Name: in vivo luminal measurement of distension-evoked urothelial atp release in rodents
Uploaded: 2022-09-07T13:20:00
Description: Watch this Scientific Journal Video about In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents at JoVE.com

This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) to JMB (DK117884).

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&#39;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...

<ol>
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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 lev...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>amplifier</td>
			<td>World Precision Instruments (WPI)</td>
			<td>SYS-TBM4M</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP assay kit</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>FLAA-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>data acquisition system/ software</td>
			<td>DataQ Instruments</td>
			<td>DI-1100</td>
			<td>Software included, requires Windows-based computer</td>
		</tr>
		<tr>
			<td>Hexamethonium bromide</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>H0879</td>
			<td>20 mg/kg dose</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetrus North America</td>
			<td>29404</td>
			<td></td>
		</tr>
		<tr>
			<td>lidocaine</td>
			<td>Covetrus North America</td>
			<td>2468</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock plugs</td>
			<td>Fisher Scientific</td>
			<td>NC0455253</td>
			<td></td>
		</tr>
		<tr>
			<td>luminometer (GloMax 20/20)</td>
			<td>Promega</td>
			<td>E5311</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene (PE50) tubing</td>
			<td>Fisher Scientific</td>
			<td>14-170-12B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump 33 DDS syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>703333</td>
			<td></td>
		</tr>
		<tr>
			<td>pressure transducers</td>
			<td>World Precision Instruments (WPI)</td>
			<td>BLPR2</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical instruments (scissors, hemostats, forceps, etc.)</td>
			<td>Fine Science Tools</td>
			<td>multiple numbers</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical lubricant</td>
			<td>Fisher Scientific</td>
			<td>10-000-694</td>
			<td></td>
		</tr>
		<tr>
			<td>Sur-Vet I.V. catheter&#160;</td>
			<td>Covetrus North America</td>
			<td>50603</td>
			<td>20 G x 1 inch</td>
		</tr>
		<tr>
			<td>tiltable surgical table (Plas Labs)</td>
			<td>Fisher Scientific</td>
			<td>01-288-30A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing connectors</td>
			<td>Fisher Scientific</td>
			<td>14-826-19E</td>
			<td>allows Luer-Lock connectors to attach to tubing</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>U2500</td>
			<td>0.5 g/mL conc., 1.2 g/kg dose</td>
		</tr>
	</tbody>
</table>

in vivo luminal measurement of distension-evoked urothelial atp release in rodents

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64227/64227fig01.jpg" /> 
<s...

Watch this Scientific Journal Video about In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents at JoVE.com

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

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

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

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

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.

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