Cloning of COX4 cDNA from the NZ white rabbit and expression of this gene in bladder smooth muscle following partial outlet obstruction

Abstract.

We cloned and characterized the rabbit COX4 coding sequence, then examined expression of the gene in bladder smooth muscle following partial obstruction of the bladder outlet. Obstruction was done surgically in male NZ white rabbits and bladders procured at 1, 3, 5, 7, and 14 days post-obstruction (two animals/time). RNA, then cDNA, was prepared from smooth muscle from each sample. Cloning was done from cDNA by PCR. COX4 mRNA in smooth muscle was defined by real-time RT-PCR, using cDNA from bladder samples. The rabbit COX4 coding sequence specifies a protein of 169 amino acids, including a membrane transit sequence for mitochondrial (mt) entry. Expression of COX4 in bladder smooth muscle decreased by 80% in the first day post-obstruction and remained low through 14 days post-obstruction. Cytochrome oxidase activity decreased sharply in the first 3 days post-obstruction in smooth muscle, in concert with COX4 transcript levels. Partial outlet obstruction of the rabbit urinary bladder results in attenuation in smooth muscle of mRNA synthesis from the COX4 gene. In turn, this results in attenuated activity of mt electron transport due to limited availability of nucleus-encoded components required for the assembly of cytochrome oxidase.