Our protocol measures ATP, an important transmitter in the urinary bladder, which will allow us to further interrogate the mechanisms controlling its release. Our technique measures ATP directly from the bladder lumen, making it more physiologically relevant than techniques using cultured cells or bladder tissue. Demonstrating the procedure will be Keara Healy and Stephanie Daugherty, research technicians in my laboratory.
To begin, induce initial anesthesia by placing the animal in a closed box gassed with 4%to 5%isoflurane in oxygen. Subcutaneously administer urethane bilaterally, then place the animal in a cage to allow the urethane to take effect for almost two hours. After waiting for the urethane to take effect, test for a proper plane of anesthesia by pinching the foot of the animal using forceps.
To prevent the contraction of the bladder in response to distension during the experiment, inject the animal with a ganglionic blocking agent, hexamethonium. Next, apply ophthalmic ointment to the animal's eyes to prevent drying during the experiment. Now shave the animal's abdomen and perform a midline laparotomy to expose the urinary bladder.
Using a flame, flare one end of a short length of PE50 intramedic tubing, place a 22-gauge needle into the other end of the tubing, and fill it with Krebs solution. Place a small loop of 3-0 silk suture over the dome of the bladder and perform a small cystostomy to insert the flared end of the catheter. While holding the catheter with one hand, tighten the suture loop to secure the catheter in place with the other hand.
Finish securing the catheter by tying two knots in the suture. Pull the catheter back until the flared head is in contact with the bladder wall. Test the setup for leaks by infusing a small amount of Krebs solution through the catheter.
Dip the end of an IV catheter in surgical lubricant. 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, as described in the manuscript. Rotate the catheter 90 degrees and advance gently.
Insert the catheter until the luer lock hub is five millimeters distal from the external urethral opening. 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 tying it off tightly. Secure the catheter to the tail with tape to prevent it from accidentally being pulled out.
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. Close the abdominal incision over the bladder using a 3-0 silk suture. Place an absorbent underpad on a heating pad to maintain body heat and absorb fluid.
Secure the animal on the inclined board to aid in draining intravesical fluid through the urethral catheter, then turn on the heating pad. Connect the suprapubic catheter to a three-way stop cock connecting the catheter to a syringe pump and a pressure transducer. Connect the pressure transducer to a computer through an amplifier and a data acquisition system.
Infuse Krebs solution through the suprapubic catheter at a rate of 0.1 milliliters per minute, and allow the fluid to drain through the urethral catheter for one hour to wash out any residual ATP released during the catheter implantations. After this washout period, cap the urethral catheter using a luer lock plug. Measure the pressure in the bladder and look for a slow rise in the intravesical pressure to a pressure of 30 centimeter water without a sharp increase in pressure, indicating a bladder contraction.
Remove the plug from the urethral catheter when the pressure reaches 30 centimeter water to prevent damage to the bladder. Infuse the bladder and collect the eluent from the urethral catheter. Test 100 microliter aliquots of the eluent immediately for ATP or freeze for later batch quantification.
To test the effect of the 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 eluent for ATP measurement or freezing, as described above. After each distension allow the bladder to rest and wash out for 10 to 15 minutes before taking additional samples. 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 the drug for 10 to 15 minutes to have an effect, and then collect the samples from non-distended and distended bladders, as demonstrated earlier. Place 100 microliters sample of perfusate with 50 microliters of the assay mix in the luminometer for reading. To convert from relative light units to ATP concentration, make serial dilutions of ATP in Krebs solution ranging from one micromolar to 10 picomolar in tenfold dilutions to create a standard curve, and read them in the luminometer.
Plot the resulting readings on a graph and perform a non-linear regression to extrapolate concentrations from the samples taken from the animal. Infusion of the solution into the bladder while the urethral catheter is plugged causes intravesical pressure to rise sharply as the bladder contracts. After hexamethonium administration, a more gradual rise in intravesical pressure was observed, allowing pressure to rise as high as 30 centimeter water without contraction.
Brilliant blue FCF significantly decreases the slope of the standard curve, sufficient to alter the calculated values for the concentration of ATP in the sample. Calculating the concentration of ATP in a sample using the Krebs standard containing brilliant blue FCF can result in an underestimation of the ATP concentration by 50%Distension of the bladder increases luminal ATP concentrations, which can be diminished in the presence of NTPDase apvrase, or increased when endogenous NTPD ACEs are inhibited with ARL67156. The stretch-induced ATP release from the urothelium was analyzed.
Distension-evoked ATP release is blocked by the panectin channel antagonists, brilliant blue FCF, and carbonoxolone, but not with a connexon channel antagonist, 18 alpha-glycyrrhetinic acid. It is imperative that all luminometer readings are converted to concentrations of ATP using the appropriate standard curve, as the Lucifer Luciferase reaction is highly susceptible to interference by a wide variety of different agents. Our technique could be used to study the distension-evoked release of any molecule in the lumen of the bladder, assuming that a suitable assay for that molecule exists.
