Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of the enantiomers of a novel anticonvulsant, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine, in rats

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the enantiomers of a novel anticonvulsant agent, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine (AAP-Cl), in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation was accomplished by an HPLC system consisting of a Chirex chiral column (250 mm x 4.6 mm i.d.) and a mobile phase of hexane:ethanol:tetrahydrofuran (280:20:40 (v/v)) containing trifluroacetic acid (0.3% (v/v)) and triethylamine (0.018% (v/v)) at a flow rate of 0.8 ml/min with UV detection. Male Sprague-Dawley rats were given (+)-AAP-Cl (10 and 20 mg/kg), (-)-AAP-Cl (10 mg/kg) or the racemic mixture (20 mg/kg) by i.v. bolus injection and serial blood samples were collected at different times after drug administration. (+)-AAP-Cl and (-)-AAP-Cl were separated with a resolution factor, Rs, of at least 1.4, and a separation factor, alpha, greater than 1.09. Linear calibration curves were obtained over the concentration range of 0.5-30 microg/ml in plasma for both (+)-AAP-Cl and (-)-AAP-Cl (R2 > or = 0.996) with a limit of quantitation of 100 ng/ml and the recovery was greater than 80% for both enantiomers. The accuracy and precision for both enantiomers ranged from 96 to 102% (+/-0.2-7%) at upper and lower concentrations. The plasma concentration-time profiles of the enantiomers of AAP-Cl were best described by a two-compartment open model with a mean terminal half-life of about 5h, volume of distribution at steady state of 3 l/kg and clearance of about 0.6l/(hkg) in rats. There was no significant difference between the pharmacokinetic parameters of (+)-AAP-Cl and (-)-AAP-Cl, suggesting that the disposition of AAP-Cl in rats is not enantioselective. In addition, no chiral inversion of (+)-AAP-Cl to (-)-AAP-Cl or vice versa was observed. The results of this investigation have shed some light on the mechanism of action and disposition of AAP-Cl in rats.     

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.     

Central injection of nicotine increases hepatic and splenic interleukin 6 (IL-6) mRNA expression and plasma IL-6 levels in mice: involvement of the peripheral sympathetic nervous system.

Accumulating evidence suggests that plasma levels of interleukin 6 (IL-6), a major cytokine stimulating the synthesis of acute-phase proteins, are intimately regulated by the central nervous system. Nicotine, one of the major drugs abused by humans, has been shown to affect immunological functions. In the present study, effects of intracerebroventricular (i.c.v.) injection of nicotine on plasma IL-6 levels were investigated in mice. Nicotine administered i.c.v. dose-dependently increased plasma IL-6 levels; the lowest effective dose was 0.3 ng/mouse and the maximal effect was attained with the dose of 105 ng/mouse. The nicotine (105 ng/mouse, i.c.v.)-induced plasma IL-6 levels peaked at 3 h and approached basal levels 6 h after injection. Mecamylamine, a nicotinic receptor antagonist, blocked nicotine-induced plasma IL-6 levels. Depletion of peripheral norepinephrine with 6-hydroxydopamine [100 mg/kg, intraperitoneal (i. p.)] inhibited the nicotine-induced plasma IL-6 levels by 57%, whereas central norepinephrine depletion with 6-hydroxydopamine (50 microgram/mouse, i.c.v.) had no effect. Pretreatment with prazosin (alpha1-adrenergic antagonist; 1 mg/kg, i.p.), yohimbine (alpha2-adrenergic antagonist; 1 mg/kg, i.p.), and ICI-118,551 (beta2-adrenergic antagonist; 2 mg/kg, i.p.), but not with betaxolol (beta1-adrenergic antagonist; 2 mg/kg, i.p.), inhibited nicotine-induced plasma IL-6 levels. Among the peripheral organs, including the pituitary, adrenals, heart, lung, liver, spleen, and lymph nodes, nicotine (105 ng/mouse, i.c.v.) increased IL-6 mRNA expression only in the liver and spleen, which was inhibited by peripheral norepinephrine depletion. These results suggest that stimulation of central nicotinic receptors induces plasma IL-6 levels and IL-6 mRNA expression in the liver and spleen via the peripheral sympathetic nervous system, alpha1-, alpha2-, and beta2-adrenoreceptors being involved.     

Interferon-gamma and interleukin 4 inhibit interleukin 1beta-induced delayed prostaglandin E(2)generation through suppression of cyclooxygenase-2 expression in human fibroblasts

Interleukin (IL-)1 stimulates prostaglandin E(2)(PGE(2)) generation in fibroblasts, and preferential couplings between particular phospholipase A(2)(PLA(2)) and cyclooxygenase (COX) isozymes are implicated with IL-1-induced delayed PGE(2)generation. The regulatory effects of interferon (IFN)-gamma and IL-4 on IL-1beta-induced COX, PLA(2)isoforms expression and terminal delayed PGE(2)generation were examined in three types of human fibroblasts. These human fibroblasts constitutively expressed cytosolic PLA(2)(cPLA(2)) and COX-1 enzymes, and exhibited delayed PGE(2)generation in response to IL-1beta. IL-1beta also stimulated expression of cPLA(2)and COX-2 only, while constitutive and IL-1beta-induced type IIA and type V secretory PLA(2)s (sPLA(2)s) expression could not be detected. A COX-2 inhibitor and cPLA(2)inhibitor markedly suppressed the IL-1beta-induced delayed PGE(2)generation, while a type IIA sPLA(2)inhibitor failed to affect it. IFN-gamma and IL-4 dramatically inhibited the IL-1beta-induced delayed PGE(2)generation; these cytokines apparently suppressed IL-1beta-stimulated COX-2 expression and only weakly suppressed cPLA(2)expression in response to IL-1beta. These results indicate that IL-1beta-induced delayed PGE(2)generation in these human fibroblasts mainly depends on de novo induction of COX-2 and cPLA(2), irrespective of the constitutive presence of COX-1, and that IFN-gamma and IL-4 inhibit IL-1beta-induced delayed PGE(2)generation by suppressing, predominantly, COX-2 expression.     

Nesfatin-1(30-59) but not the N- and C-terminal fragments, nesfatin-1(1-29) and nesfatin-1(60-82) injected intracerebroventricularly decreases dark phase food intake by increasing inter-meal intervals in mice

Nesfatin-1 is an 82 amino acid N-terminal fragment of nucleobindin2 that was consistently shown to reduce dark phase food intake upon brain injection in rodents. We recently reported that nesfatin-1(1-82) injected intracerebroventricularly (icv) reduces dark phase feeding in mice. Moreover, intraperitoneal injection of mid-fragment nesfatin-1 (nesfatin-1(30-59)) mimics the food intake-reducing effects of nesfatin-1(1-82), whereas N-terminal (nesfatin-1(1-29)) and C-terminal fragments (nesfatin-1(60-82)) did not. We therefore characterized the structure-activity relationship of nesfatin-1 injected icv to influence the dark phase meal pattern in mice. Mouse nesfatin-1(1-29), nesfatin-1(30-59), nesfatin-1(60-82) or vehicle was injected icv in freely fed C57Bl/6 mice immediately before the dark phase and food intake was monitored using an automated episodic feeding monitoring system. Nesfatin-1(30-59) (0.1, 0.3, 0.9 nmol/mouse) induced a dose-related reduction of 4-h food intake by 28%, 49% and 49% respectively resulting in a 23% decreased cumulative 24-h food intake compared to vehicle at the 0.3 nmol/mouse dose (p<0.05). The peak reduction occurred during the 3rd (-96%) and 4th hour (-91%) post injection and was associated with a reduced meal frequency (0-4h: -47%) and prolonged inter-meal intervals (3.1-times) compared to vehicle (p<0.05), whereas meal size was not altered. In contrast, neither nesfatin-1(1-29) nor nesfatin-1(60-82) reduced dark phase food intake at equimolar doses although nesfatin-1(60-82) prolonged inter-meal intervals (1.7-times, p<0.05). Nesfatin-1(30-59) is the active core of nesfatin-1(1-82) to induce satiety indicated by a reduced meal number during the first 4h post injection. The delayed onset may be indicative of time required to modulate other hypothalamic and medullary networks regulating nocturnal feeding as established for nesfatin-1.     

Endogenous opioids: role in prostaglandin-dependent and -independent fever

This study evaluated the participation of mu-opioid-receptor activation in body temperature (T(b)) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid mu-receptor-antagonist cyclic d-Phe-Cys-Try-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP; 0.1-1.0 microg) reduced fever induced by LPS (5.0 microg/kg) but did not change T(b) at ambient temperatures of either 20 degrees C or 28 degrees C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0-10.0 mg/kg, 3.0-30.0 microg, and 1-100 ng, respectively) produced a dose-dependent increase in T(b). Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 microg icv) reduced the fever induced by intracerebroventricular administration of TNF-alpha (250 ng), IL-6 (300 ng), CRF (2.5 microg), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PGF(2alpha) (500.0 ng) but not the fever induced by IL-1beta (3.12 ng) or PGE(2) (125.0 ng) or the second phase of the fever induced by PGF(2alpha). Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE(2) levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1beta and prostaglandins) recruit the opioid system to cause a mu-receptor-mediated fever.     

Kupffer cell-generated PGE2 triggers the febrile response of guinea pigs to intravenously injected LPS

Because the onset of fever induced by intravenously (i.v.) injected bacterial endotoxic lipopolysaccharides (LPS) precedes the appearance in the bloodstream of pyrogenic cytokines, the presumptive peripheral triggers of the febrile response, we have postulated previously that, in their stead, PGE2 could be the peripheral fever trigger because it appears in blood coincidentally with the initial body core temperature (Tc) rise. To test this hypothesis, we injected Salmonella enteritidis LPS (2 microg/kg body wt i.v.) into conscious guinea pigs and measured their plasma levels of LPS, PGE2, TNF-alpha, IL-1beta, and IL-6 before and 15, 30, 60, 90, and 120 min after LPS administration; Tc was monitored continuously. The animals were untreated or Kupffer cell (KC) depleted; the essential involvement of KCs in LPS fever was shown previously. LPS very promptly (<10 min) induced a rise of Tc that was temporally correlated with the elevation of plasma PGE2. KC depletion prevented the Tc and plasma PGE2 rises and slowed the clearance of LPS from the blood. TNF-alpha was not detectable in plasma until 30 min and in IL-1beta and IL-6 until 60 min after LPS injection. KC depletion did not alter the times of appearance or magnitudes of rises of these cytokines, except TNF-alpha, the maximal level of which was increased approximately twofold in the KC-depleted animals. In a follow-up experiment, PGE2 antiserum administered i.v. 10 min before LPS significantly attenuated the febrile response to LPS. Together, these results support the view that, in guinea pigs, PGE2 rather than pyrogenic cytokines is generated by KCs in immediate response to i.v. LPS and triggers the febrile response.     

Adrenocorticotropin secretion rates following histamine injection in adult and newborn dogs.

The metabolic clearance rate (MCR) for ACTH in adult dogs was previously shown not to vary significantly with varying plasma ACTH concentrations or among dogs. This is confirmed here for pups aged 1-7 days. Hence, ACTH secretion rates can be continuously calculated from a continuous function of plasma ACTH vs. time. Each of seven adult dogs under Nembutal anesthesia received two or three intravenous (i.v.) injections of histamine with increasing doses. The first injections in each dog ranged from 7 to 50 mug/kg, while the last dose was 62-108 mug/kg. A total of 16 injections were given. Twelve pups (two litters of six) aged 1-7 days each received one injection of histamine of 76-116 mug/kg (i.v.). ACTH concentrations in plasma were determined by an adrenal cell suspension bioassay before, and 6 times after each injection. Nine pups also underwent determinations of their MCR for ACTH, with plateau concentrations determined at three times during an ACTH infusion. Continuous curves of ACTH secretion rates were calculated for all 28 histamine injections, showing that all except the 1-day-old pups secrete considerable ACTH when stressed. Compared to adult dogs, the pups show lower secretion rate peaks and shorter periods of rapid secretion. Changes in plasma glucocorticoids also suggest that the adrenal cortex of newborn dogs can respond to ACTH by increased glucocorticoid secretion.     

ANP(1-28), BNP(1-32) and CNP(1-22) increase the severity of picrotoxin-kindled seizure syndrome in rats

The administration of rANP(1-28) in doses of 1.0 and 2.0 nmol (but not 0.2 nmol) into the lateral cerebral ventricle (i.c.v) of rats preliminarily kindled with picrotoxin resulted in an increase of the severity of picrotoxin-kindled convulsions 24 hrs after injection of the peptide. I.c.v. injection of pBNP(1-32) also resulted in a proepileptic effect when it was applied in the same doses with a similar time course; the increased seizure severity was observed 48 hrs after injection of pBNP in a dose of 2 nmol. I.c.v. administration of CNP(1-22) in a dose of 2 nmol induced an increase in the severity of picrotoxin-kindled convulsions 24 and 48 hrs after application of the peptide. None of the peptides influenced the seizure syndrome immediately after the injections. It is presumed that the delayed proepileptic properties of the three natriuretic peptides could be caused by some of their stable fragments which accumulate during their metabolism.     

Forearm muscle metabolism studied using (31)P-MRS during progressive exercise to fatigue after Acz administration.

The effects of acetazolamide (Acz)-induced carbonic anhydrase inhibition (CAI) on muscle intracellular thresholds (T) for intracellular pH (pH(i)) and inorganic phosphate-to-phosphate creatine ratio (P(i)/PCr) and the plasma lactate (La(-)) threshold were examined in nine adult male subjects performing forearm wrist flexion exercise to fatigue. Exercise consisted of raising and lowering (1-s contraction, 1-s relaxation) a cylinder whose volume increased at a rate of 200 ml/min. The protocol was performed during control (Con) and after 45 min of CAI with Acz (10 mg/kg body wt iv). T(pH(i)) and T(P(i)/PCr), determined using (31)P-labeled magnetic resonance spectroscopy (MRS), were similar in Acz (722 +/- 50 and 796 +/- 75 mW, respectively) and Con (855 +/- 211 and 835 +/- 235 mW, respectively). The pH(i) was similar at end-exercise (6.38 +/- 0.10 Acz and 6.43 +/- 0.22 Con), but pH(i) recovery was slowed in Acz. In a separate experiment, blood was sampled from a deep arm vein at the elbow for determination of plasma lactate concentration ([La(-)](pl)) and T(La(-)). [La(-)](pl) was lower (P < 0.05) in Acz than Con (3.7 +/- 1.7 vs. 5.0 +/- 1.7 mmol/l) at end-exercise and in early recovery, but T(La(-)) was higher (1,433 +/- 243 vs. 1,041 +/- 414 mW, respectively). These data suggest that the lower [La(-)](pl) seen with CAI was not due to a delayed onset or rate of muscle La(-) accumulation but may be related to impaired La(-) removal from muscle.     

In vivo changes in plasma acute phase protein levels in the rat induced by slow release of IL-1, IL-6 and TNF

Administration of large doses of cytokines by injection is required to induce changes in acute phase protein levels. Comparisons were made in the rat of the effects of administering recombinant human cytokines by injection with continuous release from implanted osmotic minipumps. Continuous release of interleukin-1beta (0.2-2.1 ng h(-1)) induced dose-related changes in the plasma levels of albumin, seromucoid proteins, haptoglobin and caeruloplasmin; interleukin-1alpha had similar effects but required higher doses (2-21 ng h(-1)). Tumour necrosis factor alpha (50 ng h(-1)) only significantly increased seromucoid levels, whereas IL-6 (3-30 ng h(-1)) induced haptoglobin and caeruloplassynthesis. This method provides a better technique for studying the in rive effects of cytokines which may be relevant to the release mechanisms in inflammation.     

In vivo evidence that the rise in plasma IL 6 following injection of a fever-inducing dose of LPS is mediated by IL 1 beta

Although it has often been speculated that Interleukin (IL) 1 alpha and IL 1 beta are circulating endogenous pyrogens (EP), there are few data demonstrating an elevation of these cytokines in the plasma of febrile animals. We hypothesized that IL 1 is released locally and may act to stimulate the release of another pyrogen, IL 6, which circulates to the brain to cause fever. The major purpose of the present study was to determine whether pretreatment of rats with antiserum to IL 1 beta, which attenuates lipopolysaccharide (LPS) induced fever, also results in an attenuation of the rise in plasma and cerebrospinal fluid (CSF) concentrations of IL 6. Our results show that injection of IL 1 beta produced dose-dependent rises in temperature and increases in plasma and CSF IL 6 activity, and that pretreatment of rats i.v. with antiserum to IL 1 beta produced a 55% decrease in the fever caused by LPS injection, a 68% decrease in plasma IL 6, and a 67% decrease in CSF IL 6. These data confirm the findings of previous studies that IL 1 beta is required for a portion of LPS-induced fever and also provide the first in vivo demonstration that the rise of IL 6 in rats injected with a fever-inducing dose of LPS can be significantly blocked by antiserum to IL 1 beta. Overall, the data in our study can be interpreted as being consistent with the hypothesis that the pyrogenic effect of IL 1 beta is mediated mainly through the release of IL 6, but conclusive confirmation of this hypothesis must await studies with antibodies to IL 6.     

The direct effects of catecholamines on hepatic glucose production occur via alpha(1)- and beta(2)-receptors in the dog

The role of alpha- and beta-adrenergic receptor subtypes in mediating the actions of catecholamines on hepatic glucose production (HGP) was determined in sixteen 18-h-fasted conscious dogs maintained on a pancreatic clamp with basal insulin and glucagon. The experiment consisted of a 100-min equilibration, a 40-min basal, and two 90-min test periods in groups 1 and 2, plus a 60-min third test period in groups 3 and 4. In group 1 [alpha-blockade with norepinephrine (alpha-blo+NE)], phentolamine (2 microg x kg(-1) x min(-1)) was infused portally during both test periods, and NE (50 ng x kg(-1) x min(-1)) was infused portally at the start of test period 2. In group 2, beta-blockade with epinephrine (beta-blo+EPI), propranolol (1 microg x kg(-1) x min(-1)) was infused portally during both test periods, and EPI (8 ng x kg(-1) x min(-1)) was infused portally during test period 2. In group 3 (alpha(1)-blo+NE), prazosin (4 microg x kg(-1) x min(-1)) was infused portally during all test periods, and NE (50 and 100 ng x kg(-1) x min(-1)) was infused portally during test periods 2 and 3, respectively. In group 4 (beta(2)-blo+EPI), butoxamine (40 microg x kg(-1) x min(-1)) was infused portally during all test periods, and EPI (8 and 40 ng x kg(-1) x min(-1)) was infused portally during test periods 2 and 3, respectively. In the presence of alpha- or alpha(1)-adrenergic blockade, a selective rise in hepatic sinusoidal NE failed to increase net hepatic glucose output (NHGO). In a previous study, the same rate of portal NE infusion had increased NHGO by 1.6 +/- 0.3 mg x kg(-1) x min(-1). In the presence of beta- or beta(2)-adrenergic blockade, the selective rise in hepatic sinusoidal EPI caused by EPI infusion at 8 ng x kg(-1) x min(-1) also failed to increase NHGO. In a previous study, the same rate of EPI infusion had increased NHGO by 1.6 +/- 0.4 mg x kg(-1) x min(-1). In conclusion, in the conscious dog, the direct effects of NE and EPI on HGP are predominantly mediated through alpha(1)- and beta(2)-adrenergic receptors, respectively.     

Pyrogenic Responses to Staphylococcal Enterotoxins A and B in Cats

Pyrogenic responses, ranging up to 4.8 F, were induced in cats by oral administration of highly purified staphylococcal enterotoxin B in doses from 10 to 100 mug/kg. Fever was a more sensitive indicator of intoxication than was emesis. Highly purified preparations of enterotoxin A, whether administered intravenously (0.01 to 1.0 mug/kg), orally (10 to 25 mug/kg), or into the cerebral ventricles (0.005 to 0.020 mug in 0.20 ml), were also pyrogenic in cats. Tolerance to the pyrogenic activity was produced by repeated intravenous injection of a given dose of enterotoxin A but not by repeated intracerebroventricular injection. Enterotoxin A was more potent than enterotoxin B after intravenous injection in causing both fever and emesis. Cross-tolerance could not be demonstrated between enterotoxin A and enterotoxin B or Salmonella typhosa endotoxin. This lack of cross-tolerance plus the inability of large oral doses (100 to 4,700 mug/kg) of endotoxin to cause fever or emesis indicate that the reported responses were attributable to the specific toxins administered and not to contamination by other pyrogens.     

Preoptic nitric oxide attenuates endotoxic fever in guinea pigs by inhibiting the POA release of norepinephrine

Lipopolysaccharide (LPS) administration induces hypothalamic nitric oxide (NO); NO is antipyretic in the preoptic area (POA), but its mechanism of action is uncertain. LPS also stimulates the release of preoptic norepinephrine (NE), which mediates fever onset. Because NE upregulates NO synthases and NO induces cyclooxygenase (COX)-2-dependent PGE(2), we investigated whether NO mediates the production of this central fever mediator. Conscious guinea pigs with intra-POA microdialysis probes received LPS intravenously (2 mug/kg) and, thereafter, an NO donor (SIN-1) or scavenger (carboxy-PTIO) intra-POA (20 mug/mul each, 2 mul/min, 6 h). Core temperature (T(c)) was monitored constantly; dialysate NE and PGE(2) were analyzed in 30-min collections. To verify the reported involvement of alpha(2)-adrenoceptors (AR) in PGE(2) production, clonidine (alpha(2)-AR agonist, 2 mug/mul) was microdialyzed with and without SIN-1 or carboxy-PTIO. To assess the possible involvement of oxidative NE and/or NO products in the demonstrated initially COX-2-independent POA PGE(2) increase, (+)-catechin (an antioxidant, 3 mug/mul) was microdialyzed, and POA PGE(2), and T(c) were determined. SIN-1 and carboxy-PTIO reduced and enhanced, respectively, the rises in NE, PGE(2), and T(c) produced by intravenous LPS. Similarly, they prevented and increased, respectively, the delayed elevations of PGE(2) and T(c) induced by intra-POA clonidine. (+)-Catechin prevented the LPS-induced elevation of PGE(2), but not of T(c). We conclude that the antipyretic activity of NO derives from its inhibitory modulation of the LPS-induced release of POA NE. These data also implicate free radicals in POA PGE(2) production and raise questions about its role as a central LPS fever mediator.     

Circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response produced by peripheral E. coli challenge.

Peripheral administration of a variety of inflammatory stimuli, such as endotoxin or cytokines, induces an orchestrated set of brain-mediated events referred to as the sickness response. The mechanism for how immune products signal the brain is not clear, but accumulating evidence supports the existence of neural as well as blood-borne pathways. Although endotoxin or cytokine administration results in sickness responses, few data exist regarding the role of circulating endotoxin or cytokines in the induction of sickness during a real bacterial infection. Thus the present studies examined whether subcutaneously administered Escherichia coli can activate sickness responses and whether circulating endotoxin and/or proinflammatory cytokines are a prerequisite for these responses. Male Sprague-Dawley rats were injected subcutaneously with one of three doses (2.5 x 10(7), 2.5 x 10(8), 2.5 x 10(9) colony-forming units) of replicating E. coli, a ubiquitous bacterial strain, or vehicle. Core body temperature (Tc) and activity were measured for 3 days after the injection. A second set of groups of animals were killed 3, 6, 12, 18, 24, and 48 h after the injection, and blood samples and brains were collected. Injections dose dependently and consistently increased Tc and decreased activity, with increases in Tc beginning 4 h after the injection. In addition, E. coli significantly increased serum interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha and brain IL-1beta levels beginning at the 6-h time point. Corticosterone and endotoxin were first elevated in the circulation at 3 and 18 h after the injection, respectively. Because fever onset preceded brain cytokine induction, we also examined cytokine levels in the serum, brain, and inflammation site 2 and 4 h after injection. Cytokines were elevated at the inflammation site but were not detectable in the serum or brain at 2 and 4 h. We conclude that subcutaneous injection of replicating E. coli induces a consistent and naturalistic infection that includes features of the sickness response as well as increases in circulating, brain, and inflammation site tissue cytokines. In addition, injection of replicating E. coli produces a robust fever and corticosterone response at a time when there are no detectable increases in circulating cytokines or endotoxin. These results suggest that elevated levels of circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response. Therefore, fever, activity reduction, and corticosterone elevation induced by E. coli infection may have been evoked by a neural, rather than a humoral, pathway from the periphery to the brain.     

The differential effects of galanin-(1-30) and -(3-30) on anterior pituitary hormone secretion in vivo in humans

Intravenous injection of galanin increases plasma growth hormone (GH) and prolactin (PRL) concentrations. In the rat, the effects of galanin on GH appear to be mediated via the hypothalamic galanin receptor GAL-R(1), at which galanin-(3-29) is inactive. In contrast, the effect of galanin on PRL is mediated via the pituitary-specific galanin receptor GAL-R(W), at which galanin-(3-29) is fully active. We investigated the effects of an intravenous infusion of human galanin (hGAL)-(1-30) and -(3-30) on anterior pituitary hormone levels in healthy females. Subjects were infused with saline, hGAL-(1-30) (80 pmol. kg(-1). min(-1)), and hGAL-(3-30) (600 pmol. kg(-1). min(-1)) and with boluses of gonadotropin-releasing hormone, thyrotropin-releasing hormone, and growth hormone-releasing hormone (GHRH). Both hGAL-(1-30) and -(3-30) potentiated the rise in GHRH-stimulated GH levels [area under the curve (AUC), saline, 2,810 +/- 500 vs. hGAL-(1-30), 4,660 +/- 737, P < 0.01; vs. hGAL-(3-30), 6, 870 +/- 1,550 ng. min. ml(-1), P < 0.01]. In contrast to hGAL-(1-30), hGAL-(3-30) had no effect on basal GH levels (AUC, saline, -110 +/- 88 vs. hGAL 1-30, 960 +/- 280, P < 0.002; vs. hGAL-(3-30), 110 +/- 54 ng. min. ml(-1), P = not significant). These data suggest that the effects of galanin on basal and stimulated GH release are mediated via different receptor subtypes and that the human equivalent of GAL-R(W) may exist.     

Brown fat UCP1 is not involved in the febrile and thermogenic responses to IL-1beta in mice

The activity of brown adipose tissue (BAT), a site of nonshivering metabolic thermogenesis, has been reported to increase after interleukin (IL)-1beta/lipopolysaccharide injection. To clarify the possible contribution of BAT thermogenesis to whole body febrile response, we investigated febrile and thermogenic response to IL-1beta using mice deficient in uncoupling protein-1 (UCP1), a key molecule for BAT thermogenesis. In wild-type (WT) mice, IL-1beta injection (5 microg/kg ip) increased body temperature (+1.82 degrees C at 20 min), decreased physical activity (-37% at 1 h), and produced a slight and insignificant rise (+15% at 1 h) in oxygen consumption (Vo(2)). Vo(2) dependent on metabolic thermogenesis (DeltaVO2 thermogenesis) calculated by correcting the effect of physical activity was increased after IL-1beta injection (726 +/- 200 ml x h(-1) x kg(-1) at 1 h). Almost the same responses were observed in UCP1-deficient mice, showing 638 +/- 87 ml x h(-1) x kg(-1) of DeltaVO2 thermogenesis at 1 h. In contrast, CL316,243, a selective activator of BAT thermogenesis, increased body temperature, decreased physical activity, and produced a significant rise in Vo2 in WT mice, showing 1,229 +/- 35 ml x h(-1) x kg(-1) of DeltaVO2 thermogenesis at 1 h. These changes were not observed in UCP1-deficient mice. These results, conflicting with a previously proposed idea of a role of BAT in fever, suggest a minor contribution of BAT thermogenesis to IL-1beta-induced fever. In support of this, we found no effect of IL-1beta on triglyceride content and UCP1 mRNA level in BAT, in contrast with apparent effects of CL316,243.     

Enteric neural pathways mediate the anti-inflammatory actions of glucagon-like peptide 2

Glucagon-like peptide-2 (GLP-2) is an important regulator of nutritional absorptive capacity with anti-inflammatory actions. We hypothesized that GLP-2 reduces intestinal mucosal inflammation by activation of vasoactive intestinal polypeptide (VIP) neurons of the submucosal plexus. Ileitis or colitis was induced in rats by injection of trinitrobenzene sulfonic acid (TNBS), or colitis was induced by administration of dextran sodium sulfate (DSS) in drinking water. Subsets of animals received (1-33)-GLP-2 (50 mug/kg sc bid) either immediately or 2 days after the establishment of inflammation and were followed for 3-5 days. The involvement of VIP neurons was assessed by concomitant administration of GLP-2 and the VIP antagonist [Lys(1)-Pro(2,5)-Arg(3,4)-Tyr(6)]VIP and by immunohistochemical labeling of GLP-2-activated neurons. In all models, GLP-2 treatment, whether given immediately or delayed until inflammation was established, resulted in significant improvements in animal weights, mucosal inflammation indices (myeloperoxidase levels, histological mucosal scores), and reduced levels of inflammatory cytokines (IFN-gamma, TNF-alpha, IL-1beta) and inducible nitric oxide synthase, with increased levels of IL-10 in TNBS ileitis and DSS colitis. Reduced rates of crypt cell proliferation and of apoptosis within crypts in inflamed tissues were also noted with GLP-2 treatment. These effects were abolished with coadministration of GLP-2 and the VIP antagonist. GLP-2 was shown to activate neurons and to increase the number of cells expressing VIP in the submucosal plexus of the ileum. These findings suggest that GLP-2 acts as an anti-inflammatory agent through activation of enteric VIP neurons, independent of proliferative effects. They support further studies to examine the role of neural signaling in the regulation of intestinal inflammation.