The overall goal of this procedure is to measure the activity of KV seven potassium channels in freshly dissociated mesenteric artery smooth muscle cells, and relate that to the measurements of arterial diameter recorded under physiological conditions. This is accomplished by first surgically removing the mesenteric vascular arcade from a rat. The second step is to dissect and clean the segments of the mesenteric arteries.
Next, some segments are used to isolate individual smooth muscle cells for patch clamp electrophysiological measurements of KV seven potassium currents. The final step is the cannulation of intact arterial segments for pressure myo measurements of arterial constriction or dilation in response to the pharmacological modulators of KV seven potassium channels. Ultimately, the combination of patch clamp electrophysiology and pressure myography is used to show how an increase or a decrease in the activity of KV seven potassium channels affects mesenteric arterial constriction.
The main advantage of this technique over existing methods like the use of clone channels expressed in cell lines, is that the currents are recorded from non cultured cells that natively express the channels and the KV currents can be isolated from other classes of potassium channels that may be activated in the same cells. The pressure myography allows the functional responses to be recorded from the same tissue from which the isolated cells are derived, and the method is both more sensitive and more physiological than other preparations like arterial rings or arterial strips. Dr.Bareth Mani, a postdoctoral fellow from my laboratory, will be demonstrating the arterial cannulation and pressure myography techniques and Dr.Luba Brueggeman, a research assistant professor in my laboratory, will be demonstrating the cell isolation and patch plan procedures.
To begin this procedure, use a pair of surgical scissors to cut 10 to 12 centimeter segment at both ends of a rat's small intestine. Then isolate the intestine along with its attached vasculature. Next, pin down the segment of small intestine.
Spread the mesentary over the near side of the chamber such that the primary branch of the superior mesenteric artery is in the center and the subsequent order of branches radiate from the center. Then place the pins only on the intestinal segments visualizing through an illuminated dissecting microscope. Carefully clear the adipose tissue and the connective tissue surrounding the second to fourth order arteries close to the intestines using micro forceps and scissors.
Hold the adjacent veins to facilitate the clearing of the adipose tissue and connective tissue and avoid unnecessary stretching of the arteries. Now transfer two to three segments of the mesenteric artery into a 35 millimeter tissue culture dish containing two milliliters of ice cold isolation solution. Place the dish on ice afterward, cut the mesenteric artery segments into three to four millimeter long pieces using a PEs your pipette with a fire polished tip.
Transfer the arterial segments into a glass vial containing two milliliters of prewarm 37 degrees Celsius isolation solution. Then incubate it for 30 minutes at 37 degrees Celsius. After 30 minutes, carefully aspirate the 37 degrees Celsius isolation solution with a posterior pipette.
Add two milliliters of warm enzyme solution to it, then incubate it for 35 minutes at 37 degrees Celsius. Immediately after the 35 minute incubation in the enzyme solution, place the vial on ice, aspirate the enzyme solution and add two milliliters of ice cold isolation solution. Repeat this procedure four times.
Then in one milliliter of ice cold isolation solution, gently iterate the arterial segments with the polished posterior pipette. 10 to 20 times to release individual mesenteric artery smooth muscle cells. Verify the myocytes appearance periodically by placing a drop of the cell suspension on a 35 millimeter dish and viewing them under the microscope.
Healthy MA SMCs should have a smooth elongated appearance. Next, transfer approximately 100 to 200 microliters of cell suspension to the recording chamber with a polished pastier pipette and allow the cells to adhere for 15 minutes. In this step, fill the tip of the patch pipette with amphotericin B, free internal solution by tipping the pipette tip in it.
Then apply suction on the other end using a 10 milliliter syringe attached via a piece of tubing add info terin B containing pipette solution from the top. Using an einor micro loader pipette tip until the pipette is about half filled with solution. Remove any air bubbles by gently tapping the pipette.
After that, insert the pipette into the electrode holder. Use a micro manipulator to lower the pipette to the surface of an elongated myocyte. When resistance of the pipette increases to six to 10 mega ohms, apply suction to achieve a giga seal.
At the same time, set the holding voltage to zero millivolts while waiting for membrane perforation. Next, apply 100 millisecond voltage steps to plus 10 millivolts and then to minus 10 millivolts. Use the amplifier to compensate for the fast pipette capacitance transient concurrent with the appearance of whole cell capacitance.
Transient small sustained positive currents two to 10 PICO amp, pure and amplitude indicate the presence of functional KV seven channels. The successful perforation with amphotericin B will reduce access resistance to 35 mega ohms or less. To record the KV seven current voltage relationship, apply five second voltage steps to voltages ranging from minus 84 millivolts at which the open probability of KV seven channels is minimal to plus 16 millivolts at which the open probability of KV seven channels reaches a plateau After five seconds at each test voltage, step back to the holding voltage at minus four millivolts for 10 seconds.
In this procedure, loosely suspend two pre-made fine nylon sutures on each side. Adjacent to the glass cannula carefully transferred the dissected mesenteric artery segment from the dissecting chamber to the pressure myo graft chamber by holding one end of the segment using a pair of micro forceps. Next, visualizing through an illuminated dissecting microscope, hold one end of the artery segment and gently slide it over the left glass cannula.
Secure the end using the nylon sutures. Then gently flush the lumen fluid through the mounted vessel to remove blood and debris. The valve to the left syringe should then be closed so that this side presents a static column of fluid against which the pressure is adjusted from the right side, using the micro manipulator position the movable right glass cannula closer to the artery segment and pull the right end of the segment over it.
Finally, secure the artery segment with nylon sutures and clip off the excess suture threads. Now mount the pressure graph unit on the stage over the pressure micrograph microscope. Connect the pressure column to the right side of the graph chamber and then place the cover over the chamber.
Gradually raise the 10 milliliter syringe over a period of 15 minutes and check for any leaks in the vessel until the P one pressure transducer reads 80 millimeters mercury adjust the distance between the cannula when necessary, the distance should be adjusted in such a way that the pressurized artery segment is approximately straight and forms a shoulder like pattern where the ties are made. Check the viability of the mounted vessel by the transient superfusion with 60 millimolar potassium chloride for 30 seconds. A viable vessel constricts quickly with the addition of potassium chloride and dilates immediately upon its washout.
Next, add the concentrated test substance to the bath solution directly pipette it back and forth gently near the end of the chamber to mix the test substance in the bath solution, yielding the appropriate final concentration in the 10 milliliter chamber. Volume changes in the vessel diameter following the addition of the test substance can be monitored in the live video image and also charted in the image analysis window while the experiment is in progress. This figure shows a family of sequential current traces recorded in response to voltage steps before treatment as shown in black and following the stabilization in the presence of 100 nanomolar zinc perion in a single mesenteric artery myocyte as shown in red, here are the representative currents for control and for 100 nanomolar zinc perion.
The gray bars indicate the time interval where averaged current amplitudes were measured For current voltage plots, step voltages are indicated on the right by arrowheads. This is the time course of KV seven current enhancement by 100 nanomolar zinc perion measured with continuous voltage clamp at minus 20 millivolts.Shown. Here are the averaged current voltage curves derived from KV seven current densities recorded before during treatment with 100 nanomolar zinc perion, and during treatment with 10 micromolar of the KV seven channel blocker XC 9 9 1 zinc ion induced relaxation of mesenteric artery pre constricted with 100 picomolar arginine vasopressin.
This figure shows the time course of changes in the outer diameter of the vessel in response to the application of 100 picomolar arginine vasopressin, followed by the application of 100 nanomolar zinc ion. After watching this video, you should have a good understanding of how to prepare arteries for isolation of single cells or for pressure myo graphic measurement of arterial constriction, dilator responses. In addition, you should know how to record KV seven currents in isolation from other currents in freshly isolated arterial myocytes.
