Stimulates eHsp72 Release

4.2.1. Necrosis- versus Exocytosis-Mediated Release

There are currently two potential mechanisms for extracellular Hsp72 release. The first is that eHsp72 is released from the intracellular pool after necrotic or lytic cell death. The second is that eHsp72 is released via a receptor-mediated exocytosis mechanism. Gallucci, Loema, and Matzinger (Gallucci et al., 1999) first suggested that Hsp72 is released only in pathological circumstances such as those that result in necrotic/lytic death and not after apoptosis or programmed cell death. More recently, Basu et al. (Basu et al., 2000), Sauter et al. (Sauter et al., 2000), and Berwin et al. (Berwin et al., 2001) supported these ideas and demonstrated that indeed Hsp72 was released after necrotic/lytic but not apoptotic cell death. In these studies, cellular necrosis was induced in vitro by either repeated freeze/thaw exposures (Basu et al., 2000; Sauter et al., 2000; Berwin et al., 2001), hypotonic lysis (Sauter et al., 2000), or viral lysis (Berwin et al., 2001). Apoptosis was induced by exposure to UV (Basu et al., 2000; Sauter et al., 2000) or serum-depleted culture media (Berwin et al., 2001) and verified using flow cytometric assessment of AV and PI staining. Necrotic (lytic or messy) death versus apoptosis (controlled programmed death) was verified via cytometric assessment of annexin V (AV) + propridium iodide (PI) staining (Del Bino et al., 1999; Hammill et al., 1999; Honda et al., 2000; Lecoeur et al., 2001); necrotic (AV+PI+), apoptotic (AV+PI-), or viable (AV-PI-).

In contrast with necrotic/lytic release of eHsp72, we propose that eHsp72 released after exposure to a psychological and/or physical stressor most likely occurs via an exocytosis pathway in the absence of necrosis. This hypothesis is based on the following observations. First, there is precedent for an exocytotic eHsp72 releasing mechanism. In the brain, for example, glial cells may exocytotically release Hsp72 (Guzhova et al., 2001; Tytell, 2005). In addition, there is recent evidence that suggests that eHsp72

released during times of stress are in exosomes (Lancaster and Febbraio, 2005), small membrane vesicles secreted by various cell types including antigen-presenting cells, B cells, and T cells of the immune system (Chaput et al., 2004). Exosomes contain numerous costimulatory and antigen-presenting molecules including Hsp70, and such release does not appear to depend on the classical secretory pathway (Lancaster and Febbraio, 2005). Second, eHsp72 is elevated in the blood within 10-25 min of tailshock or restraint stressor onset (Fleshner and Johnson, 2005). The rapidity of the response suggests the classic protein induction/necrosis release pathway is not likely. Third, eHsp72 is increased in the blood after exposure to psychological stressors such as conditioned contextual fear and predatory stress (Fleshner et al., 2004), stressors that are not likely to induce necrosis. Fourth, Febbraio and colleagues (Walsh et al., 2001; Febbraio et al., 2002) reported that intense exercise (~65% V02max) increases eHsp72 in blood within 30min of exercise onset and that this occurs in the absence of cellular necrosis (Febbraio et al., 2002). Fifth, the increases in concentrations of eHsp72 released into the blood are two to six fold above baseline (pre-stress) levels. If necrotic/lytic cell death were the source of eHsp72 in the blood, it would require a large number of cells to simultaneously die a necrotic/lytic death. Nonetheless, it is still not possible to rule out necrotic release at this time because we only tested splenic necrosis and some other currently unidentified tissue in body may demonstrate greater necrosis than the spleen. In addition, stress may produce a low level of necrosis in a large number of tissues in the body, and that in combination with this global stress-induced cellular necrosis produces increases in circulation eHsp72.

Thus, we propose that release of eHsp72 via necrotic cell death does occur after exposure to some stressors; however, it likely results in a local, restricted, and tissue-specific increase in eHsp72 at the site of server tissue damage or injury. In this local fashion, eHsp72 released from necrotic cells may indeed function to facilitate local innate immune responses. In contrast with local release, we hypothesize that the observed large increases in eHsp72 in the blood after exposure to a whole-organism stressor is due to a receptor-mediated exocytosis releasing mechanism and is not dependent on necrotic/lytic cell death.

4.2.2. eHsp72 Release Involves Norepinephrine and a1ADRs

We have recently reported the role of the sympathetic nervous system, and specifically norepinephrine (NE), in the stress-induced release of eHsp72 (Johnson et al., 2005). Using pharmacological blockade and stimulation of adrenergic receptors, we completed a series of studies that tested the effect of labetalol (a1ADR and p1ADR antagonist), propranolol (PADR antagonist), and prazosin (a1ADR antagonist) on tailshock-induced release of eHsp72. The results of these studies were that labetalol and prazosin but not propranolol blocked the effect of tailshock on eHsp72. We also tested the effect of phenylephrine (a^DR agonist) and isoproterenol (PiADR agonist) in the absence of stress to release eHsp72. We found that phenylephrine but not isoproterenol released eHsp72 (Johnson et al., 2005). Interestingly, as stated above there is accumulating evidence that Hsp72 is released in exosomes and is released in a calcium-dependent fashion upon stimulation of the cell (Savina et al., 2003). Because activation of aiADR results in a rise in intracellular calcium (Schwietert et al., 1992), the release of exosomes is one potential mechanism by which catecholamines trigger the rise in eHsp72. These data support our hypothesis that eHsp72 is being induced and/or released via an a1ADR-mediated mechanism. We hypothesize that NE released from sympathetic nerve terminals binds to a1ADR and stimulates Hsp72 release.We propose that NE and not epinephrine (E) is responsible because NE binds with a higher affinity to a1ADR than does E (Hardman and Limbird, 2001), and adrenalectomy depletes ~95-99% of E (Hessman et al., 1976; Vollmer et al., 1995) yet has no effect on eHsp72 release after tailshock stress (Johnson et al., 2005).

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