Effects of inhibitors of the activity of cyclo-oxygenase-2 on the hypotension and multiple organ dysfunction caused by endotoxin: A comparison with dexamethasone

1. Endotoxaemia is associated with the expression of the inducible isoform of cyclo-oxygenase, cyclo-oxygenase-2 (COX-2), and an overproduction of arachidonic acid (AA) metabolites. The role of the AA metabolites generated by COX-2 in the circulatory failure and multiple organ dysfunction caused by endotoxin is unclear. Dexamethasone prevents the expression of COX-2 and exerts beneficial effects in animal models of shock.
2. Here we compare the effects of two inhibitors of COX-2 activity, namely NS-398 (5 mg kg⁻¹, i.p., n=7) and SC-58635 (3 mg kg⁻¹, i.p., n=9) with those of dexamethasone (3 mg kg⁻¹, i.p., n=9) on the circulatory failure and organ dysfunction caused by lipopolysaccharide (LPS, E. coli, 6 mg kg⁻¹, i.v., n=11) in the rat.
3. Endotoxaemia for 6 h caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of 6-keto-PGF_1α (indicator of the induction of COX-2) and nitrite/nitrate (indicator of the induction of iNOS).
4. Pretreatment of rats with dexamethasone attenuated the hypotension, the renal dysfunction, the hepatocellular and pancreatic injury and the induction of COX-2 and iNOS caused by LPS. In contrast, inhibition of COX-2 activity with SC-58635 or NS-398 neither attenuated the circulatory failure nor the multiple organ failure caused by endotoxin.
5. Thus, the prevention of the circulatory failure and the multiple organ injury/dysfunction caused by dexamethasone in the rat is not due to inhibition of the activity of COX-2. Our results suggest that an enhanced formation of eicosanoids by COX-2 does not contribute to the development of organ injury and/or dysfunction in rats with endotoxaemia.

     

Effects of inhibitors of the activity of poly (ADP-ribose) synthetase on the liver injury caused by ischaemia-reperfusion: a comparison with radical scavengers

1. Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS) and peroxynitrite. Excessive activation of PARS may contribute to the hepatocyte injury caused by ROS in vitro and inhibitors of PARS activity reduce the degree of reperfusion injury of the heart, skeletal muscle and brain in vivo. Here we compared the effects of various inhibitors of the activity of PARS with those of deferoxamine (an iron chelator which prevents the generation of hydroxyl radicals) and tiron (an intracellular scavenger of superoxide anion) on the degree of hepatic injury caused by ischaemia and reperfusion of the liver in the anaesthetized rat or rabbit.
2. In the rat, ischaemia (30 or 60 min) and reperfusion (120 min) of the liver resulted in significant increases in the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicating the development of liver injury. Intravenous administration of the PARS inhibitors 3-aminobenzamide (3-AB, 10 mg kg⁻¹ or 30 mg kg⁻¹), 1,5-dihydroxyisoquinoline (ISO, 1 mg kg⁻¹) or 4-amino-1,8-naphthalimide (4-AN, 3 mg kg⁻¹) before reperfusion did not reduce the degree of liver injury caused by ischaemia-reperfusion.
3. In contrast to the PARS inhibitors, deferoxamine (40 mg kg⁻¹) or tiron (300 mg kg⁻¹) significantly attenuated the rise in the serum levels of AST and ALT caused by ischaemia-reperfusion of the liver of the rat.
4. In the rabbit, the degree of liver injury caused by ischaemia (60 min) and reperfusion (120 min) was also not affected by 3-AB (10 mg kg⁻¹) or ISO (1 mg kg⁻¹).
5. These results support the view that the generation of oxygen-derived free radicals mediates the liver injury associated with reperfusion of the ischaemic liver by mechanism(s) which are independent of the activation of PARS.

     

Effects of tyrphostins and genistein on the circulatory failure and organ dysfunction caused by endotoxin in the rat: a possible role for protein tyrosine kinase

1. Here we compared the effects of various inhibitors of the activity of protein tyrosine kinase on (i) the expression of the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) caused by endotoxin (lipopolysaccharide, LPS) in cultured macrophages, (ii) the induction of iNOS and cyclo-oxygenase 2 (COX-2) protein and activity in rats with endotoxaemia, and (iii) the circulatory failure and organ dysfunction caused by LPS in the anaesthetized rat.
2. Activation of murine cultured macrophages with LPS (1 μg ml⁻¹) resulted, within 24 h, in a significant increase in nitrite (an indicator of the formation of NO) in the cell supernatant. This increase in nitrite was attenuated by the tyrphostins AG126, AG556, AG490 or AG1641 or by genistein in a dose-dependent fashion (IC₅₀: ∼15 μM). In contrast, tyrphostin A1 (an analogue of tyrphostin AG126) or daidzein (an analogue of genistein) had no effect on the rise in nitrite caused by LPS.
3. Administration of LPS (E. coli, 10 mg kg⁻¹, i.v.) caused hypotension and a reduction of the pressor responses elicited by noradrenaline (NA, 1 μg kg⁻¹, i.v.). Pretreatment of rats with the tyrphostins AG126, AG490, AG556, AG1641 or A1 attenuated the circulatory failure caused by LPS. Although genistein attenuated the vascular hyporeactivity to NA, it did not affect the hypotension caused by LPS. Daidzein did not affect the circulatory failure caused by LPS.
4. Endotoxaemia for 360 min resulted in rises in the serum levels of (i) urea and creatinine (indicators of renal failure), (ii) alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin and γ-glutamyl transferase (γGT) (indicators of liver injury/dysfunction), lipase (an indicator of pancreatic injury) as well as lactate (an indicator of tissue hypoxia). None of the tyrosine kinase inhibitors tested had a significant effect on the rise in the serum levels of urea, but the tyrphostins AG126, AG556 or A1 significantly attenuated the rises in the serum levels of creatinine caused by LPS. In addition, all tyrphostins and genistein attenuated the liver injury/failure, the pancreatic injury, the hypoglycaemia and the lactic acidosis caused by LPS. In contrast, daidzein did not reduce the organ injury/dysfunction or the lactic acidosis caused by LPS.
5. Injection of LPS resulted (within 90 min) in a substantial increase in the serum level of tumour necrosis factor α (TNFα), which was attenuated by pretreatment of LPS-rats with any of the tyrphostins used. Genistein, but not daidzein, also reduced the rise in the serum levels of TNFα caused by LPS. Endotoxaemia for 6 h also resulted in a substantial increase in the expression of iNOS and COX-2 protein and activity in the lung, which was attenuated by pretreatment of LPS-rats with the tyrphostins AG126, AG556 or genistein, but not by daidzein.
6. Thus, tyrphostins (AG126, AG490, AG556, AG1641 or A1) and genistein, but not daidzein (inactive analogue of genistein), prevent the (i) circulatory failure, (ii) the multiple organ dysfunction (liver and pancreatic dysfunction/injury, lactacidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX-2 protein and activity in rats with endotoxic shock

     

Effects of a hydroxylated metabolite of the β-adrenoreceptor antagonist,carvedilol, on post-ischaemic splanchnic tissue injury

1. Reactive oxygen species have been demonstrated to play a critical role in post-ischaemic tissue injury. The present experiment was designed to evaluate the effects of SB 211475, a hydroxylated metabolite of the new β-adrenoceptor antagonist, carvedilol, on rat splanchnic ischaemia (SI, 60 min) and reperfusion(R)-induced shock and tissue injury.
2. Administration of SB 211475 two min before R attenuated SI/R injury in a dose-dependent manner. At doses of 0.5 mg kg⁻¹ and 1.0 mg kg⁻¹, SB 211475 exerted significant anti-shock and endothelial protective effects, characterized by prolonged survival times, increased survival rates, attenuated increases in tissue myeloperoxidase activity and haematocrits, and preserved endothelium-dependent vasorelaxation.
3. Administration of 1 mg kg⁻¹ carvedilol attenuated shock-induced tissue injury and endothelial dysfunction. However, administration of 0.5 mg kg⁻¹ carvedilol had no protective effects on post-ischaemic tissue injury.
4. Previous studies have shown that SB 211475 has virtually no β-blocking activity but possesses more potent antioxidant activity than carvedilol. In the present study, SB 211475 exerted more potent protective effects than the parent compound, suggesting that this metabolite of carvedilol is superior to carvedilol with regard to its protection against post-ischaemia tissue injury.

     

Intrarenal haemodynamics and renal dysfunction in endotoxaemia: effects of nitric oxide synthase inhibition

1. This study investigated the effects of low dose endotoxin (lipopolysaccharide, LPS) on (i) systemic haemodynamics, (ii) renal blood flow (RBF), (iii) renal cortical and medullary perfusion and (iv) renal function in the anaesthetized rat. We have also investigated the effects of nitric oxide (NO) synthase (NOS) inhibition with N^G-methyl-L-arginine (L-NMMA) on the alterations in systemic and renal haemodynamics and renal function caused by endotoxin.
2. Infusion of low dose LPS (1 mg kg⁻¹ over 30 min, n=6) caused a late fall in mean arterial blood pressure (MAP, at 5 and 6 h after LPS), but did not cause an early (at 1–4 h after LPS) hypotension. The pressor effect of noradrenaline (NA, 1 μg kg⁻¹, i.v.) was significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Infusion of L-NMMA (50 μg kg⁻¹ min⁻¹ commencing 60 min before LPS and continued throughout the experiment, n=7) abolished the delayed hypotension and significantly attenuated the vascular hyporeactivity to NA (at 2–6 h).
3. Infusion of LPS (1 mg kg⁻¹ over 30 min, n=6) caused a rapid (within 2 h) decline in renal function (measured by inulin clearance) in the absence of a significant fall in MAP or renal blood flow (RBF). L-NMMA (n=7) attenuated the impairment in renal function caused by LPS so that the inulin clearance in LPS-rats treated with L-NMMA was significantly greater than in LPS-rats treated with vehicle (control) at 3–6 h after infusion of LPS.
4. Endotoxaemia also caused a significant reduction in renal cortical, but not medullary perfusion (measured as Laser Doppler flux). Infusion of L-NMMA caused a significant further fall in cortical perfusion and a significant fall in medullary perfusion in the absence of changes in RBF.
5. Infusion of LPS resulted in a progressive increase in the plasma levels of nitrite/nitrate (an indicator of the formation of NO), so that the plasma concentration of nitrite/nitrate was significantly higher than baseline at 150 to 330 min after LPS. Infusion of L-NMMA attenuated the rise in the plasma concentration of nitrite/nitrate (at 270 and 330 min, P<0.05) caused by LPS.
6. Thus, the renal dysfunction caused by injection of low dose of endotoxin in the rat occurs in the absence of significant falls in blood pressure or total renal blood flow. Inhibition of NOS activity with L-NMMA attenuates the renal dysfunction caused by endotoxin (without improving intrarenal haemodynamics), suggesting that an overproduction of NO may contribute to the development of renal injury and dysfunction by causing direct cytotoxic effects.

     

Effect of L-lysine on nitric oxide overproduction in endotoxic shock

1. An enhanced production of nitric oxide (NO) from L-arginine, related to the diffuse expression of an inducible NO synthase (iNOS), contributes to the pathogenesis of endotoxic shock. Since iNOS activity depends on extracellular L-arginine, we hypothesized that limiting cellular L-arginine uptake would reduce NO production in endotoxic shock. We investigated the effects of L-lysine, an inhibitor of L-arginine uptake through system y⁺, on NO production, multiple organ dysfunction and lactate levels, in normal and endotoxaemic rats.
2. Anaesthetized rats challenged with intravenous lipopolysaccharide (LPS, 10 mg kg⁻¹) received a 5 h infusion of either L-lysine (500 μmol kg⁻¹ h⁻¹, n=12) or isotonic saline (2 ml kg⁻¹ h⁻¹, n=11). In rats treated with saline, LPS produced a large increase in plasma nitrate and L-citrulline concentrations at 5 h, both markers of enhanced NO production. LPS also caused severe hypotension, low cardiac output and marked hyperlactataemia. All these changes were significantly reduced byL-lysine administration.
3. Endotoxaemia also caused a significant rise in the plasma levels of alanine aminotransferase (ALAT), lipase, urea and creatinine, and hence, liver, pancreatic and renal dysfunction. These changes tended to be less pronounced in rats treated with L-lysine, although the differences did not reach statistical significance.
4. Similar experiments were conducted in 10 rats challenged with LPS vehicle in place of LPS and then treated with L-lysine (500 μmol kg⁻¹  h⁻¹, n=5) or saline (2 ml kg⁻¹  h⁻¹, n=5) for 5 h. In these animals, all the haemodynamic and metabolic variables remained stable and not statistically different between both treatment groups, except for a slight rise in ALAT, which was comparable in L-lysine and saline-treated rats.
5. In conclusion, L-lysine, an inhibitor of cellular L-arginine uptake, reduces NO production and exerts beneficial haemodynamic effects in endotoxaemic rats. L-lysine also reduces hyperlactataemia and tends to blunt the development of organ injury in these animals. Contrastingly, L-lysine has no effects in the absence of endotoxin and thus appears to act as a selective modulator of iNOS activity.

     

Investigation of the mechanisms underlying the hypophagic effects of the 5-HT and noradrenaline reuptake inhibitor,sibutramine, in the rat

1. Sibutramine is a novel 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin- noradrenaline reuptake inhibitor, SNRI) which is currently being developed as a treatment for obesity. Sibutramine has been shown to decrease food intake in the rat. In this study we have used a variety of monoamine receptor antagonists to examine the pharmacological mechanisms underlying sibutramine-induced hypophagia.
2. Individually-housed male Sprague-Dawley rats were maintained on reversed phase lighting with free access to food and water. Drugs were administered at 09 h 00 min and food intake was monitored over the following 8 h dark period.
3. Sibutramine (10 mg kg⁻¹, p.o.) produced a significant decrease in food intake during the 8 h following drug administration. This hypophagic response was fully antagonized by the α₁-adrenoceptor antagonist, prazosin (0.3 and 1 mg kg⁻¹, i.p.), and partially antagonized by the β₁-adrenoceptor antagonist, metoprolol (3 and 10 mg kg⁻¹, i.p.) and the 5-HT receptor antagonists, metergoline (non-selective; 0.3 mg kg⁻¹, i.p.); ritanserin (5-HT_2A/2C; 0.1 and 0.5 mg kg⁻¹, i.p.) and SB200646 (5-HT_2B/2C; 20 and 40 mg kg⁻¹, p.o.).
4. By contrast, the α₂-adrenoceptor antagonist, RX821002 (0.3 and 1 mg kg⁻¹, i.p.) and the β₂-adrenoceptor antagonist, ICI 118,551 (3 and 10 mg kg⁻¹, i.p.) did not reduce the decrease in food intake induced by sibutramine.
5. These results demonstrate that β₁-adrenoceptors, 5-HT_2A/2C-receptors and particularly α₁-adrenoceptors, are involved in the effects of sibutramine on food intake and are consistent with the hypothesis that sibutramine-induced hypophagia is related to its ability to inhibit the reuptake of both noradrenaline and 5-HT, with the subsequent activation of a variety of noradrenaline and 5-HT receptor systems.

     

Gabapentin (neurontin) and S-(+)-3-isobutylgaba represent a novel class of selective antihyperalgesic agents

1. Gabapentin (neurontin) is a novel antiepileptic agent that binds to the α₂δ subunit of voltage-dependent calcium channels. The only other compound known to possess affinity for this recognition site is the (S)-(+)-enantiomer of 3-isobutylgaba. However, the corresponding (R)-(−)-enantiomer is 10 fold weaker. The present study evaluates the activity of gabapentin and the two enantiomers of 3-isobutylgaba in formalin and carrageenan-induced inflammatory pain models.
2. In the rat formalin test, S-(+)-3-isobutylgaba (1–100 mg kg⁻¹) and gabapentin (10–300 mg kg⁻¹) dose-dependently inhibited the late phase of the nociceptive response with respective minimum effective doses (MED) of 10 and 30 mg kg⁻¹, s.c. This antihyperalgesic action of gabapentin was insensitive to naloxone (0.1–10.0 mg kg⁻¹, s.c.). In contrast, the R-(−)-enantiomer of 3-isobutylgaba (1–100 mg kg⁻¹) produced a modest inhibition of the late phase at the highest dose of 100 mg kg⁻¹. However, none of the compounds showed any effect during the early phase of the response.
3. The s.c. administration of either S-(+)-3-isobutylgaba (1–30 mg kg⁻¹) or gabapentin (10–100 mg kg⁻¹), after the development of peak carrageenan-induced thermal hyperalgesia, dose-dependently antagonized the maintenance of this response with MED of 3 and 30 mg kg⁻¹, respectively. Similar administration of the two compounds also blocked maintenance of carrageenan-induced mechanical hyperalgesia with MED of 3 and 10 mg kg⁻¹, respectively. In contrast, R-(−)-3-isobutylgaba failed to show any effect in the two hyperalgesia models.
4. The intrathecal administration of gabapentin dose-dependently (1–100 μg/animal) blocked carrageenan-induced mechanical hyperalgesia. In contrast, administration of similar doses of gabapentin into the inflamed paw was ineffective at blocking this response.
5. Unlike morphine, the repeated administration of gabapentin (100 mg kg⁻¹ at start and culminating to 400 mg kg⁻¹) over 6 days did not lead to the induction of tolerance to its antihyperalgesic action in the formalin test. Furthermore, the morphine tolerance did not cross generalize to gabapentin. The s.c. administration of gabapentin (10–300 mg kg⁻¹), R-(−) (3–100 mg kg⁻¹) or S-(+)-3-isobutylgaba (3–100 mg kg⁻¹) failed to inhibit gastrointestinal motility, as measured by the charcoal meal test in the rat. Moreover, the three compounds (1–100 mg kg⁻¹, s.c.) did not generalize to the morphine discriminative stimulus. Gabapentin (30–300 mg kg⁻¹) and S-(+)-isobutylgaba (1–100 mg kg⁻¹) showed sedative/ataxic properties only at the highest dose tested in the rota-rod apparatus.
6. Gabapentin (30–300 mg kg⁻¹, s.c.) failed to show an antinociceptive action in transient pain models. It is concluded that gabapentin represents a novel class of antihyperalgesic agents.

     

Synergistic effects of caspase inhibitors and MK-801 in brain injury after transient focal cerebral ischaemia in mice

1. Excitotoxic and apoptotic mechanisms have been implicated in the pathophysiology of cerebral ischaemia. Both MK-801, an NMDA receptor antagonist, or peptide inhibitors of the caspase family (z-VAD.FMK and z-DEVD.FMK), protect mouse brain from ischaemic cell damage. In this study, we examined whether these drugs which act via distinct mechanisms, afford even greater neuroprotection when given in combination following 2 h MCA occlusion (filament model) and 18 h reperfusion.
2. Given alone as pretreatment, MK-801 (1, 3 and 5 mg kg⁻¹, but not 0.3 mg kg⁻¹, i.p.) decreased infarct size by 34–75%. When injected 1 h after occlusion and before reperfusion, 3 mg kg⁻¹ reduced injury but not when administered 1 h after reperfusion.
3. Pretreatment with a subthreshold dose of MK-801 (0.3 mg kg⁻¹) plus a subthreshold dose of z-VAD.FMK (27 ng) or z-DEVD (80 ng) significantly decreased infarct size by 29 and 30%, respectively, and enhanced neurological function.
4. Administering a subthreshold dose of z-VAD.FMK (27 ng) or z-DEVD.FMK (80 ng) as pretreatment extended the time window for MK-801 (3 mg kg⁻¹) by 2 h from 1 h before reperfusion to at least 1 h after reperfusion.
5. Pretreating with a subthreshold dose of MK-801 (0.3 mg kg⁻¹) extended the time window for z-DEVD.FMK (480 ng) from 1 h after reperfusion to at least 3 h after reperfusion.
6. We conclude that caspase inhibitors which putatively block apoptotic cell death and inhibit cytokine production and the NMDA antagonist MK-801 act synergistically and prolong their respective therapeutic windows in cerebral ischaemia.

     

Evaluation of BTS 67 582, a novel antidiabetic agent,in normal and diabetic rats

1. The effect of BTS 67 582, a novel antidiabetic agent, has been evaluated on plasma glucose and plasma insulin in normal and streptozotocin-induced diabetic rats.
2. BTS 67 582 (3 to 300 mg kg⁻¹, p.o.) caused a dose- and time- dependent reduction in plasma glucose and an increase in plasma insulin in both fasted and glucose-loaded normal rats. The ED₅₀ for the glucose lowering effect of BTS 67 582 in fasted rats was 37.6, 18.4 and 18.5 mg kg⁻¹ at 1, 2 and 4 h after administration respectively.
3. In streptozotocin-induced (50 mg kg⁻¹, i.v.) diabetic rats, BTS 67 582 (37–147 mg kg⁻¹, p.o.) caused significant reductions of plasma glucose following a glucose load, whereas glibenclamide (100 mg kg⁻¹, p.o.) was ineffective. BTS 67 582 significantly increased plasma insulin compared to controls whereas glibenclamide did not.
4. BTS 67 582 did not displace [³H]-glibenclamide from its binding sites in rat brain, guinea-pig ventricle or the HIT-T15 insulinoma β-cell line. BTS 67 582 does not therefore appear to modulate its action via an effect on the ‘sulphonylurea'' receptor.
5. In fasted rats, the glucose lowering effect of BTS 67 582 (100 mg kg⁻¹ p.o.) and glibenclamide (1 mg kg⁻¹, p.o.) were antagonized by diazoxide (30 mg kg⁻¹, i.p.). In addition BTS 67 582, like glibenclamide, caused a dose-dependent rightward shift of cromakalim-induced relaxation of noradrenaline precontracted rat aortic strips, suggesting the involvement of K_ATP channels.
6. In summary, BTS 67 582 produces a blood glucose-lowering effect in normal and streptozotocin-induced diabetic rats associated with increased insulin concentrations. This effect appears to be due to a blockade of ATP-sensitive potassium channel activity via a different binding site to that of glibenclamide.

     

Comparison of the effects of sibutramine and other monoamine reuptake inhibitors on food intake in the rat

1. The effects of the potent 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin-noradrenaline reuptake inhibitor, SNRI), sibutramine, on the cumulative food intake of freely-feeding male Sprague-Dawley rats during an 8 h dark period were investigated and compared to those of the selective 5-HT reuptake inhibitor (selective serotonin reuptake inhibitor, SSRI), fluoxetine; the selective noradrenaline reuptake inhibitor, nisoxetine; the 5-HT and noradrenaline reuptake inhibitors, venlafaxine and duloxetine; and the 5-HT releaser and 5-HT reuptake inhibitor, (+)-fenfluramine.
2. Sibutramine (3 and 10 mg kg⁻¹, p.o.) and (+)-fenfluramine (1 and 3 mg kg⁻¹, p.o.) produced a significant, dose-dependent decrease in food intake over the 8 h dark period. These responses became apparent within the first 2 h following drug administration.
3. Fluoxetine (3, 10 and 30 mg kg⁻¹, p.o.), and nisoxetine (3, 10 and 30 mg kg⁻¹, p.o.) had no significant effect on food intake during the 8 h dark period. However, a combination of fluoxetine and nisoxetine (30 mg kg⁻¹, p.o., of each) significantly decreased food intake 2 and 8 h after drug administration.
4. Venlafaxine (100 and 300 mg kg⁻¹, p.o.) and duloxetine (30 mg kg⁻¹, p.o.) also significantly decreased food intake in the 2 and 8 h following drug administration.
5. The results of this study demonstrate that inhibition of 5-HT and noradrenaline reuptake by sibutramine, venlafaxine, duloxetine, or by a combination of fluoxetine and nisoxetine, markedly reduces food intake in freely-feeding rats and suggest that this may be a novel approach for the treatment of obesity.

     

Citalopram-induced hypophagia is enhanced by blockade of 5-HT1A receptors: role of 5-HT2C receptors

1. The selective 5-hydroxytryptamine reuptake inhibitor citalopram (10 and 20 mg kg⁻¹, i.p.) significantly reduced food intake in male rats (CD-COBS) habituated to eat their daily food during a 4-h period.
2. The 5-HT_1A receptor antagonist WAY100635 (0.3 mg kg⁻¹) administered systemically did not modify feeding but significantly potentiated the reduction in food intake caused by 10 mg kg⁻¹ i.p. citalopram. The dose of 5 mg kg⁻¹ i.p. citalopram was not active in animals pretreated with vehicle but significantly reduced feeding in animals pretreated with WAY100635.
3. WAY100635 (0.1 μg 0.5 μl⁻¹) injected into the dorsal raphe significantly potentiated the hypophagic effect of 10 mg kg⁻¹ citalopram.
4. WAY100635 (1.0 μg 0.5 μl⁻¹) injected into the median raphe did not modify feeding or the hypophagic effect of 10 mg kg⁻¹ citalopram.
5. The 5-HT_2B/2C receptor antagonist SB206553 (10 mg kg⁻¹, p.o.) slightly reduced feeding by itself but partially antagonized the effect of WAY100635 administered systemically (0.3 mg kg⁻¹, s.c.) or into the dorsal raphe (0.1 μg 0.5 μl⁻¹) in combination with 10 mg kg⁻¹ i.p. citalopram. The hypophagic effect of 10 mg kg⁻¹ i.p. citalopram alone was not significantly modified by SB206553.
6. Brain concentrations of citalopram and its metabolite desmethylcitalopram in rats pretreated with SB206553, WAY100635 and their combination were comparable to those of vehicle-pretreated rats, 90 min after citalopram injection.
7. The hypophagic effect of citalopram was potentiated by blocking 5-HT_1A receptors. Only the effect of the WAY100635/citalopram combination seemed to be partially mediated by central 5-HT_2C receptors.

     

The profile of sabcomeline (SB-202026), a functionally selective M1 receptor partial agonist,in the marmoset

1. Sabcomeline (SB-202026, 0.03 mg kg⁻¹, p.o.), a potent and functionally selective M₁ receptor partial agonist, caused a statistically significant improvement in the performance of a visual object discrimination task by marmosets. No such improvement was seen after RS86 (0.1 mg kg⁻¹, p.o.).
2. Initial learning, which only required an association of object with reward and an appropriate response to be made, was not significantly affected. Reversal learning, which required both the extinction of the previously learned response and the acquisition of a new response strategy, was significantly improved after administration of sabcomeline (0.03 mg kg⁻¹, p.o.).
3. Sabcomeline (0.03 and 0.1 mg kg⁻¹, p.o.) had no significant effect on mean blood pressure measured for 2 h after administration in the conscious marmoset.
4. Sabcomeline (0.03 mg kg⁻¹, p.o.) caused none of the overt effects such as emesis or behaviours often seen after the administration of muscarinic agonists, e.g. face rubbing and licking.
5. This is the first study to demonstrate cognitive enhancement by a functionally selective M₁ receptor partial agonist in a normal (i.e. non-cognitively impaired) non-human primate and this effect was seen at a dose which did not cause side effects.
6. Perseverative behaviour and deficient acquisition of new information are seen in patients with Alzheimer''s disease (AD). Therefore the data suggest that sabcomeline might be of therapeutic benefit in the treatment of AD.

     

Andrographolide suppresses the expression of inducible nitric oxide synthase in macrophage and restores the vasoconstriction in rat aorta treated with lipopolysaccharide

1. We investigated whether andrographolide, a diterpenoid lactone found at Andrographis paniculata, influences the induction of the inducible nitric oxide synthase (iNOS) in RAW264.7 cells activated by bacterial endotoxin (LPS), as well as in the rats with endotoxic shock and in aortic rings treated with LPS.
2. Incubation of RAW264.7 cells with andrographolide (1 to 50 μM) inhibited the LPS (1 μg ml⁻¹)-induced nitrite accumulation in concentration- and time-dependent manners. Maximum inhibition was observed when andrographolide was added together with LPS and decreased progressively as the interval between andrographolide and LPS was increased to 20 h.
3. Western blot analysis demonstrated that iNOS expression was markedly attenuated in the presence of andrographolide for 6–24 h, suggesting that andrographolide inhibited iNOS protein induction.
4. Thoracic aorta incubation with LPS (300 ng ml⁻¹) for 5 h in vitro exhibited a significant decrease in the maximal contractile response to phenylephrine (10⁻⁹–10⁻⁵ M). Andrographolide (30 μM) restored the contractile response to control level.
5. In anaesthetized rats, LPS (10 mg kg⁻¹, i.v.) caused a fall in mean arterial blood pressure (MAP) from 116±4 to 77±5 mmHg. The pressor effect of phenylephrine (10 μg ml⁻¹, i.v.) was also significantly reduced at 30, 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with andrographolide (1 mg kg⁻¹, i.v., 20 min prior to LPS) maintained a significantly higher MAP when compared to LPS-rats given with vehicle. Administration of andrographolide 60 min after LPS caused a increase in MAP and significantly reversed the reduction of the pressor response to phenylephrine.
6. Our results indicated that andrographolide inhibits nitrite synthesis by suppressing expression of iNOS protein in vitro. And, this inhibition of iNOS synthesis may contribute to the beneficial haemodynamic effects of andrographolide in endotoxic shock.

     

The role of α2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent,clozapine, in the rat

1. The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat.
2. The close structural analogues of clozapine, loxapine (0.1 mg kg⁻¹ s.c.) and iso-clozapine (1 and 3 mg kg⁻¹ s.c.) induced catalepsy in rats. In contrast, clozapine and the regio-isomer of loxapine, iso-loxapine (up to 10 mg kg⁻¹ s.c.) did not produce catalepsy, but at a dose of 1 mg kg⁻¹ significantly inhibited catalepsy induced by loxapine (0.3 mg kg⁻¹ s.c.).
3. Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D₂/5-HT_1A, D₂/5-HT_1B/1D and D₂/α₂-receptor affinity (K_D) ratios: i.e. 30–100-fold higher ratios were calculated for loxapine and iso-clozapine, whereas the ratios were less than 1 for clozapine and iso-loxapine. The ratios of affinities for D₂ to 5-HT_2A, 5-HT_2C or D₁ did not reflect the grouping of cataleptic and non-cataleptic compounds.
4. Co-treatment with the α₂-adrenoceptor antagonists, yohimbine (1–10 mg kg⁻¹ s.c.), RX 821002 (1–10 mg kg⁻¹ s.c.) and MK-912 (0.3 and 1 mg kg⁻¹ s.c.) dose-dependently inhibited the cataleptic response to loxapine (0.3 mg kg⁻¹). Yohimbine (1–10 mg kg⁻¹ s.c.) also dose-dependently inhibited the cateleptic response to haloperidol (0.3 mg kg⁻¹ s.c.). The α₂-adrenoceptor antagonists had no effect per se.
5. Neither yohimbine (10 mg kg⁻¹) nor RX821002 (3 mg kg⁻¹) altered the cataleptic response to the D₁ receptor antagonist, SCH 23390 (1 mg kg⁻¹ s.c.), while, like clozapine, both compounds abolished the response to the 5-HT_2A receptor antagonist, MDL 100,151 (3 mg kg⁻¹ s.c.).
6. The present data strongly implicate α₂-adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

     

T-type and L-type calcium channel blockers exert opposite effects on renin secretion and renin gene expression in conscious rats

1. This study aimed to investigate and to compare the effects of pharmacological T-type calcium channel and of L-type calcium channel blockade on the renin system. To this end, male healthy Sprague-Dawley rats were treated with the T-channel blocker mibefradil or with the L-channel blocker amlodipine at doses of 5 mg kg⁻¹, 15 mg kg⁻¹ and 45 mg kg⁻¹ per day for four days and their effects on plasma renin activity (PRA) and kidney renin mRNA levels were determined.
2. Whilst amlodipine lowered basal systolic blood pressure at 5 mg kg⁻¹, mibefradil had no effect on basal blood pressure in the whole dose range examined. Amlodipine dose-dependently induced up to 7 fold elevation of PRA and renin mRNA levels. Mibefradil significantly lowered PRA and renin mRNA levels at 5 mg kg⁻¹ and moderately increased both parameters at a dose of 45 mg kg⁻¹, when PRA and renin mRNA levels were increased by 100% and 30%, respectively. In primary cultures of renal juxtaglomerular cells neither amlodipine nor mibefradil (0.1–10 μM) changed renin secretion.
3. In rats unilateral renal artery clips (2K-1C) mibefradil and amlodipine at doses of 15 mg kg⁻¹ day⁻¹ were equally effective in lowering blood pressure. In contrast mibefradil (5 mg kg⁻¹ and 15 mg  kg⁻¹ day⁻¹) significantly attenuated the rise of PRA and renin mRNA levels, whilst amlodipine (15 mg kg⁻¹) additionally elevated the rise of PRA and renin mRNA levels in response to renal artery clipping.
4. These findings suggest that T-type calcium channel blockers can inhibit renin secretion and renin gene expression in vivo, whilst L-type calcium channel blockers act as stimulators of the renin system. Since the inhibitory effect of T-type antagonists is apparent in vivo but not in vitro, one may infer that the effect on the renin system is indirect rather than directly mediated at the level of renal juxtaglomerular cells.

     

Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse

1. The release of cytokines following administration of endotoxin and the contribution of nitric oxide (NO) to the subsequent haemodynamic profile were investigated in the conscious mouse.
2. Administration of endotoxin (E. Coli, 026 : B6, 12.5 mg kg⁻¹, i.v.) elevated the concentration of tumour necrosis factor-α (TNF-α) in the plasma within 0.5 h, reaching a maximum at 2 h and returning to control concentrations by 4 h. In addition, the concentration of interleukin-6 (IL-6) in the plasma was also elevated within 1 h, reaching a maximum at 3 h and remaining elevated throughout the 12 h of study.
3. Endotoxin (12.5 mg kg⁻¹, i.v.) induced the expression of a Ca²⁺-independent (inducible) NO synthase in the mouse heart and elevated the concentrations of nitrite and nitrate in the plasma within 4 h, reaching a maximum at 12 h. This was accompanied by a progressive fall in blood pressure over the same period.
4. The vasopressor effect of noradrenaline (0.5–4 μg kg⁻¹ min⁻¹, i.v.) administered as a continuous infusion was significantly attenuated 7 h after endotoxin (12.5 mg kg⁻¹, i.v).
5. The NO synthase inhibitor N^G-monomethyl-L-arginine HCl (L-NMMA; 1–10 mg kg⁻¹, i.v. bolus) reversed the fall in blood pressure when administered 7 h after endotoxin (12.5 mg kg⁻¹, i.v.).
6. In an attempt to maintain a constant blood concentration, L-NMMA was administered as a continuous infusion (10 mg kg⁻¹ h⁻¹, i.v.), beginning 4 h after a lower dose of endotoxin (6 mg kg⁻¹, i.v.). Such treatment prevented the fall in blood pressure and the elevation of nitrite and nitrate in the plasma throughout the 18 h of observation.
7. The fall in blood pressure following endotoxin (3 mg kg⁻¹, i.v.) was significantly reduced throughout the 18 h of observation in homozygous mutant mice lacking the inducible NO synthase.
8. In summary, we have developed a model of endotoxin shock in the conscious mouse in which an overproduction of NO by the inducible NO synthase is associated with the haemodynamic disturbances. This model, which exhibits many of the characteristics of septic shock in man, will enable the study of the pathology of this condition in more detail and aid the investigation of potential therapeutic agents both as prophylactics and, more importantly, as treatments.

     

Antidepressant-like effects of endogenous histamine and of two histamine H1 receptor agonists in the mouse forced swim test

1. Effects of substances which are able to alter brain histamine levels and two histamine H₁ receptor agonists were investigated in mice by means of an animal model of depression, the forced swim test.
2. Imipramine (10 and 30 mg kg⁻¹, i.p.) and amitriptyline (5 and 15 mg kg⁻¹, i.p.) were used as positive controls. Their effects were not affected by pretreatment with the histamine H₃ receptor agonist, (R)-α-methylhistamine, at a dose (10 mg kg⁻¹, i.p.) which did not modify the cumulative time of immobility.
3. The histamine H₃ receptor antagonist, thioperamide (2–20 mg kg⁻¹, s.c.), showed an antidepressant-like effect, with a maximum at the dose of 5 mg kg⁻¹, which was completely prevented by (R)-α-methylhistamine.
4. The histamine-N-methyltransferase inhibitor, metoprine (2–20 mg kg⁻¹, s.c.), was effective with an ED₅₀ of 4.02 (2.71–5.96) mg kg⁻¹; its effect was prevented by (R)-α-methylhistamine.
5. The histamine precursor, L-histidine (100–1000 mg kg⁻¹, i.p.), dose-dependently decreased the time of immobility [ED₃₀ 587 (499–712) mg kg⁻¹]. The effect of 500 mg kg⁻¹ L-histidine was completely prevented by the selective histidine decarboxylase inhibitor, (S)-α-fluoromethylhistidine (50 mg kg⁻¹, i.p.), administered 15 h before.
6. The highly selective histamine H₁ receptor agonist, 2-(3-trifluoromethylphenyl)histamine (0.3–6.5 μg per mouse, i.c.v.), and the better known H₁ agonist, 2-thiazolylethylamine (0.1–1 μg per mouse, i.c.v.), were both dose-dependently effective in decreasing the time of immobility [ED₅₀ 3.6 (1.53–8.48) and 1.34 (0.084–21.5) μg per mouse, respectively].
7. None of the substances tested affected mouse performance in the rota rod test at the doses used in the forced swim test.
8. It was concluded that endogenous histamine reduces the time of immobility in this test, suggesting an antidepressant-like effect, via activation of H₁ receptors.

     

Evidence for the involvement of 5-HT4 receptors in the 5-hydroxytryptamine-induced pattern of migrating myoelectric complex in sheep

1. The effects induced by 5-hydroxytryptamine (5-HT) on gastrointestinal myoelectric activity in conscious sheep were recorded through electrodes chronically implanted and analysed by computer. The 5-HT receptors and the cholinergic neuronal pathways involved in these actions were investigated.
2. The intravenous (i.v.) administration of 5-HT (2, 4 and 8 μg kg⁻¹ min⁻¹, 5 min) induced an antral inhibition concomitant with a duodenal activity front that migrated to the jejunum, followed by a period of intestinal inactivity. This myoelectric pattern closely resembled that observed in the phases III and I of the migrating myoelectric complex (MMC) in sheep. The 0.5 μg kg⁻¹ min⁻¹ dose evoked the same pattern in only two out of the six animals used. Likewise, the 1 μg kg⁻¹ min⁻¹ dose similarly affected four of the six animals. In addition, a transient stimulation was observed in the antrum and jejunum when the two highest doses were used.
3. The 5-HT₁ antagonist, methiothepin (0.1 mg kg⁻¹), the 5-HT₂ antagonists, ritanserin (0.1 mg  kg⁻¹) and ketanserin (0.3 mg  kg⁻¹), the 5-HT₃ antagonists, granisetron (0.2 mg kg⁻¹) and ondansetron (0.5 mg kg⁻¹), as well as the 5-HT₄ antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (0.2 mg kg⁻¹), did not modify the spontaneous gastrointestinal myoelectric activity. However, the cholinoceptor antagonists, atropine (0.2 mg kg⁻¹) and hexamethonium (2 mg kg⁻¹), inhibited gastrointestinal activity.
4. When these antagonists were injected i.v. 10 min before 5-HT (2 or 4 μg kg⁻¹ min⁻¹, 5 min), only {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808, atropine and hexamethonium were able to modify the 5-HT-induced actions, all of them being completely blocked by the three antagonists.
5. Our data show that 5-HT initiates a MMC-like pattern in the gastrointestinal area in sheep through 5-HT₄ receptors. Furthermore, these actions are mediated by cholinergic neural pathways involving muscarinic and nicotinic receptors. However, our results do not indicate a role for either 5-HT₁, 5-HT₂ or 5-HT₃ receptors in the 5-HT-induced effects.

     

Improved metabolic status and insulin sensitivity in obese fatty (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats treated with the thiazolidinedione,MCC-555

1. We examined the effect of chronic (21 days) oral treatment with the thiazolidinedione, MCC-555 ((±)-5-[{6-(2-fluorbenzyl)-oxy-2-naphy}methyl]-2,4-thiazolidinedione) on metabolic status and insulin sensitivity in obese (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats which display an impaired glucose tolerance (IGT) or overt diabetic symptoms, respectively.
2. MCC-555 treatment to obese Zucker rats (10 and 30 mg kg⁻¹) and diabetic ZDF rats (10 mg kg⁻¹) reduced non-esterified fatty acid concentrations in both rat strains and reduced plasma glucose and triglyceride concentrations in the obese Zucker rats. Liver glycogen concentrations were significantly increased by chronic MCC-555 treatment in both obese Zucker rats (30 mg kg⁻¹ day⁻¹) and diabetic ZDF rats (10 mg kg⁻¹ day⁻¹), as compared with vehicle-treated lean and obese rats and there was a significant increase in hepatic glycogen synthase activity in MCC-555-treated diabetic ZDF rats as compared to vehicle-treated controls.
3. During a euglycaemic hyperinsulinaemic clamp, MCC-555-treated obese Zucker rats and diabetic ZDF rats required significantly higher glucose infusion rates to maintain stable glucose concentrations (2.01±0.19 mg min⁻¹ and 6.42±1.03 mg min⁻¹, respectively) than vehicle-treated obese controls (0.71±0.17 mg min⁻¹ and 2.09±0.71 mg min⁻¹; P<0.05), demonstrating improved insulin sensitivity in both Zucker and ZDF rats. MCC-555 treatment also enhanced insulin-induced suppression of hepatic glucose production in ZDF rats as measured using infusions of [6-³H]-glucose under clamp conditions.
4. In conclusion, we have demonstrated that MCC-555 improves metabolic status and insulin sensitivity in obese Zucker and diabetic ZDF rats. MCC-555 may prove a useful compound for alleviating the metabolic disturbances and IGT associated with insulin resistance in man.