Name: exploring arterial smooth muscle kv7 potassium channel function using patch clamp electrophysiology and pressure myography
Uploaded: 2012-09-14T13:00:00
Description: Watch this Scientific Journal Video about Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography at JoVE.com

This work was funded by a grant from National Heart, Lung, and Blood Institute (NIH R01-HL089564) to KLB and pre-doctoral fellowships from the American Heart Association (09PRE2260209) and Arthur J. Schmitt Foundation to BKM.

1. Surgical Excision of the Small Intestinal Mesenteric Vascular Arcade
<ol>
 <li>Anesthetize a 300-400 g Sprague-Dawley rat with isoflurane (4%) administered by inhalation.</li>
 <li>Perform a midline laparotomy to expose the small intestinal mesentery. Exteriorize the small and large intestine through the abdominal incision with great care to avoid trauma to the exposed bowel and mesentery. Gently fan the mesentery out over sterile gauze.</li>
 <li>Surgically excise the small intestine and a portion of the lar...

<ol>
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The methods and experimental approaches described here are quite robust and can produce clear and reproducible results when applied with meticulous attention to detail. Good electrophysiological recordings and constriction/dilation of arterial segments are dependent on the health of the cells and artery segments, respectively. Cell preparations can vary from day to day, even using the same protocol. Isolation Solutions can be used for up to 2 weeks, but if the quality of the cell preparation is low on two consequen...

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Sigma</td>
 <td>S5886</td>
 <td>Dissecting 			Solution: 145 
 Bath solution for Electrophysiology*: 			140 
 Internal solution for electrophysiology: 10 
 Isolation 			solution for myocytes*: 140 
 Bath solution for pressure 			myography: 145 
 Lumen solution for pressure myography: 145</td>
 </tr>
 <tr>
 <td>Potassium 			chloride</td>
 <td>Sigma</td>
 <td>P5405</td>
 <td>Dissecting 			Solution: 4.7 
 Bath solution for Electrophysiology*: 			5.36 
 Internal solution for electrophysiology: 135 
 Isolation 			solution for myocytes*: 5.36 
 Bath solution for pressure 			myography: 4.7 
 Lumen solution for pressure myography: 4.7</td>
 </tr>
 <tr>
 <td>Potassium 			EGTA</td>
 <td>Sigma</td>
 <td>E4378</td>
 <td>Internal 			solution for electrophysiology: 0.05</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H9136</td>
 <td>Bath 			solution for Electrophysiology*: 10 
 Internal solution for 			electrophysiology: 10 
 Isolation solution for myocytes*: 10</td>
 </tr>
 <tr>
 <td>Disodium 			hydrogen phosphate</td>
 <td>Sigma</td>
 <td>S5136</td>
 <td>Isolation 			solution for myocytes*: 0.34</td>
 </tr>
 <tr>
 <td>Potassium 			hydrogen phosphate</td>
 <td>Sigma</td>
 <td>P5655</td>
 <td>Isolation 			solution for myocytes*: 0.44</td>
 </tr>
 <tr>
 <td>Magnesium 			Chloride</td>
 <td>Sigma</td>
 <td>M2393</td>
 <td>Bath 			solution for Electrophysiology*: 1.2 
 Internal solution for 			electrophysiology: 1 
 Isolation solution for myocytes*: 1.2</td>
 </tr>
 <tr>
 <td>Calcium 			Chloride</td>
 <td>Sigma</td>
 <td>C7902</td>
 <td>Bath 			solution for Electrophysiology*: 2 
 Isolation solution for 			myocytes*: 0.05</td>
 </tr>
 <tr>
 <td>Sodium 			phosphate</td>
 <td>Fisher 			Scientific</td>
 <td>BP331-1</td>
 <td>Dissecting 			Solution: 1.2 
 Bath solution for pressure myography: 1.2 
 Lumen 			solution for pressure myography: 1.2</td>
 </tr>
 <tr>
 <td>Magnesium 			Sulfate</td>
 <td>Sigma</td>
 <td>M2643</td>
 <td>Dissecting 			Solution: 1.17 
 Bath solution for pressure myography: 1.17 
 Lumen 			solution for pressure myography: 1.17</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Fisher 			Scientific</td>
 <td>BP308</td>
 <td>Dissecting 			Solution: 3 
 Bath solution for pressure myography: 3 
 Lumen 			solution for pressure myography: 3</td>
 </tr>
 <tr>
 <td>Pyruvic 			acid</td>
 <td>Sigma</td>
 <td>P4562</td>
 <td>Dissecting 			Solution: 2 
 Bath solution for pressure myography: 2 
 Lumen 			solution for pressure myography: 2</td>
 </tr>
 <tr>
 <td>EDTA 			dihydrate</td>
 <td>Research 			Organics</td>
 <td>9572E</td>
 <td>Dissecting 			Solution: 0.02 
 Bath solution for pressure myography: 0.02 
 Lumen 			solution for pressure myography: 0.02</td>
 </tr>
 <tr>
 <td>D-Glucose</td>
 <td>Sigma</td>
 <td>G7021</td>
 <td>Dissecting 			Solution: 5 
 Bath solution for Electrophysiology*: 10 
 Internal 			solution for electrophysiology: 20 
 Isolation solution for 			myocytes*: 10 
 Bath solution for pressure myography: 5 
 Lumen 			solution for pressure myography: 5</td>
 </tr>
 <tr>
 <td>Bovine 			serum albumin</td>
 <td>Sigma</td>
 <td>A3912</td>
 <td>Dissecting 			Solution: 1% 
 Lumen solution for pressure myography: 1%</td>
 </tr>
 <tr>
 <td>pH</td>
 <td></td>
 <td></td>
 <td>Dissecting 			Solution: 7.4 
 Bath solution for Electrophysiology*: 			7.3 
 Internal solution for electrophysiology: 7.2 
 Isolation 			solution for myocytes*: 7.2 
 Bath solution for pressure 			myography: 7.4 
 Lumen solution for pressure myography: 7.4</td>
 </tr>
 <tr>
 <td>Osmolarity</td>
 <td></td>
 <td></td>
 <td>Dissecting 			Solution: 300 
 Bath solution for Electrophysiology*: 			298 
 Internal solution for electrophysiology: 298 
 Isolation 			solution for myocytes*: 298 
 Bath solution for pressure 			myography: 300 
 Lumen solution for pressure myography: 300</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>*11</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Components of solutions used in the experiment.</td>
 </tr>
 </tbody>
</table>

exploring arterial smooth muscle kv7 potassium channel function using patch clamp electrophysiology and pressure myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Watch this Scientific Journal Video about Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography at JoVE.com

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

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