while it is possible to mount well inserts into an Ussing chamber and apply pressure to the apical side to cause stretch, it is difficult to apply enough pressure to mimic conditions inside the bladder during pathology (i.e., pressures of 30 cm H₂O or above). Whole bladder tissue can also be mounted in an Ussing chamber for stretch experiments, but this removes the bladder from the organism along with the trophic factors maintaining urothelial cell health and, hence, urothelial barrier function. Therefore, the most physiologically relevant way to study the release of ATP from the urothelium in response to stretch or pressure is in vivo. The surgical techniques needed to set up the experiment are identical to those commonly used in animal cystometry and, therefore, should be easily performed by anyone familiar with that technique.

In this protocol, we will describe the technique used to examine luminal ATP in female Sprague Dawley rats weighing approximately 200-250 g, as the transurethral catheterization described below is much easier in females; however, transurethral catheterization can also be performed in male rodents¹⁰. As transurethral catheterization has now been performed in mice of both sexes as well¹¹, these experiments can easily be adapted for mice or rats of either sex or of varying sizes, depending on the needs of the research team.

Protocol

All procedures carried out in rodents must adhere to the applicable guidelines and be approved by the local institutional ethics review committee. The experiments performed for this manuscript were carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh School of Medicine. See Figure 1 for a modified version of the standard rodent cystometry setup used in this protocol.

1. Laboratory animals

1. Maintain the rats in social housing (multiple rodents in one cage) with a 12 h light/dark cycle and ad libitum access to water and food pellets.

2. Anesthesia and ganglionic block

1. Induce initial anesthesia by placing the animal in a closed box gassed with 4%-5% isoflurane in O₂ (1 L/min).
2. Anesthetize the animal using urethane.
   1. Inject urethane subcutaneously bilaterally (1/2 dose on each side of the animal) at a dose of 1.2 g/kg. Place the animal in a cage to allow the urethane to take effect, which generally takes 2 h.
   2. Alternatively, administer urethane intraperitoneally (i.p.) by injecting the full dose in two separate doses ~10 min apart.
3. After waiting for the appropriate time for the urethane to take effect (s.c.: 2 h, i.p.: 30 min), test for a proper plane of anesthesia by pinching the foot of the animal using forceps. If a reflex is observed, administer an additional dose of urethane (0.05-0.1 mL i.p.), wait for 15 min, and test again. Continue to monitor the animal for the proper plane of anesthesia throughout the procedure.
4. To prevent a contraction of the bladder during distension, inject the animal with a ganglionic blocking agent, such as hexamethonium (20 mg/kg, i.p.).
5. Apply ophthalmic ointment to the animal's eyes to prevent drying during the experiment.

3. Surgical procedure-suprapubic bladder catheterization

1. Shave the abdomen of the animal and perform a midline laparotomy to expose the urinary bladder.
2. Prepare a catheter by flaring one end of a short (~10-15 cm) length of PE50 intramedic tubing using a flame. Place a 22 G needle into the other end of the tubing and fill with Krebs solution (see Table of Materials for the composition).
3. Place a small loop of 3-0 silk suture over the dome of the bladder and perform a small cystostomy (using fine scissors or an 18 G needle) large enough to insert the flared end of the catheter made above. With one hand holding the catheter in place, use the other hand to tighten the loop of the suture to secure the catheter in place. Finish securing the catheter by tying two knots in the suture and pulling the catheter back until the flared head is in contact with the bladder wall.
   1. Alternatively, secure the catheter using a classic purse-string suture technique, as previously described for cystometry¹².
      NOTE: It is imperative not to introduce air bubbles into the bladder during this procedure.
4. Test the setup for leaks by infusing a small amount of Krebs solution through the catheter. If the fluid leaks out of the cystostomy, resecure the catheter with additional suture around the cystostomy.

4. Transurethral catheterization

1. Dip the end of a 20 G x 1" I.V. catheter (with the needle removed) in surgical lubricant.
2. Hold the external urethral meatus gently with a pair of forceps and insert the tip of the catheter into the urethral orifice in the direction of the tail until the tip causes the wall of the adjacent vaginal opening to deform. Rotate the catheter 90° (bringing the Luer-Lock end of the catheter toward the tail) and gently advance. Fully insert the catheter until the Luer-Lock hub is approximately 5 mm distal from the external urethral opening.
   NOTE: Do not insert the catheter too far, which may cause the tip to poke the inside wall of the bladder. If resistance is felt while advancing the catheter, stop and begin again or risk puncturing the urethra. See the discussion section on tips to increase successful catheterization.
3. Secure the catheter and prevent leakage around the catheter by looping a short length of 3-0 silk suture around the external urethral meatus and tie it off tightly. Secure the catheter to the tail with tape to prevent it from accidentally being pulled out.
4. Once catheterized, gently infuse Krebs solution into the bladder through the suprapubic bladder catheter and confirm that the fluid flows out of the urethral catheter and not around it. If necessary, retie the suture around the external urethral orifice.
5. Close the abdominal incision over the bladder using a 3-0 silk suture.

5. Experimental setup

1. Secure the animal on a board capable of being inclined to aid in the draining of intravesical fluid through the urethral catheter. Place a heating pad and absorbent underpad between the animal and the board to maintain body heat and absorb fluid draining from the urethral catheter.
2. Connect the suprapubic catheter to a three-way stopcock, which connects the catheter to a syringe pump and a pressure transducer. Connect the pressure transducer to a computer by way of an amplifier and a data acquisition system.
   NOTE: Care should be taken to prevent air bubbles from forming in the tubing connecting the syringe pump, transducer, and bladder catheter.
3. Calibrate the bladder pressure recording using the procedure suggested by the manufacturer of the pressure transducer and/or data acquisition software.
4. Infuse Krebs solution through the suprapubic catheter at a rate of 0.1 mL/min and allow the fluid to drain from the urethral catheter for 1 h to wash out any residual ATP released during the catheter implantations.
5. After this washout period, cap the urethral catheter using a Luer-Lock plug and measure the pressure in the bladder. Look for a slow rise in the intravesical pressure to a pressure of 30 cm H₂O without a sharp increase in pressure, which would indicate a bladder contraction (see Figure 2). Remove the plug from the urethral catheter when the pressure reaches 30 cm H₂O to prevent damage to the bladder.
   ​NOTE: If bladder contractions continue to occur after 1 h, give an additional dose of hexamethonium (5 mg/kg dose i.p.).

6. Collection of samples

1. Infuse the bladder at 0.1 mL/min and collect the eluate from the urethral catheter. Test 100 µL aliquots of the eluate immediately for ATP (see below) or freeze for later batch quantification.
2. To test the effect of bladder distension on luminal ATP concentrations, cap the urethral catheter with the plug and monitor bladder pressure until it reaches the desired level. Then, uncap the urethral catheter and collect the eluate for ATP measurement or freezing, as described above.
3. After each distension, allow the bladder to rest and wash out for 10-15 min before taking additional samples. Take a total of 3-5 predistension samples and 3-5 samples at each desired distention pressure to demonstrate repeatability.
4. To test the effect of drugs on the release of ATP, switch the Krebs solution infusing the bladder to Krebs containing the drug of choice. Perfuse at 0.1 mL/min for 10-15 min for the drug to have an effect, and then collect the samples from non-distended and distended bladders as described in steps 6.1 and 6.2.

7. Quantifying ATP from collected samples

1. Quantify ATP in the collected 100 µL samples using a commercially available luciferin/luciferase assay kit following the manufacturer's instructions and a luminometer.
   1. To quantify ATP, combine 100 µL samples of perfusate with 50 µL of the assay mix and place them in the luminometer for reading. To convert the Relative Light Units (RLUs) reported by the luminometer to a concentration of ATP, make serial dilutions of ATP in Krebs solution ranging from 1 µM to 10 pM in 10-fold dilutions to create a standard curve and read them in the luminometer. Plot the resulting readings on a graph and perform a non-linear (quadratic) regression to extrapolate concentrations from the samples taken from the animal.
      ​NOTE: It is important to make ATP standards for any drug solution tested in the experiment, as many drugs interfere with the luciferin/luciferase reaction, which must be corrected for.

8. Euthanasia of animals

1. When the experiment is completed and all the samples are collected, humanely euthanize the animal according to USDA guidelines and the National Research Council's Guide for the Care and Use of Laboratory Animals.
   1. Remove the anesthetized animal from the experimental setup, place it in a closed box, and gas it with 100% CO₂. Ensure that the fill rate is equal to 30%-70% of the chamber volume per minute (e.g., 3-7 L/min for a box with a 10 L volume). Continue the CO₂ flow for at least 1 min after respiration ceases.
2. Use a secondary form of euthanasia to ensure death.
   1. Perform a thoracotomy as a secondary form of euthanasia by grasping the small flap of skin at the caudal end of the sternum and cutting a small hole in the skin and musculature at the diaphragm with a sharp pair of scissors. Complete the thoracotomy by inserting the scissors into the opening and cutting rostrally through the rib cage and exposing the thoracic cavity.
      NOTE: The euthanized animal should be disposed of according to institutional guidelines.

Representative Results

The described protocol allows for the accurate measurement of urothelial ATP release in vivo from the lumen of the bladder, using a modified version of the standard rodent cystometry setup (see Figure 1). This allows the researcher to examine the effects of drugs on stretch-mediated ATP release in a physiological setting.

Figure 1: Experimental setup. (A) Image showing the experimental setup, with the various equipment labeled. The area of the animal outlined in a red rectangle is shown in B. (B) Photograph depicting the abdomen of the rat following surgery. Note the suprapubic catheter inserted and tied into the bladder dome, as well as the urethral catheter inserted through the external orifice. The urethral catheter is plugged in the photo to show that bladder pressure is increased during perfusion through the bladder catheter. During the experiment, the abdominal opening would be sutured shut, but was left open here to allow visualization of the urinary bladder. Please click here to view a larger version of this figure.

Of primary importance for the successful measurement of stretch-mediated ATP release in the rodent bladder is ensuring that the micturition reflex is blocked, allowing the bladder to be distended beyond the threshold pressure normally sufficient to activate the micturition reflex. As shown in Figure 2A, when the urethral catheter is plugged, infusion of the solution into the bladder causes intravesical pressure to rise sharply as the bladder contracts. As we are interested in the effects of passive stretch, not contraction, on urothelial ATP release, the animal is treated with a ganglionic blocker, hexamethonium, to prevent the micturition reflex. As evidenced in Figure 2B, when hexamethonium is administered, instillation of the solution into the bladder with the urethral catheter blocked results in a much more gradual rise in intravesical pressure, allowing pressures to rise as high as 30 cm H₂O without contraction. This allows for measurement of luminal ATP release at pressures high enough to be relevant to pathology, such as those observed in an underactive bladder, partial bladder outlet obstruction, or during detrusor-sphincter dyssynergia after spinal cord injury. While it may require a supplemental dose of hexamethonium to completely block the micturition reflex, care should be taken to not administer enough to result in an overdose. This could lead to significantly decreased cardiac output and compromise the quality of the experiment. It should be noted that the same inhibitory effect on the bladder reflex can be obtained by either cutting the pelvic nerves bilaterally or severing the spinal cord at level L4 or above. This, of course, increases the difficulty of the experimental setup and the time required to complete the experiment, significantly.

Figure 2: Pressure recordings from rats. Before (A) and after (B) ganglionic block. Arrows indicate when the urethral catheter was plugged. Notice that the pressure in the top trace rapidly increases when pressure reaches the threshold to induce micturition, while the pressure in the bottom trace gradually rises. Please click here to view a larger version of this figure.

Another very important consideration for these experiments is to ensure proper conversion of the luminometer readings (normally expressed in RLUs) to a concentration of ATP using standard curves. The luciferin/luciferase reaction is very susceptible to interfering agents¹³. This interference can come from a direct interaction with luciferin/luciferase. For example, a number of compounds can act as competitive or non-competitive inhibitors of luciferase; one study suggested that ~3% of compounds in the Molecular Libraries Small Molecule Repository could inhibit firefly luciferase at concentrations below 11 µM¹⁴. Moreover, luciferin is susceptible to oxidizing agents, reducing the amount of substrate available for the luminescence-producing reaction¹⁵. It is also possible to interfere with the luciferin/luciferase reaction by altering the availability of the reaction's co-factor, magnesium. For example, one common technique for mimicking mechanical stretch in in vitro ATP experiments is to induce cell swelling by reducing the osmolarity of the perfusing solution. However, many researchers accomplish this reduction by diluting the solution with water, reducing the concentration of magnesium available to act as a co-factor in the luciferin/luciferase reaction. Additionally, some drugs are sold as magnesium salts, which can greatly increase the amount of magnesium in the solution, also leading to measurable differences in luminescence readings. Finally, it is possible for a drug solution to interfere with the ability to accurately measure the light produced by the luciferin/luciferase reaction. Brilliant Blue FCF (BB-FCF, also known as erioglaucine or FD&C Blue dye #1) is a specific inhibitor of pannexin-mediated ATP release¹⁶. At effective doses, Brilliant Blue FCF solution has a distinct blue hue, which absorbs light at a peak of around 600 nm¹⁷. This is within the range of wavelengths of light that is emitted by firefly luciferase; therefore, light emission in experiments with BB-FCF is greatly reduced. Thus, it is imperative that standard curves using known quantities of ATP dissolved in solutions containing each experimental drug are used to quantify the ATP release during the experiment. As shown in Figure 3, BB-FCF (100 µM) significantly decreases the slope of the standard curve, which is sufficient to significantly alter the calculated values for the concentration of ATP in the sample. As shown at the bottom of Figure 3, using the Krebs standard to calculate the concentration of ATP in a sample containing BB-FCF can result in an underestimation of the ATP concentration by ~50%. In some instances, using the wrong standard curve could obscure a drug-mediated change in ATP release, or make it erroneously appear as if a change has occurred. In the experiment depicted in Figure 3, for example, using the Krebs standard for all samples would have made it appear that Brilliant Blue FCF reduced ATP release by ~67% (16.6 to 5.5 nM) when, in actuality, it had only decreased ~42% (16.6 to 9.6 nM).

Figure 3: Effects of experimental drugs on ATP standard curve. Note that the pannexin channel antagonist, Brilliant Blue, significantly alters the RLUs measured for any given concentration of ATP, which would result in an underestimation of the ATP measured, if not corrected for. Abbreviation: RLUs = Relative Light Units; BB-FCF = Brilliant Blue FCF. Please click here to view a larger version of this figure.

One other consideration to keep in mind is that ATP is commonly released from tissues as a result of damage, and that these surgical procedures and urethral catheter insertion will result in abnormally high ATP readings if taken too soon after setup. This procedure describes a 1 h washout period after the experiment is set up, which should not be skipped. We have found that after 1 h, ATP concentrations measured in samples taken when the bladder is not distended are relatively low (~10-30 nM) while samples taken before the washout period can read an order of magnitude higher. Short, 5 min washouts should also be performed between distensions as ATP levels can remain elevated for a short period. A good rule of thumb would be to measure ATP in multiple samples at the beginning of the experiment and after each distension and only proceeding when ATP readings level out. In Figure 4, we show a representation of a typical experiment with measurements taken at various time points. Note the high readings taken pre-washout (samples #1 and #5) versus those taken post washout (samples #2 and #6).

Figure 4: Representation of a typical experiment. A stylized representation of a typical pressure recording. Each number represents a timepoint when a sample was taken and measured for ATP: 1) immediately after setup is completed, before the washout period, 2) after a 1 h washout period, 3) immediately preceding a distension, 4) during distension, 5) immediately following a distension, and 6) after a short washout period following the distension. The inset table shows representative RLU values and ATP concentrations for each sample. Abbreviation: RLUs = Relative Light Units. Please click here to view a larger version of this figure.

Figure 5 shows representative graphs of experimental results examining the mechanism of ATP release from the urothelium. As shown in Figure 5A, increasing pressure causes a significant increase in the concentration of ATP in the lumen of the bladder, with pressures of 30 cm of water increasing concentrations almost 2.5x of non-distended controls. Intravesical perfusion of 2 U/mL of apyrase, an enzyme that catalyzes the hydrolysis of ATP, significantly decreases the measured concentration of ATP in the bladder lumen. Conversely, intravesical perfusion of ARL67156, an inhibitor of NTPDases present on the urothelium, significantly increases luminal ATP concentrations. Figure 5B depicts the experiments into the mechanism of stretch-induced ATP release from the urothelium. Perfusion with the pannexin channel antagonists Brilliant Blue FCF (BB-FCF, 100 µM) or carbenoxolone (CBX, 100 µM) significantly reduces the release of ATP into the bladder lumen at all pressures. Luminal ATP concentrations are not diminished when the connexin blocker 18α-glycyrrhetinic acid is perfused, indicating that distension-evoked ATP release is mediated by pannexin channels and not connexin channels.

Figure 5: Representative measurements of distension-evoked ATP release in the lumen of the urinary bladder. (A) Distention of the bladder increases luminal ATP concentrations, which can be diminished in the presence of the NTPDase apyrase (2 U/mL) or increased when endogenous NTPDasesare inhibited with ARL67156 (10 µM). (B) Distension-evoked ATP release is blocked by the pannexin channel antagonists Brilliant Blue FCF (BB-FCF, 100 µM) and carbenoxolone (CBX, 100 µM) but not the connexin channel antagonist 18α-glycyrrhetinic acid (18α-GA, 50 µM). Data are presented as the mean with standard error bars. ** p < 0.05 between the two bars indicated by the line, ## p < 0.05 as compared to Krebs controls at the same distension. This figure is reprinted with permission from Beckel et al.⁸. Please click here to view a larger version of this figure.

Discussion

The majority of research into urothelial ATP release is conducted in cultured cells, using either immortalized cell lines or primary cultures of rodent urothelial cells. While these models have the benefit of being relatively high throughput (i.e., one culture/passage can make many plates/dishes of cells), their physiological relevance is diminished due to: 1) the inability of urothelial cells to grow polarized unless they are grown on special supports and 2) the difficulty in exposing cultured cells to physiological levels of stretch/pressure. One way to deal with these limitations is by removing the bladder from a rodent, cutting it open, and placing it in an Ussing chamber, inserting the bladder tissue between two half chambers filled with physiological solution. This allows the researcher to subject the urothelium, in its native polarized state, to distension by adding volume to the urothelial side of the chamber, causing stretch of the bladder tissue in the chamber. However, it is difficult to relate this stretch to the distension seen by the urothelium in vivo. Moreover, distension-evoked ATP in the Ussing chamber is released into the large volume of physiological fluid contained in the chamber, diluting the concentration of ATP several-fold. Using this procedure, bladder pressure is measured directly, so that distension can be kept in a range relevant for physiology or pathophysiology. Moreover, since we are measuring ATP concentration directly from the luminal fluid and not from an arbitrary and excessive amount of fluid used to maintain tissue in vitro, the concentration measured does not have to be corrected. Thus, the procedure described in this paper allows the researcher to examine ATP release in the bladder lumen under conditions that much more closely resemble physiological or pathophysiological conditions of the bladder.

The technique described is very similar to standard cystometry techniques used to study the effects of drugs on reflex bladder activity and should be easily carried out by anyone who routinely carries out those experiments. For those researchers new to bladder catheterization, however, there are a few caveats to be aware of. First, transurethral catheterization can be difficult for the inexperienced due to the curvature of the rodent urethra. This can also be made more difficult by the tightness of the external urethral sphincter, even under urethane anesthesia. It is imperative that the researcher does not try to force the catheter into the urethra, as it will most likely cause increased inflammation and swelling, which will in turn make the urethra even harder to successfully catheterize. Additionally, it is possible to poke the catheter through the wall of the urethra, ruining the entire experiment.

We have developed some tips and tricks for increasing the rate of success. For example, it is sometimes necessary to give the animal another short dose of inhaled isoflurane (0.5%-1.0%) to relax the urethral sphincter. This should be performed sparingly and with care, as the combination of urethane and isoflurane can result in respiratory depression. Another trick is to dip the tip of the catheter into a 2% lidocaine solution before inserting it into the external urethral orifice; this will often cause the sphincter to relax after a few minutes. Emptying the bladder by gently pressing on the abdomen of the animal can also help relax the urethral sphincter. Finally, it may be necessary to use a smaller gauge catheter; we sometimes use a 24 G catheter, especially with smaller animals. Keep in mind, however, that it may be necessary to reduce the infusion rate into the bladder when using a smaller gauge transurethral catheter, to match the slower drainage from the urethral catheter. Failure to do so would cause bladder pressure to rise, despite the urethral catheter not being capped.

It should be noted that urethane is used as an anesthetic in this protocol. Urethane is commonly used in laboratories studying the lower urinary tract, since it spares the micturition reflex whereas other anesthetics do not. Because of this, distension of the bladder above a pressure of ~7-10 cm H₂O under urethane anesthesia will trigger a micturition reflex. Since we are interested in ATP release in response to passive distension, this protocol calls for blocking bladder contractions using the ganglionic blocker hexamethonium. This allows the measurement of ATP release in response to the higher intravesical pressures (i.e., 30 cm H₂O) commonly seen in bladder pathology involving outlet obstruction. Therefore, the use of a urethane/hexamethonium allows for the measurement of distension-evoked luminal ATP release for long periods of time (the duration of action of both drugs is measured in hours). However, some labs may prefer to use an anesthetic that does not spare the micturition reflex, such as ketamine, and eliminate the need for a ganglionic blocker. This will also work well for this procedure, although it will require more frequent (or continuous) administration due to the shorter duration of action of the anesthetic.

One noteworthy difference between this technique to measure ATP and the common cystometry setup urology researchers may be familiar with is the use of buffered Krebs solution as a perfusate instead of the standard isotonic saline solution. ATP is a rather labile molecule and is stabilized somewhat in a buffered solution. Additionally, the composition of Krebs solution is much closer to that of urine than saline, which further adds clinical relevance to these experiments. This is an important consideration, as the control of urothelial ATP release has been shown to be modulated by calcium permeable channels, and the lack of calcium in normal saline may negatively influence the ATP release. The instability of ATP in solution is also why we suggest that samples be immediately quantified using the luciferin-luciferase reaction. However, if the researcher is unable to read the samples immediately, the samples should be frozen as quickly as possible to limit the degradation of ATP.

One limitation of this technique is that it only measures luminal ATP release and cannot measure ATP release from the serosal side of the urothelium. It is believed that ATP is released from the serosal side to directly influence afferent excitability; however, direct measurement of this release is difficult. In this case, using cells grown on permeable membrane supports such as Transwell plates or surgically removing the urothelium for Ussing chamber experiments is the preferred technique. However, given the apparent importance of luminal ATP in bladder pathology, this experimental model is a useful tool to elucidate the cellular mechanisms controlling urothelial ATP release during pathology.

Materials

Name	Company	Catalog Number	Comments
amplifier	World Precision Instruments (WPI)	SYS-TBM4M
ATP assay kit	Sigma-Aldrich, Inc.	FLAA-1KT
data acquisition system/ software	DataQ Instruments	DI-1100	Software included, requires Windows-based computer
Hexamethonium bromide	Sigma-Aldrich, Inc.	H0879	20 mg/kg dose
Isoflurane	Covetrus North America	29404
lidocaine	Covetrus North America	2468
Luer Lock plugs	Fisher Scientific	NC0455253
luminometer (GloMax 20/20)	Promega	E5311
Polyethylene (PE50) tubing	Fisher Scientific	14-170-12B
Pump 33 DDS syringe pump	Harvard Apparatus	703333
pressure transducers	World Precision Instruments (WPI)	BLPR2
surgical instruments (scissors, hemostats, forceps, etc.)	Fine Science Tools	multiple numbers
surgical lubricant	Fisher Scientific	10-000-694
Sur-Vet I.V. catheter	Covetrus North America	50603	20 G x 1 inch
tiltable surgical table (Plas Labs)	Fisher Scientific	01-288-30A
Tubing connectors	Fisher Scientific	14-826-19E	allows Luer-Lock connectors to attach to tubing
Urethane	Sigma-Aldrich, Inc.	U2500	0.5 g/mL conc., 1.2 g/kg dose