8-bromocyclic-gmp and Fever

In spite of considerable research attention focused on clarifying the mechanisms by which the mammalian respiratory rhythm is generated, little attention has been given to examining how this neuronal circuit can be protected from heat stress. Hyperthermia has a profound effect on neuronal circuits including the circuit that generates breathing in mammals. As temperature of the brainstem increases, respiratory frequency concomitantly rises. If temperature continues to increase respiratory arrest (apnea) and death can occur. Previous research has implicated protein kinase G (PKG) activity in regulating neuronal thermosensitivity of neuronal circuits in invertebrates. Here we examine if pharmacological manipulation of PKG activity in a brainstem slice preparation could alter the thermosensitivity of the fictive neonatal mouse respiratory rhythm. We report a striking effect following alteration of PKG activity in the brainstem such that slices treated with the PKG inhibitor KT5823 recovered fictive respiratory rhythm generation significantly faster than control slices and slices treated with a PKG activator (8-Br-cGMP). Furthermore, slices treated with 8-Br-cGMP arrested fictive respiration at a significantly lower temperature than all other treatment groups. In a separate set of experiments we examined if altered PKG activity could regulate the response of slices to hypoxia by altering the protective switch to fictive gasping. Slices treated with 8-Br-cGMP did not switch to the fictive gasp-like pattern following exposure to hypoxia whereas slices treated with KT5823 did display fictive gasping. We propose that PKG activity inversely regulates the amount of stress the neonatal mammalian respiratory rhythm can endure.

Topics: Action Potentials; Animals; Animals, Newborn; Brain Stem; Carbazoles; Cell Hypoxia; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Fever; In Vitro Techniques; Mice; Mice, Inbred Strains; Microelectrodes; Neurons; Periodicity; Protein Kinase Inhibitors; Respiration; Temperature

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO-cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non-selective NOS inhibitor (N(G)-monomethyl-l-arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or a cGMP analogue (8-bromo-cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8-bromo-cGMP increased the response. These findings indicate that the LC NO-cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO-cGMP pathway in the LC.

Topics: Animals; Cyclic GMP; Endotoxins; Fever; Guanylate Cyclase; Locus Coeruleus; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroso Compounds; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

We tested the hypothesis that nitric oxide (NO) acts in the anteroventral preoptic region (AVPO) modulating fever. To this end, body core temperature (T(c)) of rats was monitored by biotelemetry before and after pharmacological modulation of the NO pathway. Nitrite/nitrate and cGMP in the anteroventral third ventricular region (AV3V), where the AVPO is located, were also determined. Intra-AVPO microinjection of the NO synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA, 12.5 microg) did not affect basal T(c), but it enhanced the early stage of lipopolysaccharide (LPS) fever, indicating that NO plays an antipyretic role in the AVPO. In agreement, intra-AVPO microinjection of the NO donor sodium nitroprusside (5 microg) reduced T(c). The antipyretic effect of NO seems to be mediated by cGMP because 1) NO has been shown to activate soluble guanylate cyclase, 2) intra-AVPO microinjection of 8-bromo-cGMP (8-BrcGMP) reduced T(c), and 3) the changes in AV3V levels of nitrite/nitrate and cGMP were similar in the course of fever. Additionally, we observed that nitrite/nitrate and cGMP levels decreased in the AV3V after, but not before, the onset of LPS fever, showing that the activity of the NO-cGMP pathway is reduced in the AV3V after intraperitoneal LPS, a mechanism that could contribute to the genesis and maintenance of fever. It was also observed that the efficacy of 8-BrcGMP in reducing T(c) in the AVPO is increased after LPS, emphasizing that the NO-cGMP pathway is antipyretic. This response could explain why intra-AVPO L-NMMA enhanced the early stage of LPS fever, even though the activity of the NO pathway before the onset of fever was unchanged. In summary, these data support an antipyretic role of the NO-cGMP pathway in the AVPO.

Topics: Animals; Cyclic GMP; Enzyme Inhibitors; Fever; Injections, Intraperitoneal; Lipopolysaccharides; Male; Microinjections; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrites; Nitroprusside; omega-N-Methylarginine; Preoptic Area; Rats; Rats, Wistar; Solubility

The present study aimed to test the hypothesis that a decrease in preoptic cAMP mediates fever. To this end, body core temperature (T(c)) of unanesthetized, freely moving rats was monitored by biotelemetry before and after pharmacological modulation of the cAMP pathway, and cAMP levels in the anteroventral third ventricular region (AV3V), where the preoptic region (POA) is located, were determined. We observed that intra-POA administration of the cAMP agonist dibutyryl-cAMP (Db-cAMP, 40 microg) reduced T(c). PGE(2) (the proximal mediator of fever, 200 ng) raised T(c) with a concomitant decrease in AV3V cAMP levels from 22.7+/-1.8 to 17.0+/-1.0 fmol/microg protein. Moreover, PGE(2)-induced fever was impaired by the phosphodiesterase inhibitor aminophylline. In order to verify the interaction between the cAMP- and cGMP-dependent pathways in the POA, we then co-injected Db-cAMP and 8-Br-cGMP into the POA. As a result, 8-Br-cGMP augmented the drop in T(c) evoked by Db-cAMP. Lastly, we observed that intra-POA co-microinjection of the protein kinase A inhibitor (Rp-cAMPS, 1 microg) with the protein kinase G inhibitor (Rp-cGMPS, 1 microg), mimicking the effects of reduced production of cAMP and cGMP, respectively, produced a fever-like response. In summary, the present data support that a decrease in the levels of cAMP and cGMP in the POA is associated with the genesis of fever.

Topics: Aminophylline; Animals; Body Temperature Regulation; Bucladesine; Cyclic AMP; Cyclic GMP; Dinoprostone; Drug Interactions; Fever; Injections, Intraventricular; Lipopolysaccharides; Male; Microinjections; Neurons; Preoptic Area; Rats; Rats, Wistar; Second Messenger Systems; Thionucleotides