Protective effect of oral -arginine administration on gentamicin-induced renal failure in rats

We investigated the effects of orally supplemented -arginine, the substrate of nitric oxide (NO) and N^ω-nitro- -arginine methyl ester ( -NAME), a nitric oxide-synthase inhibitor in gentamicin-induced renal failure. Rats were given gentamicin (100 mg/kg/day s.c.), gentamicin and -arginine (2 g/l, drinking water), gentamicin and -NAME (100 mg/l, drinking water) or gentamicin plus -arginine and -NAME. After 8 days, the gentamicin group developed marked renal failure, characterized by a significantly decreased creatinine clearance and increased blood creatinine, fractional excretion of sodium, fractional excretion of lithium, urine gamma glutamyl transferase, systolic blood pressure and daily urine volume when compared to controls. Renal histological analysis confirmed tubular necrosis. -arginine administration caused normalization of these parameters, whereas -NAME led to aggravation of the failure. Concomittant administration of -NAME and -arginine to gentamicin-treated rats caused no significant changes when compared to the rats receiving gentamicin alone. We conclude that -arginine supplementation has beneficial effects in gentamicin-induced renal failure in rats and that these effects are reversed by the NO-synthase inhibitor, -NAME.     

Nitric Oxide Modulates the Acute Increase of Gastrointestinal Transit Induced by Endotoxin in Rats: a Possible Role for Tachykinins

Because of the evidence that endogenous nitric oxide (NO) plays an essential role in the physiological regulation of gastrointestinal motility we have investigated, by use of the NO synthase inhibitor, N^G-nitro-l -arginine methyl ester (l -NAME), the role of endogenous NO in the acute endotoxin-induced changes of gastrointestinal transit. Pre-treatment with E. coli endotoxin (100 μg kg^?, i.v.) induced a significant increase in the gastrointestinal transit of a charcoal suspension in anaesthetized rats. Previous administration of the NO synthase inhibitor, l -NAME (10 mg kg^?, i.v.) significantly prevented the effects of endotoxin. l -arginine (200 mg kg^?, i.v.) and the substance P antagonist [d -Pro², d -Trp^7,9]-substance P (SPA), significantly reversed the effects of l -NAME on gastrointestinal transit in rats treated with endotoxin. Pre-treatment with dexamethasone (5 mg kg^?, s.c., twice), an inhibitor of the expression of inducible NO synthase, did not affect the increase in the gastrointestinal transit through constitutive NO synthesis. The results suggest that constitutive nitric oxide is involved in the increase of gastrointestinal transit induced by endotoxin and that the reduction in transit induced by l -NAME in endotoxin-treated rats is mediated by endogenous tachykinins.     

Constitutive nitric oxide modulates the injurious actions of vasopressin on rat intestinal microcirculation in acute endotoxaemia

The administration of the nitric oxide (NO) synthase inhibitor, N^G-nitro-l-arginine methyl ester (L-NAME, 5 mg/kg s.c.) concurrently with Escherichia coli endotoxin (3 mg/kg i.v.) increased vascular permeability and caused mucosal damage in the rat intestine 1 h later. The vasopressin V₁ receptor antagonist, [Mca¹,Tyr(Me)²,Arg⁸]vasopressin (0.01–0.2 μg/kg s.c., 15 min before endotoxin) dose-dependently reduced this damage. These results suggest a beneficial role of NO, counteracting the injurious vascular actions of endogenous vasopressin, in maintaining intestinal mucosal integrity in acute endotoxaemic states.     

Antinociceptive action of myricitrin: involvement of the K+ and Ca2+ channels

The present study was designed to investigate the mechanisms involved in the antinociception afforded by myricitrin in chemical models of nociception in mice. Myricitrin given by intrathecal (i.t.) or intracerebroventricular (i.c.v.) route produced dose-related antinociception when evaluated against acetic acid-induced visceral pain in mice. In addition, the intraperitoneal administration of myricitrin caused significant inhibition of biting behaviour induced by i.t. injection of glutamate, substance P, capsaicin, interleukin 1 β (IL-1β) and tumor necrosis factor-α (TNF-α). The antinociception caused by myricitrin in the acetic acid test was fully prevented by i.t. pre-treatment with pertussis toxin, a Gi/o protein inactivator, and by i.c.v. injection of calcium chloride (CaCl₂). In addition, the i.t. pre-treatment of mice with apamin, a blocker of small (or low)-conductance calcium-gated K⁺ channels and tetraethylammonium, a blocker of voltage-gated K⁺ channels significantly reversed the antinociception induced by myricitrin. The charybdotoxin, a blocker of large (or fast)-conductance calcium-gated K⁺ channels and glibenclamide, a blocker of the ATP-gated K⁺ channels had no effect on myricitrin-induced antinociception. Calcium uptake analysis revealed that myricitrin inhibited ⁴⁵Ca²⁺ influx under a K⁺-induced depolarization condition. However, calcium movement was modified in a non-depolarizing condition only when the highest concentration of myricitrin was used. In summary, our findings indicate that myricitrin produces consistent antinociception in chemical models of nociception in mice. These results clearly demonstrate an involvement of the Gi/o protein dependent mechanism on antinociception caused by myricitrin. The opening of voltage- and small-conductance calcium-gated K⁺ channels and the reduction of calcium influx led to the antinociceptive of myricitrin.     

脑室注射硝普钠、L-精氨酸、NG-硝基-L-精氨酸对清醒状态大鼠心血管活动的影响

给大鼠侧脑室内注射(icv)硝普钠(SNP)、NO的前体物质L-精氨酸(L-Arg)和精-精二肽(Arg-Arg),NO合成酶(NOS)抑制剂N^G-硝基-L-精氨酸(NNLA),观察清醒状态大鼠血压和心率的变化,探讨脑内NO对清醒状态大鼠心血管活动的调节作用。实验结果表明,icvSNP(8,16,32μg)使血压升高,并呈剂量效应关系,同时使心率加快。icvL-Arg(200μg)或不同剂量的精-精二肽也使血压升高,心率加快;icvNNLA(100μg)则使血压下降,心率减慢。以上结果提示,在一定范围内提高脑内NO,对心血管活动有正性调节作用,降低脑内NO则对心血管活动有负性调节作用。     

Effect of LP-805, a releaser of endothelium-derived nitric oxide,on systemic vasodilatation in vivo

Summary We have investigated relations between hypotensive responses to LP-805, a newly synthesized vasodilator, and the production of nitric oxide (NO), in anesthetized rats. LP-805 (0.1–0.5 mg/kg, i.v.) or acetylcholine (ACh) (0.3 – 3.0 g/kg, i.v.) caused a dose-dependent transient decrease in diastolic blood pressure. The decrease induced by 0.3 mg/kg LP-805 (i.v.) was partially inhibited by pretreatment with N^G-nitro-l-arginine (LNNA), a specific inhibitor of endothelial NO synthase, but the responses to lower or higher doses of LP-805 (0.1 or 0.5 mg/kg, i.v.) were not affected. The dose-dependent decrease in diastolic blood pressure, caused by LP-805, was not affected by pretreatment with l- or d-arginine. The dose-dependent decrease in diastolic blood pressure caused by ACh was not affected by pretreatment with L-NNA or with l- or d-arginine. The hypotensive response to 20-min infusions of LP-805 (100 g/kg per min) wassignificantly inhibited by pretreatment with L-NNA (10 mg/kg, i.v.). The half-recovery times (T_1/2) of LP-805 or ACh-induced depressor responses were shortened by pretreatment with L-NNA. They were prolonged by l-arginine, but not by d-arginine. This shortening, by L-NNA, of the half-recovery time after LP-805 or ACh was reversed by l-arginine, but not by d-arginine. The T_/2 of the LP-805-induced hypotensive response was not affected by pretreatment with indomethacin (1 mg/kg, i.v.). In the presence of L-NNA (10 mg/kg, i.v.), the T_/2 of the LP-805-induced hypotensive response was not affected by pretreatment with indomethacin. The results suggest that the LP-805-induced hypotensive response may be related to direct or indirect activation of NO synthase in vascular endothelial cells, and to release of endothelium-derived NO. Correspondence to M. Inazu at the above address     

Study of in vivo and in vitro resting vasodilator nitric oxide tone in normotensive and genetically hypertensive rats

The effects of N^G-nitro- -arginine ( -NNA) on mean arterial pressure and the effects of both -NNA and methylene blue on isolated aorta tone, were studied in order to elucidate potential alterations in vasodilator resting nitric oxide (NO) tone in genetic hypertension. -NNA produced a significantly greater increase of mean arterial pressure in spontaneously hypertensive rats (SHR) than in Wistar Kyoto (WKY) rats; in both cases, -arginine completely inhibited the -NNA hypertensive effect. Neither ganglion blockade with hexamethonium nor cyclooxygenase inhibition with indomethacin significantly modified the effect of -NNA in both rat strains. In intact aorta rings, after submaximally contraction with KCl (25 mM), both -NNA and methylene blue induced strong dose-dependent contractions. The maximum contractions were, however, significantly greater in WKY rats than in SHR. The mechanical elimination of endothelium markedly inhibited both -NNA and methylene blue maximum contractions. In intact rings, -arginine completely inhibited the -NNA effects in both rat strains; in rubbed rings, the -arginine inhibitory effects were strong in WKY rats but not important and erratic in SHR. -Arginine had no effect on the contractions induced only by KCl in any of the preparations. In WKY rat-rubbed rings, sodium nitroprusside was significantly more effective in relaxing the contractions in response to 25 mM KCl than the contractions in response to methylene blue. These results indicate that contractions induced by -NNA and methylene blue in isolated aorta are principally due to the inhibition of an important endothelial resting vasodilator NO tone. They also show that hypertension reduces the resting vasodilator NO tone in isolated rat aorta, in spite of enhancing the total vasodilator NO tone in anaesthetized rat.     

Role of nitric oxide in penile erection and yawning induced by 5-HT1c receptor agonists in male rats

The effect of the intracerebroventricular (i.c.v.) administration of N^G-nitro-l,-arginine methyl ester and N^G-monomethyl-l.-arginine, two inhibitors of nitric oxide (NO) synthase, on penile erection and yawning induced by 1-(3-chlorophenyl)-piperazine (m-CPP)- and N-(3-trifluoromethylphenyl)-piperazine (TFMPP), two selective 5HT_1c receptor agonists, was studied in male rats. Both NO synthase inhibitors (50–500 g i.c.v.) prevented dose-dependently the behavioural responses induced by m-CPP (0.5 mg/kg s.c.) or by TFMPP (I mg/kg s.c.), but N^G-nitro-l-arginine methyl ester was about 4–5 times more potent than N^G-monomethyl-l,-arginine. The D-isomer of N_G-monomethyl-l-arginine, which does not inhibit nitric oxide synthase, was ineffective. The inhibitory effect of N^G-nitro-l-arginine methyl ester on m-CPP- and TFMPP-induced responses was prevented by the administration of l-arginine (1 mg i.c.v.). In contrast, N^G-nitro-l-arginine methyl ester (20 g) was ineffective when injected in the paraventricular nucleus of the hypothalamus, a brain area that plays a key role in the expression of these behavioural responses. m-CPP- and TFMPP-induced penile erection and yawning was prevented also by the i.c.v. administration of LY 83583 (50–200 g) or methylene blue (50–400 g), two inhibitors of guanylate cyclase but not by reduced hemoglobin (50–400 g), a NO scavenger. The results suggest that central nitric oxide is involved in the expression of penile erection and yawning induced by 5-HT_1c receptor agonists.     

Effects of water deprivation and angiotensin II intracerebroventricular administration on brain nitric oxide synthase activity

Intracranial administration of -arginine causes a reduction of the water intake induced by water deprivation or by intracerebroventricular (i.c.v.) injection of angiotensin II (angiotensin II), through the release of nitric oxide (NO) in the central nervous system. We studied the effects of i.c.v. angiotensin II (120 ng/rat) in association with i.c.v. -arginine (2.5–10 μg/rat) on blood pressure. We also studied the effects of both peripheral and central angiotensin II injection (1.5–6 mg kg⁻¹ i.p. and 30–120 ng rat⁻¹ i.c.v., respectively) on NO synthase activity in the cortex, diencephalon and brainstem, after water deprivation (24 h), conditions producing activation of the renin-angiotensin system. -arginine dose dependently antagonized the increase in blood pressure induced by i.c.v. angiotensin II (P<0.001). Peripheral administration of angiotensin II produced a dose-dependent reduction of NO synthase activity in the brainstem and cortex (P<0.001), but not in the diencephalon. Water deprivation produced similar effects on brain NO synthase activity. Angiotensin II i.c.v. injection caused NO synthase activity reduction in all brain regions studied (P<0.001). Our findings suggest that NO and angiotensin II could play opposite roles in brain regulation of blood pressure and drinking behaviour.     

Role of ATP-sensitive K+ channels in antinociception induced by R-PIA,an adenosine A1 receptor agonist

The influence of several K⁺ channel-acting drugs on antinociception induced by the adenosine A₁ receptor agonist (–)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) was evaluated with a tail flick test in mice. The subcutaneous administration of R-PIA (0.5–8 mg/kg) induced a dose-dependent antinociceptive effect. The ATP-sensitive K⁺ (K_ATP) channel blocker gliquidone (2–8 g/mouse, i.c.v.) produced a dose-dependent displacement to the right of the R-PIA dose-response line, whereas the K_ATP channel opener cromakalim (32 g/mouse, i.c.v.) shifted it to the left. Several K_ATP channel blockers dose-dependently antagonized the antinociceptive effect of R-PIA, the order of potency being gliquidone > glipizide > glibenclamide (i.e., the same order of potency shown by these drugs in blocking K_ATP channels in neurons). In contrast, the K⁺ channel blockers 4-aminopyridine and tetraethylammonium did not antagonize the effect of R-PIA. These data suggest that antinociception produced by adenosine A₁ receptor agonists is mediated by the opening of ATP-sensitive K⁺ channels. The present results, together with those of previous studies, further support a role for K⁺ channel opening in the antinociceptive effect of agonists of receptors coupled to G_i/G_o proteins. Correspondence to: José M. Baeyens at the above address     

Negative modulation of nitric oxide production by neurotensin as a putative mechanism of the diuretic action of SR 48692 in rats

1. We investigated the effect of the non-peptide neurotensin (NT) antagonist SR 48692 on renal function in rats and the involvement of nitric oxide (NO) in the diuretic action of this compound.
2. In fed animals, SR 48692 dose-dependently (0.5 to 12.5 mg kg⁻¹, p.o., 0.03 to 1 mg kg⁻¹, i.p. and 0.1 to 1 μg/rat, i.c.v.) increased urine output and urinary excretion of Na⁺, K⁺ and Cl⁻ and reduced urine osmolality. The diuretic activity was also evident in water-deprived, fasted animals and in fasted, water-loaded rats.
3. NT (0.1 μg/rat, i.c.v.) had no effect on urine output in fed rats, but reduced the diuretic action of SR 48692 (1 μg/rat, i.c.v.). The opposite result was obtained in fasted, water-loaded animals: NT dose-dependently (0.01 and 0.1 μg/rat, i.c.v.) inhibited diuresis and this effect was significantly inhibited by i.c.v. SR 48692. In this experimental condition, SR 48692 did not further increase the on-going diuresis.
4. The NO synthesis inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME; 30 mg kg⁻¹, i.p.) alone had no effect on urine output in fed rats but prevented the diuretic action of i.c.v. or i.p. SR 48692; L-arginine (1 g kg⁻¹, i.p.) but not D-arginine (1 g kg⁻¹, i.p.) restored the SR 48692-dependent increase in diuresis. L-NAME had no effect on furosemide-stimulated diuresis.
5. Systemically administered L-NAME or i.c.v. NT in fasted, water-loaded rats significantly reduced water diuresis but this effect was no longer seen in animals given i.p. L-arginine. Rats receiving i.c.v. NT, whose diuresis was significantly reduced, also excreted less nitrates and nitrites in urine.
6. Increased diuresis after central or systemic administration of SR 48692 to fed rats was paralleled by increased urinary excretion of nitrates and nitrites, this being consistent with peripheral enhancement of NO production after NT-receptor blockade by SR 48692. The increase in diuresis after furosemide also involved an increase of nitrates and nitrites in urine, but this effect was about half that attained with an equipotent diuretic dose of SR 48692.
7. In fed rats, the NO donor isosorbide-dinitrate, reduced systolic blood pressure (unlike SR 48692 which did not affect blood pressure) but also dose-dependently (1 and 5 mg kg⁻¹, i.p.) stimulated urine output.
8. The overall effects of SR 48692 strongly support a link between the actions of endogenous NT, AVP and peripheral NO production in the modulation of renal excretion of water, Na⁺, K⁺ and Cl⁻.

     

Intracerebroventricular administration of nitric oxide-sensitive guanylyl cyclase inhibitors induces catalepsy in mice

Rationale Catalepsy is a preclinical test that predicts extrapyramidal symptoms in humans. It models symptoms of acute extrapyramidal side effects induced at the beginning of antipsychotic treatment. Nitric oxide (NO) plays a role in a series of neurobiological functions underlying behavior. For example, inhibition of NO synthesis disrupts rodent exploratory behavior and induces catalepsy. Although several effects mediated by NO involve the activation of soluble guanylyl cyclase (sGC), the transduction mechanism of the catalepsy-inducing effect of NO has not yet been investigated. Objectives The study was designed to test if intracerebroventricular (i.c.v.) microinjection of NO-sensitive inhibitors of sGC (NO-sGC) induces catalepsy in mice similar to that induced by NO synthase (NOS) inhibitors. Exploratory behavior was tested in the open field. In addition, the effects of a NOS inhibitor on oxidative metabolites of NO were measured in the striatum. Materials and methods Drug effects were examined in the hanging-bar test after the following i.c.v. treatments: oxadiazolo-quinoxalin (ODQ, 30–300 nmol) or methylene blue (MB, 3–100 nmol), selective and nonselective sGC inhibitors, respectively, or 7-nitroindazole (7-NI, 3–90 nmol) and G-nitro-l-arginine methyl ester (l-NAME, 3–90 nmol), selective and nonselective neuronal NOS inhibitors. To test if the effects were related to interference with the NO system, additional groups received 7-NI (30 nmol), ODQ (100 nmol), or L-NAME (90 nmol) preceded by l-arginine (l-arg, 30–100 nmol, i.c.v. 30 min before). A possible interference of ODQ and 7-NI on exploratory behavior was tested in an open field. The concentration of nitrites and nitrates (NO _x ) in striatum homogenates was measured by the Griess reaction. Results Both NO-sGC and NOS inhibitors induced catalepsy in mice that lasted for at least 2 h. The range of effective doses of these drugs, however, was limited, and the dose–effect curves had an inverted U shape. The cataleptic effect induced by l-NAME was inversely correlated with NO _x products in the striatum. The cataleptic effect of 7-NI and ODQ was prevented by pretreatment with l-arginine. No drug changed exploratory behavior in the open field. Conclusion This study showed that pharmacological disruption of the endogenous NO-sGC signaling in the central nervous system induces long-lasting catalepsy in mice. Moreover, the cataleptic effect of NOS inhibition correlates with the decrease in NO _x products formation in the striatum. The results give further support to the hypothesis that NO plays a role in motor behavior control mediated, at least in part, by cyclic guanosine monophosphate production in the striatum.     

Evidence for the involvement of the nitric oxide–cGMP pathway in the antinociception of morphine in the formalin test

The effect of inhibition of nitric oxide synthesis and guanylate cyclase on the peripheral antinociceptive effect of morphine was assessed by using the formalin test in the rat. Saline, N^G-monomethyl-

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-arginine, a nitric oxide synthesis inhibitor (50 μg) and methylene blue, a guanylate cyclase inhibitor (500 μg), did not exhibit any antinociceptive activity. However, morphine (10 μg) produced a significant antinociceptive effect in phases 2a and 2b, which was reduced by pretreatment with either N^G-monomethyl-

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-arginine or methylene blue. These results suggest that the local administration of morphine induces antinociception by the activation of the

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-arginine–nitric oxide–cGMP pathway.     

Excitatory regulation of noradrenergic neurons by <Emphasis Type="SmallCaps">l</Emphasis>-arginine/nitric oxide pathway in the rat locus coeruleus in vivo

To elucidate conflicting findings about the role of l-arginine/nitric oxide (NO) pathway in the locus coeruleus (LC), we investigated the effects of different drugs affecting NO concentrations by single-unit extracellular recordings from LC neurons in vivo and in vitro. In anesthetized rats, central (3.8–15.3 nmol i.c.v.) and local (16.5–66 pmol into the LC) administrations of the NO donor sodium nitroprusside, but not those of the inactive analogue potassium ferricyanide (16.5–66 pmol into the LC), increased by 65–84% the firing rate of LC neurons. In brain slices, low concentrations (50–200 μM) of diethylamine/NO complex, a short-lived NO releaser, also increased the neuron firing rate, although higher drug concentrations (400–800 μM) caused slowly reversible reductions of the firing activity. On the other hand, the NO synthase inhibitors N^ω-nitro-l-arginine methyl ester (l-NAME) (148–371 nmol i.c.v.) and N^ω-nitro-l-arginine (l-NA) (46 nmol i.c.v.) gradually decreased the firing rate of LC neurons, whereas the NO synthase substrate l-arginine (0.71–1.42 μmol i.c.v. and 0.6–4.8 nmol into the LC) increased the neuron activity. The latter effect was not mimicked by the vehicle or the less active isomer d-arginine (0.6–4.8 nmol into the LC). Unexpectedly, pretreatment with high concentrations of l-NAME (371 nmol and 18.5 μmol i.c.v.) or l-NA (45.6 nmol i.c.v. and 0.24 nmol into the LC) failed to block the effect of l-arginine. The glutamate receptor antagonist kynurenic acid (1 μmol i.c.v.) strongly reduced the effect of l-arginine but not that of sodium nitroprusside. These data confirm in vivo a direct excitatory effect of NO on LC neurons and suggest a tonic regulation of noradrenergic neurons by NO in vivo. l-arginine also excites LC neurons, but this effect may be caused by a nitric-oxide-unrelated glutamate-receptor-mediated mechanism.     

Possible involvement of l-arginine-nitric oxide pathway in modulating regional blood flow to brown adipose tissue of rats

To evaluate whether the l-arginine-nitric oxide (NO) pathway is involved in the regulation of regional blood flow to brown adipose tissue (BAT), the effects of two specific NO synthase inhibitors, N^G-nitro-l-arginine methyl ester (l-NAME) and N^G-monomethyl-l-arginine (l-NMMA), on the blood flow to interscapular brown adipose tissue (IBAT) were studied in urethane-anesthetized rats. Regional blood flow in MAT was measured with laser-Doppler flowmetry.An intravenous injection of l-NAME and l-NMMA, but not of either d-enantiomer, caused a transient and dose-dependent increase in IBAT blood flow. Dose-response curves for these NO synthase inhibitors showed that l-NAME was more potent than l-NMMA in increasing IBAT blood flow. We also observed a concomitant pressor effect accompanied by a slight decrease in heart rate following intravenous injection of l-NAME and l-NMMA. An elevation of IBAT blood flow and blood pressure induced by both l-NAME and l-NMMA was reversed by l-arginine in an enantiomerically specific manner. The increase in IBAT blood flow induced by NO synthase inhibitors was of shorter duration and less sensitive to l-arginine than the increase in blood pressure.Our results show that the WAY blood flow is increased by inhibition of NO synthase and that the response of IBAT vasculature to NO synthase inhibitors is different from that of the resistance vessels which regulate blood pressure. The involvement of l-arginine-NO pathways in modulating microcirculation in IBAT is suggested. Correspondence to: Y. Uchida at the above address     

The rostroventromedial medulla is not involved in α2-adrenoceptor-mediated antinociception in the rat

The aim of the current study was to investigate the role of the rostroventromedial medulla (RVM) in α₂-adrenoceptor-mediated antinociception. Medetomidine or clonidine, selective α₂-adrenoceptor agonists were microinjected into the RVM in unanesthetized rats with a chronic guide cannula. The antinociceptive effects were evaluated using the tail-flick and hot-plate tests. For comparison, medetomidine was microinjected into the cerebellum or the periaqueductal gray (PAG). To study the role of medullospinal pathways, the tail-flick latencies were also measured in spinalized rats. The reversal of the antinociception induced by intracerebral microinjections of medetomidine was attempted by s.c. atipamezole, a selective α₂-adrenoceptor antagonist. The reversal of the antinociception induced by systemic administration of medetomidine was attempted by microinjections of 5% lidocaine or atipamezole into the RVM. When administered into the RVM, medetomidine produced a dose-dependent (1–30 μg) antinociception in the tail-flick and hot-plate tests, which antinociceptive effect was completely reversed by atipamezole (1 mg/kg, s.c.). Also clonidine produced a dose-dependent (3–30 μg) antinociception following microinjection into the RVM. Microinjections of medetomidine into the cerebellum or the PAG produced an identical dose-response curve in the tail-flick test as that obtained following microinjection into the RVM. In spinalized rats the antinociceptive effect (tail-flick test) induced by medetomidine microinjected into the RVM was not less effective than in intact rats. Lidocaine (5%) or atipamezole (5 μg) microinjected into the RVM did not attenuate the antinociception induced by systemically administered medetomidine (100 μg/kg, s.c.). The adapting skin temperature of the tail was increased in a nonmonotonic fashion following medetomidine. The results indicate that the RVM is not a site which is critical for the α₂-adrenergic antinociception. The antinociception following intracerebral microinjections of medetomidine into the RVM, PAG or the cerebellum in the current study can be explained by a spread of the α₂-adrenoceptor agonist into the spinal level to activate directly spinal α₂-adrenoceptors. Also, the antinociception following systemic administration of medetomidine can be explained by spinal α₂-adrenergic mechanisms. The medetomidine-induced increase of the adapting skin temperature may have attenuated the medetomidine-induced increases in the response latencies to noxious heat.     

The effect of cyclosporin A on morphine tolerance and dependence: involvement of L-arginine/nitric oxide pathway

Cyclosporin A is known to decrease nitric oxide (NO) production in nervous tissues. The effects of systemic cyclosporine A on the induction and expression of morphine tolerance and dependence, acute morphine-induced antinociception, and the probable involvement of the L-arginine/nitric oxide pathway in these effects were assessed in mice. Cyclosporin A (20 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and a combination of the two at lower and per se non-effective doses (5 and 3 mg/kg, respectively) showed a similar pattern of action, inhibiting the induction of tolerance to morphine-induced antinociception and increasing the antinociception threshold in the expression phase of morphine tolerance. These agents also inhibited the expression of morphine dependence as assessed by naloxone-precipitated withdrawal signs, while having no effect on the induction of morphine dependence. L-Arginine, at a per se non-effective dose (60 mg/kg), inhibited the effects of Cyclosporin A. Moreover, acute administration of Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) enhanced the antinociception induced by acute administration of morphine (5 mg/kg), while chronic pretreatment with Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) for 2 days (twice daily) did not affect morphine-induced antinociception. The inducible nitric oxide synthase inhibitor, aminoguanidine (100 mg/kg), did not alter morphine antinociception, tolerance or dependence. In conclusion, decreasing NO production through constitutive nitric oxide synthase may be a mechanism through which cyclosporin A differentially modulates morphine tolerance, dependence and antinociception.     

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2 and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (<EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B₂ receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B₂ receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development.     

Analysis of the mechanisms underlying the endothelium-dependent antivasoconstriction of puerarin in rat aorta

Puerarin, a major isoflavonoid compound from the Chinese herb, Ge-gen (Pueraria lobata), has effective treatment on myocardial and cerebral ischemia, glaucoma and sudden deafness in clinical setting in China. Our present work showed that puerarin (50, 150, 450 μM) concentration-dependently inhibited phenylephrine or KCl-induced contraction only in endothelium-intact rat aortic rings. In Ca²⁺-free solution, the antivasoconstriction of puerarin on phenylephrine was totally deprived. N ^G-nitro-l-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, indomethacin and the three K⁺ channel blockers, glibenclamide, tetraethylammonium and Ba²⁺ displayed significant inhibitory effects on the antivasoconstriction of puerarin. 8-bromo-cGMP significantly strengthened the action of puerarin. Puerarin (10–160 μM) concentration-dependently induced the NO production in the rat aortic cells. These findings suggested that the antivasoconstriction elicited by puerarin is endothelium-dependent. NO/NO–cGMP pathway, PGI₂ and the opening of K⁺ channels sensitive to glibenclamide, tetraethylammonium, and Ba²⁺, which might be triggered by the extracellular Ca²⁺ influx in the endothelium, appear to contribute to the antivasoconstriction of puerarin.     

L-arginine exerts a dual role in nociceptive processing in the brain: involvement of the kyotorphin-Met-enkephalin pathway and NO-cyclic GMP pathway.

1. Intracerebroventricular (i.c.v.) administration of L-arginine (L-Arg), at 10-100 micrograms per mouse, produced antinociception in mice, as assessed by the tail flick test; this antinociception was reversed by pretreatment (s.c.) with naltrindole (NTI), a delta-selective opioid antagonist, and by co-administered L-leucyl-L-arginine (Leu-Arg), a kyotorphin (endogenous Met-enkephalin releaser) receptor antagonist. 2. L-NG-nitroarginine methyl ester (L-NAME), a NO synthase inhibitor, but not D-NG-nitroarginine methyl ester, given i.c.v. at 3-10 micrograms per mouse, exhibited antinociceptive activity that was resistant to naloxone (s.c.), NTI (s.c.) and Leu-Arg (i.c.v.). 3. The L-NAME (i.c.v.)-induced antinociception was not reversed by L-Arg (i.c.v.), which was antinociceptive by itself, but was abolished by combined injection of L-Arg plus Leu-Arg (i.c.v.) or by L-Arg (i.c.v.) after NTI (s.c.). 4. Methylene blue (MB), a soluble guanylate cyclase inhibitor, at 0.1-1 microgram per mouse, produced antinociception by i.c.v. administration. The antinociception induced by MB (i.c.v.) or L-NAME (i.c.v.) was reversed by co-administered dibutyryl cyclic GMP. 5. These findings suggest that L-Arg plays a dual role in nociceptive processing in the brain, being antinociceptive via the kyotorphin-Met-enkephalin pathway and nociceptive via the NO-cyclic GMP pathway.