Defining the Phosphodiesterase Superfamily Members in Rat Brain Microvessels

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of the second messengers, cAMP and cGMP, by controlling their rates of degradation. The functions of the PDEs include modulation of vascular tone and permeability to ensure an appropriate blood supply and molecular delivery to local tissues. Eleven PDE families have been identified, each having several different isoforms and splice variants.1 On the other hand, expression of individual PDE family members may be tissue-specific. Following the well-accepted definition for nomenclature, a PDE family member written in lower case letters in italics refers to the nucleotide while those written in nonitalicized capital letters refer to the protein.1 Expression of the cGMP-stimulated PDE2A is differential in human venous and capillary endothelial cells.2 PDE4a and PDE4b express at both transcriptional and translational levels; simultaneously, pde4d mRNA is detectable but PDE4D protein cannot be identified with the isoform-specific antibody in rat pulmonary microvascular endothelial cells.3 Activities of the cAMP PDE and the cGMP PDE were identified in bovine brain microvessels three decades ago,4 and more recently the expression of PDE1 and PDE5 was characterized in guinea pig basilar arteries.5 Nevertheless, there is limited information concerning detailed expressions of the PDE superfamily components in brain microvessels. The brain-blood-barrier (BBB) maintains the homeostasis of the brain. Studies using dyes and electron-dense tracers indicate that the primary BBB occurs at the level of the intracerebral microvasculature.6 Vascular endothelial cells at the blood–brain interface are sealed together at their edges by tight junctions and make up the walls of microvessels. The tight junctions of these cells are composed of smaller transmembrane protein subunits, frequently biochemical dimers. There is increasing evidence that PDEs may control the BBB through modulating cAMP or cGMP levels. Elevation of intracellular cAMP levels increases the electrical resistance of endothelial monolayers by stabilizing intercellular junctional complexes7 and thus reduces BBB permeability. Decreased cAMP and increased calcium levels are linked to increased permeability in the endothelial cells.8−10 Conversely, agents that increase endothelial cAMP levels prevent barrier injury in the lung injury models.11,12 Evidence from other organs, such as kidney, suggests PDE involvement in osmotic water permeability. PDE function underlies arginine vasopressin-regulated water reabsorption in the principal cells of the renal collecting duct by terminating protein kinase A (PKA) signaling through hydrolysis of localized cAMP.13 A recent report indicates that PDE4D tethers EPAC1 in a vascular endothelial cadherin (VE-Cad)-based signaling complex and controls cAMP-mediated vascular permeability.14 PDEs also play a pivotal role in regulation of cerebral blood flow (CBF) at both physiological and pathophysiological states. Noticeably, the cyclic nucleotide-related signal transduction pathways are particularly important to the vasodilating function of cerebral arteries and arterioles.15 Mechanistically, a basic level of NO produced in the endothelium activates the soluble guanylate cyclase to generate cGMP. The elevated cGMP inhibits PDE3 and may in turn lead to an increase in intracellular cAMP.16 In newborn pigs, PDE inhibitors applied topically dilated pial arterioles with concomitant increases in cAMP and/or cGMP levels in cerebral spinal fluid.17 Furthermore, cAMP and/or cGMP appear to be involved in arterial vasodilation in response to hypercapnia.17 Regulation of PDEs on CBF may interact with the nitric oxide (NO) pathway and histamine receptor(s).18,19 NO- and prostanoid-induced vascular relaxations largely rely on cGMP and cAMP generation, respectively.15 Regulation of the cell signaling pathways by PDEs may involve activation of protein kinases and ion channels as well as changes of intracellular Ca2+ in modulation of cerebral arterial tones.17,20 In the present study, we profiled gene expression in brain microvessels using microarray technology, highlighting PDE superfamily components. We used immunofluorescent markers to define the location of PDE4D and PDE5A in brain microvessels by costaining with an endothelial marker, rat endothelial cell antigen-1 (RECA-1), and a vascular machinery marker, α-smooth muscle actin (α-SMA). Apparently, defining the PDE superfamily components in brain microvessels would be an essential step facilitating future studies to explore their biological role and application value.