Organ culture mimics the effects of hypoxia on membrane potential, K(+) channels and vessel tone in pulmonary artery.
OBJECTIVE
Blood vessel culture is gaining interest for use with transfection-based techniques, but alters the contractile properties of the vessels. The present study tested the effects of culture on the intrinsic tone of rat pulmonary arteries (PAs) and examined the function and expression of K(+) channels regulating the resting membrane potential (E(m)) and tone of pulmonary artery smooth muscle cells (PASMCs).


METHODS
Rat intrapulmonary arteries were isolated and cultured under standard and modified conditions. Contractile responses of fresh and cultured PA were compared using vessel myograph. Electrophysiology experiments on isolated PASMCs used the patch-clamp technique. K(+) channel expression was quantified using reverse transcription and real-time PCR.


RESULTS
After 4 days in culture vessels contracted to phenylephrine, but relaxation to carbachol was significantly impaired. Contractile responses to 10 mM KCl, 4-aminopyridine and tetraethylammonium increased, and vessels developed an uncharacteristic relaxation response to Ca(2+)-free solution, nifedipine and levcromakalim. PASMCs from cultured vessels were depolarized and K(+) currents reduced, in association with down-regulation of K(v)1.5, K(v)2.1 and TWIK-related acid-sensitive K(+) channel-1 mRNA. These changes were partially reversed by increased oxygenation of the culture medium or removing the endothelium before culture.


CONCLUSIONS
Culture of PA for 3-4 days induced loss of functional K(+) channels, depolarization of PASMCs, Ca(2+) influx, intrinsic tone and spontaneous constrictions, similar to the effects of chronic hypoxia. This limits the use of cultured vessels for studying excitation-contraction coupling, although oxygenating the culture medium and removing the endothelium can help to retain normal smooth muscle function.
Manoury
Boris
B
The University of Manchester, Manchester, UK.
Etheridge
Sarah L
SL
Reid
Joy
J
Gurney
Alison M
AM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015687:Cell Hypoxia; D004730:Endothelium, Vascular / Q000502:physiology; D008297:Male; D008564:Membrane Potentials; D009119:Muscle Contraction; D009129:Muscle Tonus; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009924:Organ Culture Techniques; D010100:Oxygen / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley
D015221:Potassium Channels; D012333:RNA, Messenger; D010100:Oxygen; D002118:Calcium
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