Release of ATP in the central nervous system during systemic inflammation: real-time measurement in the hypothalamus of conscious rabbits.

Receptors for extracellular ATP (both ionotropic and metabotropic) are widely expressed in the CNS both in neurones and glia. ATP can modulate neuronal activity in many parts of the brain and contributes to the central nervous control of several physiological functions. Here we show that during the systemic inflammatory response the extracellular concentrations of ATP increase in the anterior hypothalamus and this has a profound effect on the development of the thermoregulatory febrile response. In conscious rabbits we measured ATP release in real time with novel amperometric biosensors and monitored a marked increase in the concentration of ATP (4.0 +/- 0.7 microm) in the anterior hypothalamus in response to intravenous injection of bacterial endotoxin - lipopolysaccharide (LPS). No ATP release was observed in the posterior hypothalamus. The release of ATP coincided with the development of the initial phase of the febrile response, starting 18 +/- 2 min and reaching its peak 45 +/- 2 min after LPS injection. Application of the ATP receptor antagonists pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid, Brilliant Blue G or periodate oxidized ATP dialdehyde to the site of ATP release in the anterior hypothalamus markedly augmented and prolonged the febrile response. These data indicate that during the development of the systemic inflammation, ATP is released in the anterior hypothalamus to limit the magnitude and duration of fever. This release may also have a profound effect on the hypothalamic control of other physiological functions in which ATP and related purines have been implicated to play modulatory roles, such as food intake, hormone secretion, cardiovascular activity and sleep.

P2 receptor blockade attenuates fever and cytokine responses induced by lipopolysaccharide in rats.

Adenosine 5'-triphosphate (ATP) has been shown to induce release of cytokines implicated in fever, including interleukin(IL)-1beta, IL-6, and tumour necrosis factor-alpha (TNF-alpha). The role of ATP-mediated purinergic signalling in fever and cytokine release during systemic inflammation was investigated by studying the effects of P2 receptor antagonists suramin, pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and Brilliant Blue G (BBG) on changes in body temperature and the increases in plasma levels of IL-1beta, IL-6, and TNFalpha induced by bacterial lipopolysaccharide (LPS) in rats. LPS (Escherichia coli; 50 microg kg(-1))-induced febrile response was attenuated by suramin (25 mg kg(-1) and 100 mg kg(-1)), PPADS (25 mg kg(-1)), and a more selective P2X(7) receptor antagonist BBG (100 mg kg(-1)) injected intraperitoneally before the induction of fever. The increase in plasma concentrations of IL-1beta and IL-6, measured 1 h after LPS treatment, was reduced by PPADS (25 mg kg(-1)) and BBG (100 mg kg(-1)). LPS-induced increase in plasma TNF-alpha concentration was also markedly attenuated by BBG (100 mg kg(-1)), but not by PPADS (25 mg kg(-1)). These data indicate that purinergic signalling plays an important role in the mechanisms responsible for the LPS-induced febrile response and increases in the levels of circulating cytokines. We suggest that ATP acting via P2X(7) receptors induces release of pyrogenic cytokines to mediate fever during systemic inflammation.

Fever in systemic inflammation: roles of purines.

Extracellular purine nucleotide and nucleoside signalling molecules, such as ATP and adenosine, acting through specific receptors (P2 and P1, respectively) play significant roles in the mechanisms underlying the febrile response. A variety of P2 and P1 receptor subunits have been identified in the hypothalamus, the area of the brain that orchestrates the febrile response. Importantly, both ATP and adenosine have been shown to modulate release and/or action of cytokines that are implicated in fever, as well as to be involved in the central mechanisms of cardiovascular and respiratory control. Our data indicate that at the level of the anterior hypothalamus extracellular ATP is involved in the control of the development of fever. A population of warm-sensitive neurones in the anterior hypothalamus is likely to be the site of action of ATP on body temperature. ATP-induced cytokine release does not appear to play a significant role in the hypothalamic mechanisms leading to the development of the febrile response. However, the blockade of fever by P2 receptor antagonists given systemically suggests that ATP-mediated signalling may play a role in the release of pyrogenic cytokines in the periphery. At the level of the anterior hypothalamus adenosine appears to be released tonically, and acts to maintain body temperature under afebrile conditions. There is also evidence that adenosine-mediated signalling may play a role in the hypothalamic mechanisms controlling the degree of body temperature increase during fever. Our investigations have identified possible mechanisms by which purines modulate the febrile response. The actions of purines on body temperature during fever are most likely "site specific" (brain vs. periphery), may or may not involve their effect on cytokine release and/or action, and are likely to involve P2 and P1 receptors of different subtypes. Further extensive studies are needed to elucidate these mechanisms in greater detail and may lead to the development of new approaches for modifying febrile, cytokine and acute-phase responses to infection.

Role of alpha(2)-macroglobulin in fever and cytokine responses induced by lipopolysaccharide in mice.

Alpha(2)-macroglobulin (alpha(2)M) is not only a proteinase inhibitor in mammals, but it is also a specific cytokine carrier that binds pro- and anti-inflammatory cytokines implicated in fever, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha). To define the role of alpha(2)M in regulation of febrile and cytokine responses, wild-type mice and mice deficient in alpha(2)M (alpha(2)M -/-) were injected with lipopolysaccharide (LPS). Changes in body temperature as well as plasma levels of IL-1beta, IL-6, and TNF-alpha and hepatic TNF-alpha mRNA level during fever in alpha(2)M -/- mice were compared with those in wild-type control mice. The alpha(2)M -/- mice developed a short-term markedly attenuated (ANOVA, P < 0.05) fever in response to LPS (2.5 mg/kg ip) compared with the wild-type mice. At 1.5 h after injection of LPS, the plasma concentration of TNF-alpha, but not IL-1beta or IL-6, was significantly lower (by 58%) in the alpha(2)M -/- mice compared with their wild-type controls (ANOVA, P < 0.05). There was no difference in hepatic TNF-alpha mRNA levels between alpha(2)M -/- and wild-type mice 1.5 h after injection of LPS. These data support the hypotheses that 1) alpha(2)M is important for the normal development of LPS-induced fever and 2) a putative mechanism of alpha(2)M involvement in fever is through the inhibition of TNF-alpha clearance. These findings indicate a novel physiological role for alpha(2)M.

Involvement of purinergic signalling in central mechanisms of body temperature regulation in rats.

1. P2 purinoreceptors are present in hypothalamic and brainstem nuclei that are involved in the regulation of body temperature (T(b)). The role of ATP acting on these P2 receptors in thermoregulation was investigated by studying the effects of the stable ATP analogue alpha,beta-methyleneATP (alpha,beta-meATP) and P2 receptor antagonists suramin and pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on T(b) when injected intracerebroventricularly (i.c.v.) via a pre-implanted cannula in conscious rats at various ambient temperatures and during lipopolysaccharide (LPS)-induced fever. 2. Depending on ambient temperature, alpha,beta-meATP (0.2 micromol, i.c.v.) induced a fall in T(b) (-3.3 degrees C, P<0.05), no changes in T(b) when compared to pre-injection levels, or an increase in T(b) ( approximately 1.0 degrees C, P<0.05) in rats maintained at 10 degrees C, 25 degrees C and 30 degrees C ambient temperature, respectively. 3. Suramin (7 nmol, i.c.v.) induced a lasting (up to 6 h) increase in T(b) (on average 1.2 degrees C, P<0.05) in rats kept at 25 degrees C or 30 degrees C, but failed to induce any rise in T(b) in rats at 10 degrees C ambient temperature. An increase in T(b) was also observed in rats (25 degrees C ambient temperature) treated with PPADS (0.2 micromol, i.c.v.). 4. alpha,beta-meATP (0.2 micromol) injected i.c.v. or directly into the anterior hypothalamus caused a profound fall in T(b) (by 0.9 degrees C and 1.0 degrees C, respectively; P<0.05) during LPS (E.coli; 50 microg kg(-1))-induced fever in rats at 25 degrees C ambient temperature. Fever was initiated more rapidly in rats treated with suramin (7 nmol) or PPADS (70 nmol), however its late phase was unaffected. Suramin (7 nmol) and PPADS (70 nmol) injected at the time when fever was already developed (2.5 h after LPS injections) did not alter febrile T(b). 5. These data indicate that purinergic signalling may play a significant role in central mechanisms of T(b) regulation at various ambient temperatures and during fever.