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

Dette arbejde blev finansieret af en bevilling fra National Heart, Lung, og Blood Institute (NIH R01-HL089564) til KLB og PhD-stipendier fra American Heart Association (09PRE2260209) og Arthur J. Schmitt Foundation til BKM.

1. Kirurgisk excision af tyndtarmens Mesenterial Vaskulær Arcade <ol><li> Bedøve en 300-400 g Sprague-Dawley-rotte med isofluran (4%) administreres ved inhalation. </li><li> Udfør en midtlinie laparotomi at eksponere tyndtarmens mesenterium. Exteriorize den lille og store tarmen gennem abdominal incision med stor omhu for at undgå traumer på udsatte tarm og mesenterium. Forsigtigt lufte mesenteriet ud over steril gaze. </li><li> Kirurgisk punktafgiftspligtige tyndtarmen og en del af tyktarmen, herunder caecum (bl...

<ol>
	<li>Passmore, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNQ/M+Currents+in+Sensory+Neurons:+Significance+for+Pain+Therapy.">KCNQ/M Currents in Sensory Neurons: Significance for Pain Therapy.</a> J. Neurosci. 23, 7227-7236 (2003).</li><li>Falloon, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+small+artery+sensitivity+and+morphology+in+pressurized+and+wire-mounted+preparations.">Comparison of small artery sensitivity and morphology in pressurized and wire-mounted preparations.</a> Am. J. Physiol. Heart Circ. Physiol. 268, H670-H678 (1995).</li><li>Dunn, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+resistance+artery+sensitivity+to+agonists+under+isobaric+compared+with+isometric+conditions.">Enhanced resistance artery sensitivity to agonists under isobaric compared with isometric conditions.</a> Am. J. Physiol. Heart Circ. Physiol. 266, H147-H155 (1994).</li><li>Buus, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+sensitivity+of+rat+mesenteric+small+arteries+to+agonists+when+studied+as+ring+preparations+or+as+cannulated+preparations.">Differences in sensitivity of rat mesenteric small arteries to agonists when studied as ring preparations or as cannulated preparations.</a> Br. J. Pharmacol. 112, 579-587 (1994).</li><li>Abdelhalim, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+big+endothelin-1+in+comparison+with+endothelin-1+on+the+microvascular+blood+flow+velocity+and+diameter+of+rat+mesentery+in+vivo.">Effects of big endothelin-1 in comparison with endothelin-1 on the microvascular blood flow velocity and diameter of rat mesentery in vivo.</a> Microvasc. Res. 72, 108-112 (2006).</li><li>Altura, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-response+relationships+for+arginine+vasopressin+and+synthetic+analogs+on+three+types+of+rat+blood+vessels:+possible+evidence+for+regional+differences+in+vasopressin+receptor+sites+within+a+mammal.">Dose-response relationships for arginine vasopressin and synthetic analogs on three types of rat blood vessels: possible evidence for regional differences in vasopressin receptor sites within a mammal.</a> J. Pharmacol. Exp. Ther. 193, 413-423 (1975).</li><li>Henderson, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasopressin-induced+vasoconstriction:+two+concentration-dependent+signaling+pathways.">Vasopressin-induced vasoconstriction: two concentration-dependent signaling pathways.</a> J. Appl. Physiol. 102, 1402-1409 (2007).</li><li>Mackie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+KCNQ+potassium+channels+as+novel+targets+for+the+control+of+mesenteric+artery+constriction+by+vasopressin,+based+on+studies+in+single+cells,+pressurized+arteries,+and+in+vivo+measurements+of+mesenteric+vascular+resistance.">Vascular KCNQ potassium channels as novel targets for the control of mesenteric artery constriction by vasopressin, based on studies in single cells, pressurized arteries, and in vivo measurements of mesenteric vascular resistance.</a> J. Pharmacol. Exp. Ther. 325, 475-483 (2008).</li><li>Brueggemann, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+selective+cyclooxygenase-2+inhibitors+on+vascular+smooth+muscle+ion+channels+may+account+for+differences+in+cardiovascular+risk+profiles.">Differential effects of selective cyclooxygenase-2 inhibitors on vascular smooth muscle ion channels may account for differences in cardiovascular risk profiles.</a> Mol. Pharmacol. 76, 1053-1061 (2009).</li><li>Brueggemann, L. I., Kaneez, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamp Technique. , (2012).</li><li>Berra-Romani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TTX-sensitive+voltage-gated+Na++channels+are+expressed+in+mesenteric+artery+smooth+muscle+cells.">TTX-sensitive voltage-gated Na+ channels are expressed in mesenteric artery smooth muscle cells.</a> Am. J. Physiol. Heart Circ. Physiol. 289, H137-H145 (2005).</li></ol>

De metoder og eksperimentelle metoder beskrevet her er ganske robust og kan producere klare og reproducerbare resultater, når de anvendes med omhyggelig opmærksomhed på detaljer. Gode ​​elektrofysiologiske optagelser og konstriktion / dilatation af arterielle segmenter er afhængige af sundhed celler og arterie segmenter, hhv. Cellepræparater kan variere fra dag til dag, selv efter samme protokol. Isolation Solutions kan anvendes i op til 2 uger, men hvis kvaliteten af ​​cellen præparatet er lav af to deraf...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Catalog Number </td><td> Kommentarer </td></tr><tr><td> Sodium Chloride </td><td> Sigma </td><td> S5886 </td><td> Dissecting Løsning: 145 Badopløsning for Elektrofysiologi *: 140 Intern opløsning elektrofysiologi: 10 Isolation løsning for myocytter *: 140 Badopløsning for tryk myography: 145 Lumen opløsning tryk myography: 145 </td></tr><tr><td> Kaliumchlorid </td><td> Sigma </td><td> P5405 </td><td> Dissecting Løsning: 4,7 Badopløsning for Elektrofysiologi *: 5,36 Intern opløsning elektrofysiologi: 135 Isolation løsning for myocytter *: 5,36 Badopløsning for tryk myography: 4,7 Lumen opløsning tryk myography: 4,7 </td></tr><tr><td> Kalium EGTA </td><td> Sigma </td><td> E4378 </td><td> Intern opløsning elektrofysiologi: 0,05 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H9136 </td><td> Badopløsning for Elektrofysiologi *: 10 Intern opløsning elektrofysiologi: 10 Isolation løsning for myocytter *: 10 </td></tr><tr><td> Dinatriumhydrogenphosphat </td><td> Sigma </td><td> S5136 </td><td> Isolation løsning for myocytter *: 0,34 </td></tr><tr><td> Kaliumhydrogenphosphat </td><td> Sigma </td><td> P5655 </td><td> Isolation løsning for myocytter *: 0,44 </td></tr><tr><td> Magnesium Chloride </td><td> Sigma </td><td> M2393 </td><td> Badopløsning for Elektrofysiologi *: 1,2 Intern opløsning elektrofysiologi: 1 Isolation løsning for myocytter *: 1,2 </td></tr><tr><td> Calciumchlorid </td><td> Sigma </td><td&gt; C7902 </td><td> Badopløsning for Elektrofysiologi *: 2. Isolation løsning for myocytter *: 0,05 </td></tr><tr><td> Natriumphosphat </td><td> Fisher Scientific </td><td> BP331-1 </td><td> Dissekere Løsning: 1,2 Badopløsning for tryk myography: 1,2 Lumen løsning til tryk myography: 1,2 </td></tr><tr><td> Magnesiumsulfat </td><td> Sigma </td><td> M2643 </td><td> Dissecting Løsning: 1,17 Badopløsning for tryk myography: 1,17 Lumen opløsning tryk myography: 1,17 </td></tr><tr><td> MOPS </td><td> Fisher Scientific </td><td> BP308 </td><td> Dissecting Løsning: 3 Badopløsning for tryk myography: 3 Lumen opløsning tryk myography: 3 </td></tr><tr><td> Pyrudruesyre </td><td> Sigma </td><td> P4562 </td><td> Dissecting Løsning: 2 Badopløsning fortryk myography: 2 Lumen opløsning tryk myography: 2 </td></tr><tr><td> EDTA-dihydrat </td><td> Research Organics </td><td> 9572E </td><td> Dissecting Løsning: 0,02 Badopløsning for tryk myography: 0,02 Lumen opløsning tryk myography: 0,02 </td></tr><tr><td> D-glucose </td><td> Sigma </td><td> G7021 </td><td> Dissecting Solution: 5 Badopløsning for Elektrofysiologi *: 10 Intern opløsning elektrofysiologi: 20 Isolation løsning for myocytter *: 10 Badopløsning for tryk myography: 5 Lumen opløsning tryk myography: 5 </td></tr><tr><td> Bovint serumalbumin </td><td> Sigma </td><td> A3912 </td><td> Dissekere Løsning: 1% Lumen løsning til tryk myography: 1% </td></tr><tr><td> pH </td><td></td><td></td><td> Dissekering Solution: 7,4 Badopløsning for Elektrofysiologi *: 7,3 Intern opløsning elektrofysiologi: 7,2 Isolation løsning for myocytter *: 7,2 Badopløsning for tryk myography: 7,4 Lumen opløsning tryk myography: 7,4 </td></tr><tr><td> Osmolaritet </td><td></td><td></td><td> Dissecting Løsning: 300 Badopløsning for Elektrofysiologi *: 298 Intern opløsning elektrofysiologi: 298 Isolation løsning for myocytter *: 298 Badopløsning for tryk myography: 300 Lumen opløsning tryk myography: 300 </td></tr><tr><td></td><td></td><td></td><td> * 11 </td></tr><tr><td></td><td></td><td></td><td> Tabel 1. Komponenter i opløsninger, der anvendes i forsøget. </td></tr></tbody></table>

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

Kontraktion eller relaksation af glatte muskelceller i væggene i modstandskar bestemmer arteriediameteren og derved styrer strømmen af ​​blod gennem karret og bidrager til det systemiske blodtryk. Kontraktionen Processen reguleres primært af cytosolisk calciumkoncentration ([Ca2 +] cyt), som igen styres af en række forskellige ion-transportører og kanaler. Ionkanaler er almindelige mellemprodukter i signaltransduktionsveje aktiveres af vasoaktive hormoner for at bevirke vasokonstriktion eller vasodilatation. Og ionkanaler er ofte ramt af terapeutiske midler enten bevidst (f.eks kalciumblokkere anvendes til at fremkalde vasodilatation og lavere blodtryk) eller utilsigtet (f.eks at fremkalde uønskede kardiovaskulære bivirkninger). Kv7 (KCNQ) spænding-aktiverede kaliumkanaler er for nylig blevet impliceret som vigtige fysiologiske og terapeutiske Targets for regulering af glatte muskelkontraktion. For at belyse de specifikke roller Kv7 kanaler i både fysiologiske signaltransduktion og i aktionerne af terapeutiske midler, er vi nødt til at undersøge, hvordan deres aktivitet moduleres på celleniveau samt vurdere deres bidrag inden for rammerne af det intakte arterie. Rotte mesenteriske arterier et nyttigt modelsystem. Arterierne kan let dissekeres, renses for bindevæv, og anvendt til fremstilling af isolerede arterielle myocytter til patch-clamp-elektrofysiologi, eller kanyle og sættes under tryk for målinger af vasokonstriktor / vasodilator reaktioner under relativt fysiologiske betingelser. Her beskriver vi de metoder, der anvendes til begge typer af målinger og give nogle eksempler på, hvordan det eksperimentelle design kan integreres til at give en bedre forståelse af de roller, disse ionkanaler i reguleringen af ​​vaskulær tonus.

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

Målinger af Kv7 (KCNQ) kalium kanal aktivitet i isolerede arterielle myocytter (ved hjælp af patch clamp elektrofysiologiske teknikker) parallelt med målinger af kvælerslange / dilator reaktioner (med tryk myography) kan afsløre vigtige oplysninger om de roller, Kv7 kanaler i vaskulære glatte muskulatur fysiologi og farmakologi.

Udforskning arteriernes glatte muskulatur Kv7 kaliumkanal Funktion hjælp Patch Clamp Elektrofysiologi og Pressure Myography

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

Research

JoVE Journal

Biology

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Focal Ca2+ Transient Detection in Smooth Muscle

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

Giant liposomfremstilling for billedbehandling og Patch-Clamp Elektrofysiologi

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Vurdere Murine Resistance Artery Funktion Brug Pressure Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

One-channel Cell-attached Patch-clamp Recording

Én-kanals Cell-attached Patch-clamp optagelse

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Rekonstituering af et transmembranprotein, Voltage ionkanal, KvAP, i Giant unilamelvesikler til mikroskopi og Patch clamp-forsøg

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Måling af Glat muskel funktion i den isolerede vævsbad-ansøgninger til Farmakologi Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...