Phosphatidylinositide 3-kinases (PI3K) are a family of lipid kinases important for cell cycle control, apoptosis, proliferation, and cell motility. Recently, evidence has been accumulating that PI3K may also play a critical role in regulating vascular smooth muscle tone, contractility and remodelling in response to stress and hypertension. However, no comprehensive survey of PI3K isoform expression in vascular tissue was available. In this investigation the aim was to examine the expression of different PI3K isoforms in the rat at the mRNA level, in particular focusing on vascular tissue. To this end, we have developed primers and methods to detect RNA expression of all known mammalian PI3K catalytic and regulatory isoforms in rat tissues.

Male Wistar rats (250-350g) were killed by cervical dislocation and exsanguinations following Schedule 1 procedures (Animal Scientific Procedures Act 1986, U.K.). Various organs, aortas, and mesenteric arteries were removed and placed in oxygenated Krebs buffer held at 4 °C. After removing fat and connective tissue, organs were flash frozen in liquid nitrogen, then pulverized and suspended in Trizol reagent. Total RNA was extracted and 1 mg was used to synthesize cDNA by reverse transcription. Primers were designed for the fourteen known Class IA, Class IB, Class II, and Class III PI3K catalytic and adaptor molecule isoforms: p110, p110ß, p110, p85, p55, p50, p85ß, p55, p110, p101, C2, C2ß, C2, and VPS34. PCR reactions were carried out in a GeneAmp PCR System 2400 (Perkin Elmer) using the PCR Core System Kit (Promega) with appropriate controls, then were resolved on 2% agarose gels.

Our results confirmed that expression of some isoforms of PI3K, such as Class III VPS34, are ubiquitous in all tissues tested, whereas other isoforms appear to have some tissue specificity. The Class IA PI3K catalytic subunits and the major forms of the Class I adaptor subunits were all highly expressed in the organs tested. A similar result was obtained in aorta with the exception of p110ß, which was conspicuous in its absence. In contrast, only brain and kidney tissue displayed expression of both the splice variants of p85, p55 and p50, although p50 was also present in mesenteric arteries, but not in aorta. The Class IB catalytic subunit p110 was found in aorta and all major organ tissues, with relatively high abundance in kidney, but low expression in liver. However its corresponding regulatory subunit p101 was mainly expressed in lung and kidney, but was completely absent in aorta and mesentery arteries. Among the Class II isoforms, C2 a was abundant in all tissues tested, whereas C2ß and C2 showed more limited and variable tissue distribution.

Upon repeated trials, the relative expression levels within a tissue type were very consistent for the PI3K isoforms that appear to be ubiquitously expressed. This suggests that these isoforms of PI3K may be involved in general housekeeping functions for many cell types. Variability in expression levels of other isoforms suggests that these may be involved in more tissue- or cell-specific functions and may be subject to up- and down-regulation in response to varying conditions.

Vanhaesebroeck B., et al. (2001) Annu. Rev. Biochem. 70:535-602