| Summary: | This study aims to examine the signalling pathways underlying endothelium-dependent
responses in subcutaneous arteries of humans with Type 2 diabetes mellitus (T2DM) by
assessing the relative contributions of nitric oxide (NO), prostacyclin and endothelium
derived hyperpolarising factor (EDH) to responses to endothelium-dependent and
independent agonists. This study was approved by the Human Ethical Committee of Universiti Sains
Malaysia (USM); work conducted in this study conformed to the provisions of the
Declaration of Helsinki. Written informed consent was obtained from patients undergoing
lower limb surgical procedures. Sixteen healthy controls and twenty diabetic patients between
the ages of 18 to 65 years old were recruited among those undergoing lower limb surgical
procedures.
Acetylcholine hydrochloride, phenylephrine and sodium nitroprusside were purchased
from Sigma Chemical Co. (St. Louis, MO). l-[(2- Chlorophenyl) diphenylmethyl]-1Hpyrazole
(TRAM 34), 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-l 1,7-metheno-
7H-dibenzo \b,n\ [l,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684) and
salbutamol were purchased from Tocris Bioscience (Bristol, UK). Indomethacin, L-NAME
and prostacyclin were obtained from Cayman Chemical Company (Ann Arbor, MI). Distilled
water was used to prepare the drug solutions, except for indomethacin, TRAM-34 and UCL
1684, which were dissolved in dimethyl sulphoxide (DMSO). Concentrations are given as
final molar concentration in the bath solution. Primary antibodies against endothelial nitric
oxide synthase (eNOS; AB5589), cyclooxygenase-1 (COX-1; AB53766), cyclooxygenase-2
(COX-2; AB15191), prostacyclin synthase (PGIS; AB23668), prostacyclin (IP; AB123419)
receptor and horseradish peroxidase (HRP)-conjugated secondary antibodies (AB 6721) were
purchased from Abeam (Cambridge, UK). A rabbit polyclonal antibody to 0-actin was
purchased from Sigma Chemical Co.
Subcutaneous tissues from lower limb surgical procedures were transported to
Pharmacology Vascular laboratory in ice cold physiological salt solution. Subcutaneous
arteries were dissected free of connective tissue and fat, and then cut into rings. Care was
|
|---|