Yonsei Med J.  2006 Apr;47(2):249-254. 10.3349/ymj.2006.47.2.249.

Elevated Contractile Responses to Acetylcholine in Organ Cultured Rabbit Carotid Artery

• Lee Y
• Jung S
• Won JE
• Nam T
• Ahn D

• KMID: 1110750
• DOI: http://doi.org/10.3349/ymj.2006.47.2.249

Abstract

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum-free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.

Keyword

Organ culture; carotid artery; acetylcholine; N(G)-nitro-L-arginine methyl ester; Ca2+ regulation

MeSH Terms

Time Factors
Rabbits
Organ Culture Techniques/*methods
Nitroprusside/pharmacology
NG-Nitroarginine Methyl Ester/metabolism/pharmacology
*Muscle Contraction
Models, Statistical
Dose-Response Relationship, Drug
Culture Media, Serum-Free/metabolism
Carotid Arteries/*drug effects/metabolism/*pathology
Calcium/metabolism
Animals
Acetylcholine/*pharmacology

Figure

• 

  Fig. 1 Concentration-response relationship for the relaxant effect of acetylcholine (ACh) on the phenylnephrine (PE)-induced contractions in endothelium (EC)-intact rabbit carotid arteries. Carotid arterial rings were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) for up to 7 days. ACh was cumulatively added after PE-induced contraction had reached a steady state. The difference between steady state precontraction and basal tension was considered one hundred percent. Results are expressed as mean ± S.E. (n = 14). The inset indicates the concentration-relaxation relationship after the relaxation was normalized to the maximal relaxation at each experimental condition. *denotes significant difference from freshly isolated arteries (p < 0.01).

• 

  Fig. 2 Effect of Nitric oxide synthase inhibitor, L-NAME, on the ACh-induced relaxation responses in EC-intact, freshly isolated rabbit carotid arteries (A) and arteries cultured in serum-free DMEM for up to 7 days (B). Concentration-response curve shows ACh-induced change of tension in arteries that were: freshly prepared (○), cultured for 3 days (●), and cultured for 7 days (▲). NG-nitro-L-arginine methyl ester (L-NAME; 30 µM) was added 30 minutes before PE (1 µM) application. Results are expressed as mean ± S.E. (n = 10). *and **denote significant difference from freshly isolated arteries (p < 0.05 and p < 0.01, respectively).

• 

  Fig. 3 Concentration-response relationship of the effect of ACh on EC-denuded carotid arteries that were freshly isolated (●),cultured in serum free DMEM for 3 days (○) or cultured for 7 days (▲). ACh was cumulatively added after PE-induced contraction had reached a steady state. The difference between steady state precontraction and basal tension was considered one hundred percent. Results are expressed as mean ± S.E. (n = 10). *and **denote significant difference from freshly prepared arteries (p < 0.05 and p < 0.01, respectively).

• 

  Fig. 4 Concentration-response relationship of the effect of SNP on endothelium-denuded carotid arteries that were freshly isolated (●), cultured for 3 days (○) and 7 days (▲). Sodium nitroprusside (SNP) was cumulatively added after PE-induced contraction had reached a steady state. The difference between steady state precontraction and basal tension was considered one hundred percent. Results are expressed as mean ± S.E. (n = 15).

• 

  Fig. 5 ACh-induced changes in [Ca2+]i and contraction in EC-denuded, freshly prepared arteries and carotid arterial strips cultured in serum-free DMEM for 7 days. A) Typical recordings of simultaneous measurement of [Ca2+]i (upper panel) and tension (lower panel) in rabbit carotid arteries. L-NAME (30 µM) was added 30 minutes before application of PE (1 µM). ACh (10 µM) was added after PE-induced contraction reached steady-state conditions. B) Mean changes in [Ca2+]i induced by ACh in freshly prepared arteries and arteries cultured for 7 days. Results are expressed as mean ± S .E. (n = 6). The PE-induced change in ratio was considered one hundred percent. C) Mean changes in contraction induced by ACh in freshly prepared arteries and arteries cultured for 7 days. Results are expressed as mean ± S.E. (n = ). The PE-induced contraction was considered one hundred percent. *denotes significant difference from freshly prepared arteries (p < 0.01).

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