Transient focal ischemia in rat brain differentially regulates mRNA expression of protease‐activated receptors 1 to 4

Abstract

Degeneration or survival of cerebral tissue after ischemic injury depends on the source, intensity, and duration of the insult. In the model of focal ischemia, reduced blood flow results in a cascade of pathophysiologic events, including inflammation, excitotoxicity, and platelet activation at the site of injury. One serine protease that is associated closely with and produced in response to central nervous system (CNS) injury is thrombin. Thrombin enters the injury cascade in brain either via a compromised blood-brain barrier or possibly from endogenous prothrombin. Thrombin mediates its action through the protease-activated receptor family (PAR-1, -3, and -4). PARs belong to the superfamily of G protein-coupled receptors with a 7-transmembrane domain structure and are activated by proteolytic cleavage of their N-terminus. We showed that thrombin can be neuroprotective or deleterious when present at different concentrations before and during oxygen-glucose deprivation, an in vitro model of ischemia. We examined the change in mRNA expression levels of PAR-1 to 4 as a result of transient focal ischemia in rat brain, induced by microinjection of endothelin near the middle cerebral artery. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, after ischemic insult on the ipsilesional side, PAR-1 was found to be downregulated significantly, whereas PAR-2 mRNA levels decreased only moderately. PAR-3 was upregulated transiently and then downregulated, and PAR-4 mRNA levels showed the most striking (2.5-fold) increase 12 hr after ischemia, in the injured side. In the contralateral hemisphere, mRNA expression was also affected, where decreased mRNA levels were observed for PAR-1, -2, and -3, whereas PAR-4 levels were reduced only after 7 days. Taken together, these data suggest involvement of the thrombin receptors PAR-1, PAR-3, and PAR-4 in the pathophysiology of brain ischemia.

Inhibiting Protease-Activated Receptor 4 Limits Myocardial Ischemia/Reperfusion Injury in Rat Hearts by Unmasking Adenosine Signaling

“…This consists of cDNA reverse transcribed from mRNA pooled from three rat hearts. Forward and reverse primers used for amplifying rat PAR4 were prepared commercially, based on published data (Rohatgi et al, 2004): sense, 5Ј-GGA ATG CCA GAC GCC CAG CAT C-3Ј; and antisense, 5Ј-GGT GAG GCG TTG ACC ACG CA-3Ј (PCR product, 559 bp). The conditions for amplification were 95°C for 10 min for one cycle, 94°C for 30 s, 64°C for 90 s, 72°C for 60 s for 40 cycles, and 72°C for 10 min for 1 cycle.…”

Deficiency of PAR4 Attenuates Cerebral Ischemia/Reperfusion Injury in Mice

“…Because PAR4 is also expressed on neurons, it is also possible that inhibiting expression of PAR4 in the brain has a direct neuroprotective effect after brain ischemic injury. Other studies also showed that PAR4 mRNA levels were found to be significantly increased 12 h after ischemia in the rat injured hemisphere and this increased level was reduced only after 7 days (Rohatgi et al, 2004). Because PAR4 receptors are widely distributed and deficiency of PAR4 showed multiple effects in cerebral I/R injury, it is still difficult to determine the exact mechanisms through which PAR4 acts.…”

SCH 79797, a selective PAR1 antagonist, limits myocardial ischemia/reperfusion injury in rat hearts

“…More recently, PAR1 deficiency in a PAR1 knockout mouse has been demonstrated to be protective against renal failure in a model of renal I/R [24]. Even though PAR1 is expected to be activated by thrombin at the time of myocardial injury, little is known about its role in determining the extent of myocardial I/R injury.…”