Nitric Oxide Donor Glyceryl Trinitrate Research Articles

Vascular extracellular signal‐regulated kinase mediates migraine‐related sensitization of meningeal nociceptors

To examine changes in the response properties of meningeal nociceptors that might lead to migraine pain and examine endogenous processes that could play a role in mediating them using a clinically relevant model of migraine triggering, namely infusion of the nitric oxide (NO) donor nitroglycerin (NTG). Single-unit recordings made in the trigeminal ganglion of rats were used to test changes in the activity and mechanosensitivity of meningeal nociceptors in response to administration of the migraine trigger NTG or another NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) at doses relevant to the human model of migraine headache. Immunohistochemistry and pharmacological manipulations were used to investigate the possible role of meningeal vascular signaling in mediating the responses of meningeal nociceptors to NO. Infusion of NTG promoted a delayed and robust increase in the mechanosensitivity of meningeal nociceptors, with a time course resembling the development of the delayed migraine headache. A similar sensitization was elicited by dural application of NTG and SNAP. NTG-evoked delayed meningeal nociceptor sensitization was associated with a robust extracellular signal-regulated kinase (ERK) phosphorylation in meningeal arteries. Pharmacological blockade of meningeal ERK phosphorylation inhibited the development of NTG-evoked delayed meningeal nociceptor sensitization. The development of delayed mechanical sensitization evoked by the migraine trigger NTG is potentially of great importance as the first finding of a neurophysiological correlate of migraine headache in meningeal nociceptors. The arterial ERK phosphorylation and its involvement in mediating the NTG-evoked delayed sensitization points to an important, yet unappreciated, role of the meningeal vasculature in the genesis of migraine pain.

St. John's wort reversal of meningeal nociception: A natural therapeutic perspective for migraine pain

Despite a number of antimigraine drugs belonging to different pharmacological classes are available, there is a huge unmet need for better migraine pharmacotherapy. We here demonstrated the capability of Hypericum perforatum, popularly called St. John's wort (SJW), to relieve meningeal nociception in an animal model induced by administration of the nitric oxide (NO) donors glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). GTN and SNP produced a delayed meningeal inflammation, as showed by the upregulation of interleukin (IL)-1β and inducible NO synthase (iNOS), and a prolonged cold allodynia and heat hyperalgesia with a time-course consistent with NO-induced migraine attacks. A single oral administration of a SJW dried extract (5mg/kg p.o.) counteracted the nociceptive behaviour and the overexpression of IL-1β and iNOS. To clarify the cellular pathways involved, the expression of protein kinase C (PKC) and downstream effectors was detected. NO donors increased expression and phosphorylation of PKCγ, PKCɛ and transcription factors, such as nuclear factor (NF)-κB, cyclic AMP response element binding protein (CREB), Signal Transducer and Activator of Transcription (STAT)-1. All these molecular events were prevented by SJW and hypericin, a SJW main component. In conclusion, SJW counteracted the NO donor-induced pain hypersensitivity and meningeal activation by blocking PKC-mediated pathways involving NF-κB, CREB, STAT1. These results might suggest SJW as an innovative and safe perspective for migraine pain.

Reversal of NO-induced nociceptive hypersensitivity by St. John’s wort and hypericin: NF-κB, CREB and STAT1 as molecular targets

Hypericum perforatum, popularly called St. John's wort (SJW), is a medicinal plant mainly used as antidepressant with a favorable safety profile than standard antidepressants. Some studies have also documented other SJW bioactivities, including pain modulation. The aim of this study was to demonstrate the capability of SJW to relieve nitric oxide (NO)-induced nociceptive hypersensitivity and identify the effective component. Nociceptive hypersensitivity induced by administration of the NO donors nitroglycerin (GTN) and sodium nitroprusside (SNP) was assessed by cold and hot plate tests. The cellular pathways and molecular targets involved were investigated by Western blotting. GTN and SNP produced a prolonged allodynia and hyperalgesia in mice. A single oral administration of low doses of an SJW dried extract or purified hypericin reversed the NO donor-induced nociceptive behavior whereas hyperforin and flavoinoids were ineffective. Investigating into the cellular pathways involved, an increased CREB and STAT1 phosphorylation, and activation of NF-κB were detected within PAG and thalamus following NO donors' administration. These cellular events were prevented by SJW or hypericin. Since hypericin showed PKC blocking properties, a role of PKC as an upstream modulator of these transcription factors was hypothesized. NO donors increased expression and phosphorylation of protein kinase C (PKC) γ and ε isoforms, molecular events prevented by SJW or hypericin. SJW reversed NO-induced nociceptive hypersensitivity through the blockade of a supraspinal signaling pathway involving a PKC-dependent CREB, STAT1 and NF-κB activation due to presence of hypericin. These data indicate SJW/hypericin as a therapeutic perspective for pain treatment.

St. John's wort relieves pain in an animal model of migraine

Despite the substantial improvement that antimigraine drugs brought to migraineurs, there is the need for a long-acting and better tolerated migraine treatment than actual pharmacotherapy. St. John's wort (SJW), a medicinal plant endowed with a favourable tolerability profile, showed numerous bioactivities. We here investigated the pain-relieving property of SJW and its main components, hypericin and flavonoids, in a mouse model induced by nitric oxide (NO) donors administration. The NO donors nitroglycerin and sodium nitroprusside (SNP) induced allodynia (cold plate test) and hyperalgesia (hot plate test). Western blotting experiments were performed to detect c-Fos and protein kinase C (PKC) expression within periaqueductal grey matter (PAG). A single oral administration of an SJW dried extract (5 mg/kg p.o.) produced a prolonged relief from pain hypersensitivity. Similarly, preventive SJW administration increased the latency to the induction of hyperalgesia and reduced the duration of the painful symptomatology. Among SJW main components, hypericin showed a similar profile of activity, whereas flavonoids were devoid of any antihyperalgesic effect. To clarify the cellular pathways involved in the SJW mechanism of action, we examined the effects induced by the herbal drug on PKC. NO donors' administration upregulated and increased phosphorylation of PKCγ and PKCε isoforms within PAG that was prevented by SJW treatment. The absence of behavioural side effects or altered animals' locomotor activity by SJW was demonstrated. These results suggest SJW as a safe therapeutic perspective for migraine pain, and indicate PKC as an innovative target for antimigraine therapy.

Inhibition of the PKCγ-ε pathway relieves from meningeal nociception in an animal model: an innovative perspective for migraine therapy?

There is convincing evidence that nitric oxide (NO) may be a causative factor in the pathogenesis of migraine. We investigated the consequences of NO donors' administration on meningeal processes related to the development of migraine pain in an animal model of meningeal nociception. The administration in mice of the NO donors nitroglycerin (GTN) and sodium nitroprusside (SNP) produced a delayed meningeal upregulation of interleukin-1ß and inducible NO synthase. A thermal allodynia and hyperalgesia devoid of side effects was produced 1 to 4h after administration. To clarify the cellular pathways modulated by GTN and SNP, we examined the expression of cellular factors involved in pain modulation, such as protein kinase C (PKC) and its downstream effectors. Western blotting experiments showed an upregulation and increased phosphorylation of PKCγ and PKCε within dura mater after NO donors' administration. A dramatic PKC-dependent increase of the phosphorylation of cyclic AMP response element binding protein (CREB) and signal transducer and activator of transcription (STAT)-1 was observed, along with an activation of the nuclear factor-κB (NF-κB) pathway, as reflected by a reduction of the inhibitory protein-κ-Bα (IκBα). Furthermore, the PKC blocker, Calphostin C, prevented the GTN and SNP-induced pain hypersensitivity. These results suggest the relevance of the PKC-mediated pathway in the induction of meningeal nociception and might help clarify the etiopathology of migraines. We can suggest PKC as a new target for migraine pain.

Calcitonin gene-related peptide receptors in rat trigeminal ganglion do not control spinal trigeminal activity

Calcitonin gene-related peptide (CGRP) is regarded as a key mediator in the generation of primary headaches. CGRP receptor antagonists reduce migraine pain in clinical trials and spinal trigeminal activity in animal experiments. The site of CGRP receptor inhibition causing these effects is debated. Activation and inhibition of CGRP receptors in the trigeminal ganglion may influence the activity of trigeminal afferents and hence of spinal trigeminal neurons. In anesthetized rats extracellular activity was recorded from neurons with meningeal afferent input in the spinal trigeminal nucleus caudalis. Mechanical stimuli were applied at regular intervals to receptive fields located in the exposed cranial dura mater. α-CGRP (10(-5) M), the CGRP receptor antagonist olcegepant (10(-3) M), or vehicle was injected through the infraorbital canal into the trigeminal ganglion. The injection of volumes caused transient discharges, but vehicle, CGRP, or olcegepant injection was not followed by significant changes in ongoing or mechanically evoked activity. In animals pretreated intravenously with the nitric oxide donor glyceryl trinitrate (GTN, 250 μg/kg) the mechanically evoked activity decreased after injection of CGRP and increased after injection of olcegepant. In conclusion, the activity of spinal trigeminal neurons with meningeal afferent input is normally not controlled by CGRP receptor activation or inhibition in the trigeminal ganglion. CGRP receptors in the trigeminal ganglion may influence neuronal activity evoked by mechanical stimulation of meningeal afferents only after pretreatment with GTN. Since it has previously been shown that olcegepant applied to the cranial dura mater is ineffective, trigeminal activity driven by meningeal afferent input is more likely to be controlled by CGRP receptors located centrally to the trigeminal ganglion.

Sensitivity analysis of near-infrared functional lymphatic imaging

Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150 μg/mL ICG and 60 g/L albumin. ICG fluorescence can be detected at a concentration of 150 μg/mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time.

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

Evaluation of anti-migraine potential of Areca catechu to prevent nitroglycerin-induced delayed inflammation in rat meninges: Possible involvement of NOS inhibition

Areca catechu nut extract is a popular folk remedy for the treatment of migraine in Kerala and Tamil Nadu states of India. In order to prove the claimed utilization of plant, the effect of hydroalcoholic extract of Areca catechu nut (ANE) was investigated in nitroglycerine induced inflammation in rat meninges. In these models infusion of nitric oxide donor glyceryl trinitrate (GTN) produces augmented plasma protein extravasation (PPE) in dura mater, provides an important substrate for the development of migraine in rats. The effect on plasma protein extravasation was assessed in both the models of intravenous and topical GTN application following oral administration of ANE (250 mg/kg and 500 mg/kg) in both curative and preventive treatment and compared with that of control positive. The l-NAME (15 mg/kg, i.v.) was used as reference standard. Plasma protein extravasation was measured using fluorescein as marker and was measured using a Perkin-Elmer LS-30 luminescence spectrometer. Expression of iNOS in the spleen after intravenous injection produced PPE into the dura mater in control positive group was significantly (P<0.01) reduced to 1.553±0.02499 and 1.398±0.01887 by preventive treatment with ANE at the dose of 250 and 500 mg/kg, orally, respectively. The extravasation produced by topical GTN due to expression of iNOS in dural macrophages was also reduced to 1.555±0.03384 and 1.425±0.01204 by preventive treatment with ANE at the dose of 250 and 500 mg/kg, orally, respectively. While ANE do not showed any significant results in curative treatment in both the models of i.v. and topical GTN application. These findings collectively indicate that the extract exhibited significant inhibition of iNOS, which may be the probable mechanism for its anti-migraine activity, providing evidence, at least in part, for its folkloric use.

Comments on ‘Calcitonin gene-related peptide (CGRP) levels during glyceryl trinitrate (GTN)-induced headache in healthy volunteers’

The role of nitric oxide (NO) in migraine has been studied in the experimental glyceryl trinitrate (GTN)-infusion headache model. We hypothesized that GTN-induced headache may activate the trigeminovascular system and be associated with increased levels of sensory neuropeptides, including calcitonin gene-related peptide (CGRP). CGRP, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and somatostatin plasma levels were measured before and after placebo/sumatriptan injection and during GTN-induced headache. Following a double-blind randomized cross-over design, 10 healthy volunteers received subcutaneous sumatriptan 6 mg or placebo. This was succeeded by 20 min of GTN (0.12 mu g kg(-1) min(-1)) infusion. At baseline no subject reported headache (using verbal rating scale from 0 to 10) and the jugular CGRP-like immunoreactivity (-LI) level was 18.6 +/- 2.5 pmol/l. After a 20-min intravenous infusion of GTN 0.12 mu g kg(-1) min(-1), median peak headache intensity was 4 (range 2-6) (P 0.05). There were no changes in VIP-, NPY- or somatostatin-LI. In conclusion, the NO donor GTN appears not to induce headache via immediate CGRP release. (Less)

Oral nitric-oxide donor glyceryl-trinitrate induces sensitization in spinal cord pain processing in migraineurs: A double-blind, placebo-controlled, cross-over study

Nitric-oxide donor glyceryl-trinitrate (GTN) modulates cerebral and spinal regions that are involved in migraine and pain processing. We hypothesized that in migraineurs, the susceptibility to develop a migraine attack after GTN administration should parallel with an high sensitivity to GTN-induced change in the pain processing at spinal level. We used the temporal summation threshold (TST) of the lower limb nociceptive withdrawal reflex (NWR) and the related pain sensation to study in parallel the time-course of the effect of the GTN administration on the pain processing at spinal level in migraine and healthy subjects. Twenty-eight (21 F; 7 M; mean age 34.2 ± 8.2) migraine and 15 (11 F; 4 M; mean age 35.9 ± 8.9) healthy subjects were recruited in a double-blind, placebo-controlled, cross-over trial. Neurophysiological examinations were carried out before (baseline) and 30′, 60′, 120′, 180′ and 240′ after GTN (0.9 mg sublingual) or placebo administration during two different sessions. In migraineurs, GTN administration was associated to a significant facilitation in temporal summation of pain (reduced TST and increased painful sensation) 60′, 120′ and 180′ after drug intake when compared to baseline, to placebo condition and to controls after GTN intake. Furthermore, in migraineurs who developed migraine after GTN, a significant facilitation in temporal summation of pain was detected 60′, 120′ and 180′ after drug intake when compared to patients without clinical response. In migraineurs the susceptibility to develop migraine attack after GTN administration seems to be a specific trait of a subgroup of patients linked to a supersensitivity of the pain system to GTN.

Kynurenate Derivative Attenuates the Nitroglycerin‐Induced CamKIIα and CGRP Expression Changes

To examine the efficacy of L-kynurenine and a novel kynurenic acid derivative on the nitroglycerin-induced calmodulin-dependent protein kinase II alpha (CamKIIalpha) and calcitonin gene-related peptide (CGRP) expression changes in the rat caudal trigeminal nucleus. Systemic administration of the nitric oxide donor nitroglycerin can trigger an attack in migraineurs. In the rat, nitroglycerin activates second-order neurons in the caudal trigeminal nucleus, and increases expression of the CamKIIalpha and decreases that of the CGRP there. As glutamatergic mechanisms may be crucial in trigeminal pain processing, the aim of our study was to examine the effects of L-kynurenine, a metabolic precursor of the N-methyl D-aspartate receptor antagonist kynurenic acid, on the nitroglycerin-induced changes in CamKIIalpha and CGRP immunoreactivity. One hour before the nitroglycerin (10 mg/kg bodyweight, s.c.) injection, the animals were pretreated with L-kynurenine (300 mg/kg bodyweight, i.p.) or 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride (300 mg/kg bodyweight, i.p.), a novel kynurenic acid derivative. Four hours later, the rats were perfused transcardially and the cervical spinal cord segments were removed for immunohistochemistry. Results.- L-kynurenine and 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride pretreatment attenuated the nitroglycerin-induced changes in CamKIIalpha and CGRP immunoreactivity in the rat caudal trigeminal nucleus. These findings suggest a mechanism by which the inhibition of the excitatory amino acid receptors by kynurenic acid and its derivatives can alter trigeminal nociception.

CGRP receptor antagonist olcegepant (BIBN4096BS) does not prevent glyceryl trinitrate-induced migraine

There is a striking similarity between the migraine-provoking effect of the nitric oxide (NO) donor glyceryl trinitrate (GTN) and that of calcitonin gene-related peptide (CGRP). We tested the hypothesis that NO releases CGRP to cause the delayed migraine attack after GTN. In a double-blind-cross-over study, 13 migraine without aura (MO) patients were administered GTN 0.5 µg/kg/minute for 20 minutes and subsequently BIBN4096BS (olcegepant) 10 mg or placebo. Headache scores and development of MO were followed for 24 hours. MO developed in seven of 13 with olcegepant and in nine of 13 with placebo (p=0.68). The headache scores were similar after the two treatments (p=0.58). Thus CGRP receptor blockade did not prevent GTN-induced migraine. The present study indicates that NO does not induce migraine by liberating CGRP. The most likely explanation for our findings is that CGRP has its effect higher than NO in the cascade of events leading to MO attacks.

Hydralazine does not Ameliorate Nitric Oxide Resistance in Chronic Heart Failure

The A-HeFT trial demonstrated incremental survival with hydralazine/isosorbide dinitrate combination in African-American patients with chronic heart failure (CHF). It has been suggested that hydralazine might enhance nitric oxide (NO)-mediated effects of organic nitrates by decreasing superoxide (O (2) (-) ) formation, one of the factors inducing NO resistance. We evaluated whether hydralazine therapy potentiates nitrate-induced vasodilation and inhibition of platelet aggregation by ameliorating NO resistance. Patients (n = 14) with NYHA class II-III CHF were studied in a randomised, double-blind, placebo-controlled, crossover study of the effects of hydralazine therapy (25 mg b.d., for 1 week) on physiological responsiveness to glyceryl trinitrate (GTN). Vascular response to GTN was assessed via applanation tonometry, as change in augmentation index (AIx) over time. Platelet responsiveness to GTN and sodium nitroprusside (SNP) was determined, as inhibition of ADP-induced platelet aggregation. O (2) (-) release was evaluated during aggregation via lucigenin-derived chemiluminescence. Platelet responsiveness to the NO donors GTN and SNP was impaired, denoting the presence of severe NO resistance. Hydralazine therapy decreased systolic blood pressure by 6.8 +/- 10.5 (S.D.) mmHg (p = 0.02), and caused a reduction in AIx by 15 +/- 24% (p = 0.03). However, there were no significant changes in platelet aggregability and associated O (2) (-) release, or in platelet or vascular responses to NO donor. The results of the present study do not support the assumption that hydralazine could be viewed as a "NO enhancer"; there is no evidence of attenuation of NO resistance by hydralazine treatment.

Sildenafil and glyceryl trinitrate reduce tactile allodynia in streptozotocin-injected rats

The possible antiallodynic effect of phosphodiesterase 5 inhibitor sildenafil and nitric oxide donor glyceryl trinitrate as well as the changes in phosphodiesterase 5A2 mRNA expression in dorsal root ganglion and spinal cord of allodynic diabetic rats was assessed. Diabetes was induced by streptozotocin (50mg/kg, i.p.) in male Wistar rats. Streptozotocin injection produced hyperlglycemia, polydipsia, polyphagia and polyuria as well as long-term tactile allodynia (12 weeks) and a reduction of phosphodiesterase 5A2 mRNA expression in spinal cord of diabetic rats. Systemic administration of sildenafil (1-5.6 mg/kg, i.p.) reduced tactile allodynia in a dose-dependent manner in diabetic rats. Likewise, glyceryl trinitrate patches (0.2mg/h) also reduced tactile allodynia in diabetic rats. Moreover, both drugs reversed streptozotocin-induced phosphodiesterase 5A2 mRNA expression reduction. Our results indicate that glyceryl trinitrate and sildenafil reduce tactile allodynia in diabetic rats suggesting that nitric oxide and cyclic GMP supply is an important step in their mechanism of action of these drugs in diabetic animals. Data suggest that nitric oxide donors (as glyceryl trinitrate) and drugs which increase cyclic GMP levels (as sildenafil) could have a role in the pharmacotherapy of tactile allodynia in diabetic patients.

Nitric Oxide is Negatively Correlated to Pain during Acute Inflammation

BackgroundThe role that nitric oxide (NO) plays in modulating pain in the periphery is unclear. We show here, the results of two independent clinical studies (microdialysis and gene expression studies) and a pilot dose finding study (glyceryl trinitrate study), to study the role of NO in the early phase of acute inflammatory pain following oral surgery. The effect of ketorolac on NO production and nitric oxide synthase (NOS) gene expression was also studied.ResultsMicrodialysis samples showed significantly higher levels of NO at the first 100 min compared to the last 80 minutes in the placebo treated group. In the ketorolac group, on the other hand, NO levels gradually decreased over the first 60 min but were similar to placebo over the later 100-180 min, with no significant change in NO level over time. The levels of NO were negatively correlated to pain intensity scores. Local infusion of the NO donor glyceryl trinitrate at the site of surgery, showed a small analgesic effect that did not reach statistical significance in the sample size used. While the gene expression of iNOS and eNOS were not up-regulated, 3 hours after surgery, nNOS was downregulated in both treatment groups and eNOS gene expression was significantly lower in the ketorolac group compared to the placebo group. Further, there was a positive correlation between the change in gene expression of nNOS and eNOS in the placebo goup but not in the ketorolac group.ConclusionWe suggest that at this early stage of inflammatory pain in man, NO is analgesic in the periphery. Further, ketorolac down-regulates eNOS gene expression.

Modulatory effects of probenecid on the nitroglycerin-induced changes in the rat caudal trigeminal nucleus

Four hours after systemic administration of the nitric oxide donor nitroglycerin (10 mg/kg bodyweight, s.c.), the neurons of the rat caudal trigeminal nucleus are activated, the area covered by calcitonin gene-related peptide (CGRP)-immunoreactive fibres is decreased and the neuronal nitric oxide synthase (nNOS)- and the calmodulin-dependent protein kinase II alpha (CamKIIα)-immunopositive neurons in the same area are increased. Probenecid is a non-selective inhibitor of multidrug-resistance associated proteins and organic anion transporters thus it can modulate the transport functions in the central nervous system influencing nociception. Accordingly, the aim of the present experiments was to examine the effects of probenecid administration on the nitroglycerin-induced expressions of nNOS, CamKIIα and CGRP in the rat caudal trigeminal nucleus. Probenecid (200 mg/kg bodyweight, i.p.) pretreatment proved to mitigate the nitroglycerin-induced changes in expression in the rat caudal trigeminal nucleus. The data suggest that the changes caused by nitroglycerin in the expressions of CGRP, nNOS and CamKIIα can be influenced by probenecid modulating the inflammatory functions in the nervous system. These data may be of relevance for the pathogenesis of migraine headache.

Calcitonin gene-related peptide (CGRP) levels during glyceryl trinitrate (GTN)-induced headache in healthy volunteers

The role of nitric oxide (NO) in migraine has been studied in the experimental glyceryl trinitrate (GTN)-infusion headache model. We hypothesized that GTN-induced headache may activate the trigeminovascular system and be associated with increased levels of sensory neuropeptides, including calcitonin gene-related peptide (CGRP). CGRP, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and somatostatin plasma levels were measured before and after placebo/sumatriptan injection and during GTN-induced headache. Following a double-blind randomized cross-over design, 10 healthy volunteers received subcutaneous sumatriptan 6 mg or placebo. This was succeeded by 20 min of GTN (0.12 µg kg(-1) min(-1)) infusion. At baseline no subject reported headache (using verbal rating scale from 0 to 10) and the jugular CGRP-like immunoreactivity (-LI) level was 18.6 ± 2.5 pmol/l. After a 20-min intravenous infusion of GTN 0.12 µg kg(-1) min(-1), median peak headache intensity was 4 (range 2-6) (P < 0.05), while jugular CGRP-LI levels were unchanged (19.0 ± 2.8 pmol/l; P > 0.05). There were no changes in VIP-, NPY- or somatostatin-LI. In conclusion, the NO donor GTN appears not to induce headache via immediate CGRP release.

EFFECTS OF 1400W AND/OR NITROGLYCERIN ON RENAL OXYGENATION AND KIDNEY FUNCTION DURING ENDOTOXAEMIA IN ANAESTHETIZED RATS

1. The pathogenesis of acute renal failure (ARF) in sepsis is multifactorial. The role of nitric oxide (NO) in septic ARF has been a source of controversy. We hypothesized that endotoxaemia-induced exacerbation of inducible nitric oxide synthase (iNOS)-related NO release impairs renal oxygenation and contributes to ARF in anaesthetized rats. 2. In the present study, rats received lipopolysaccharide (2.5 mg/kg) for 30 min. Two hours later, fluid resuscitation was started (HES130; 5 mL/kg per h after a 5 mL/kg bolus) supplemented either by the NO donor nitroglycerin (NTG; 0.5 µg/kg per min after a 2 µg/kg bolus), the selective iNOS inhibitor 1400 W (3 mg/kg per h after a 3 mg/kg bolus) or both. Systemic haemodynamics and renal microvascular Po2 (µPo(2)) were recorded continuously. Furthermore, creatinine clearance, plasma NO(x) (nitrate + nitrite + S-nitrosothiols) levels and the expression of iNOS mRNA were measured. 3. Endotoxaemia reduced renal blood flow, decreased mean arterial pressure, resulted in anuria and was associated with an increase in plasma NO(x) levels and renal iNOS expression. Renal µPo2 deteriorated gradually during endotoxaemia and there was a significant decrease in renal O(2) delivery and consumption. Manipulation of NO levels had no beneficial effect on systemic haemodynamics, renal µPo(2) or creatinine clearance over standard fluid resuscitation. The application of 1400 W+NTG significantly reduced plasma NO(x) levels compared with fluid resuscitation and NTG alone. 4. Neither iNOS inhibition, NO donation nor a combination of both showed beneficial effects on systemic haemodynamics, renal oxygenation and renal function compared with fluid resuscitation alone. Our results question the proposed key role of NO in the pathogenesis of septic ARF in rats.

L-kynurenine combined with probenecid and the novel synthetic kynurenic acid derivative attenuate nitroglycerin-induced nNOS in the rat caudal trigeminal nucleus

Systemic administration of the nitric oxide (NO) donor nitroglycerin (NTG) triggers a delayed attack without aura in many migraineurs, but not in healthy volunteers. In rats, 4 h after the systemic administration of NTG (10 mg/kg bw, s.c.), the neurons of the caudal trigeminal nucleus (TNC) are activated and the expression of neuronal NO synthase (nNOS) in the same area is increased suggesting a self-amplifying process in the trigeminal system, which seems to be crucial in migraine pathogenesis. Kynurenic acid (KYNA) and its analogues may exert modulatory effects in many neuropathological conditions, probably via N-methyl- d-aspartate (NMDA) antagonism. Since NMDA receptors play a crucial role in trigeminal pain processing, the aim of our experiments was to compare the effects of l-kynurenine ( l-KYN) combined with probenecid (PROB) or with 2-(2- N, N-dimethylaminoethylamine-1-carbonyl)-1 H-quinolin-4-one hydrochloride alone, a newly synthetized KYNA derivative, on the NTG-induced nNOS expression in the rat TNC. Pretreatment with l-KYN (300 mg/kg bw, i.p.) together with PROB (200 mg/kg bw, i.p.) and KYNA derivative (300 mg/kg bw, i.p.) attenuated the NTG-induced nNOS expression in the rat TNC. Our data suggest that the stimulating effect of NTG, and thus of NO, on the expression of nNOS might be modulated by increasing the KYNA level in the brain, probably through the NMDA receptors. These data could help promote a better understanding of the pathogenesis of headaches and the action of antimigraine drugs.