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Abstract

When administered to migraine patients, nitroglycerin induces a spontaneous-like migraine attack, with a latency of several hours. Nitroglycerin acts directly and/or indirectly on the central nervous system, through the release of nitric oxide (NO). Systemic administration of the drug to the rat causes neuronal activation in selected subcortical areas, particularly in monoaminergic nuclei of the brainstem. In this study, we sought to investigate whether this activation correlates with changes in monoaminergic neurotransmission. For this purpose, we evaluated the tissue levels of catecholamines and serotonin in the hypothalamus, mesencephalon, pons and medulla of rats treated with systemic nitroglycerin or vehicle, at different time points (1, 2 and 4 h). We also evaluated the peripheral sympathetic response to the drug by measuring the concentrations of plasma catecholamines. Nitroglycerin caused an early (1 h) increase in cerebral (pons) and plasma levels of norepinephrine, followed by a delayed (4 h) decrease in medullary and pontine levels of serotonin. The initial noradrenergic activation may reflect the autonomic response to the rapid cardiovascular effects of the drug, while the delayed response may result from the interaction of nitroglycerin- released NO and 5-HT in central areas devoted to the modulation of nociception. These data might therefore help to clarify the mechanisms underlying the delayed migraine attack observed in migraine sufferers after systemic administration of nitroglycerin.

Keywords

nitroglycerin nitric oxide serotonin catecholamines nociception

Introduction

Nitroglycerin is a powerful vasodilating agent, widely used for the treatment of angina pectoris – which acts by releasing nitric oxide (NO) (1–3). A less known, but well-documented, clinical application of nitroglycerin regards the migraine field: its systemic administration to pain-free migraine patients induces a spontaneous-like migraine attack, with a latency of several hours (4–6). Such delayed response, which is absent in normal subjects, has been initially related to the vasodilatory effect of the drug. However, the long latency that characterizes this response conflicts with the very short plasma half-life of the drug. Different hypotheses have been proposed over the years and, although an exhaustive explanation has not been provided yet, it is likely that the NO released from the drug plays a direct pro-nociceptive action (7–9).

Nitroglycerin may act both directly and indirectly on the central nervous system (for review see 10). We have previously shown that systemic administration of nitroglycerin causes massive expression of Fos protein – a nonspecific marker of neuronal activation (11, 12) – in selected areas of the rat brain (13–15). The time-course of Fos expression is not the same for all the brain areas. In fact, those nuclei that are mainly involved in the central control of blood pressure (i.e. ventrolateral medulla, nucleus tractus solitarius and parabrachial nucleus) show maximal Fos expression two hours after nitroglycerin injection. In other areas involved, for example, in the processing of sensory-nociceptive stimuli (nucleus trigeminalis caudalis), integration of autonomic responses (locus coeruleus, paraventricular nucleus of the hypothalamus) or both functions (periaqueductal grey and central nucleus of the amygdala), maximal Fos expression is reached 4 h after nitroglycerin injection. Therefore, in addition to the reflex responses elicited by the well-known cardiovascular effects of the drug, other mechanisms may be involved in the delayed neuronal activation induced by nitroglycerin (10).

Due to its highly hydrophobic nature, nitroglycerin crosses the blood brain barrier and accumulates in the brain. The drug may therefore exert a direct, local effect on the central nervous system (16, 17). Systemic administration of nitroglycerin provokes a long-lasting (almost 2-h long) release of NO from the surface of the rat brain (18) and increases NO concentrations in the posterior hypothalamus (19). Whether the released NO derives exclusively from the drug, or a drug-related endogenous synthesis occurs, is presently unknown. Inhibition of NO synthase, the enzyme required for the synthesis of endogenous NO, prevents nitroglycerin-induced neuronal activation (20). Therefore, it is likely that both exogenous and endogenous NO are involved in the nitroglycerin-induced neuronal activation.

NO is involved in the central and peripheral control of pain perception (21–23). In animals, NO mediates centrally induced thermal hyperalgesia to noxious heat stimuli applied peripherally (24, 25). NO is also involved in the development of secondary hyperalgesia (26), and in the cyclic guanosine monophosphate (cGMP)-dependent hyperalgesia (27). In humans, the role of NO in nociceptive modulation is largely unknown, but increasing data are being gathered on the role of this molecule, or its metabolites, in chronic painful conditions, including migraine (8, 28–31).

Most of the brainstem neurones expressing Fos in response to nitroglycerin use monoamines (i.e. norepinephrine, epinephrine and serotonin), as neurotransmitters (15). Monoamines may therefore play a substantial role in the neuronal effects induced by the drug. This fact is of particular relevance in the migraine field: brain monoamines – particularly serotonin (5-HT) – play a major role in the pathogenesis of migraine (32–34) and NO has been recently indicated as a fundamental mediator of migraine attacks (35).

The present study was undertaken to investigate the effect of nitroglycerin on central monoaminergic neurotransmission in rats. For this purpose, we evaluated the changes in tissue levels of catecholamines – norepinephrine (NE), epinephrine (E) and dopamine (DA) – and 5-HT in the hypothalamus, mesencephalon, pons and medulla of rats treated with systemic nitroglycerin. We also evaluated the peripheral sympathetic response to the drug by measuring, at the same time points, the concentrations of plasma catecholamines.

Materials and methods

Experiments were performed on male, Sprague–Dawley adult rats (weight 250–320 g). Animals were housed 2 per cage, at 22°C on a 12-h light/dark cycle, with food and water ad libitum for at least 10 days before being used for the experiments.

Assay of brain and plasma monoamine levels

Animals were divided into two groups. Rats in group 1 (n = 21) received an i.p. injection of nitroglycerin (10 mg/kg) dissolved in alcohol, propylene glycol and water. Rats in group 2 (n = 21) received the vehicle alone and served as controls. The dose of nitroglycerin was chosen according to the results of our previous studies (13, 14).

Animals were killed by decapitation at different time intervals (1, 2 and 4 h after injection). After decapitation, brains were rapidly removed and dissected, over dry ice, into four regions: hypothalamus, mesencephalon, pons and medulla. The micropunches technique was not used because it would have required an additional freeze-thawing cycle. This would have rendered the detection of transmitters whose concentrations are usually low, such as E or DA, extremely critical. Concomitantly, truncal blood was collected and transferred to plastic tubes containing lithium heparin, as anticoagulant. Blood samples were immediately centrifuged at 1500 g, at 4°C, to obtain plasma. Tissue and plasma samples were stored at −80°C for the analysis of NE, E, DA and 5-HT levels, which was carried out – within one week – using high performance liquid chromatography (HPLC).

The day of the analysis, brain samples were homogenized using ultrasounds (Ultrasonic 2000, Artek, Farmingdale, NY, USA) in 3 ml of an ice-cold solution containing 90% of 0.1 N perchloric acid and 10% of 0.4 mm sodium metabisulfite. Homogenates were centrifuged at 17 000× g for 15 min at 4°C. Supernatants were collected and split into separate aliquots for catecholamine or 5-HT analysis.

HPLC assays

The chromatographic system consisted of a pump (System Gold 116, Beckman, San Ramon, CA, USA) equipped with a C₁₈, reversed-phase, 70 × 4.6 mm (I.D.), 3 µm column (Ultrasphere XL ODS, Beckman), fitted with a 5 × 4.6 mm precolumn (Ultrasphere XL ODS, Beckman). The pump was connected to an auto-sampler (AS 100, Bio-Rad, Richmond, CA, USA). The detection device was an electrochemical detector (Coulochem 5100A, ESA Inc., Bedford, MA, USA), with a dual electrode analytical cell, connected to a dedicated PC equipped with chromatography software (Value Chrom, Bio-Rad, Hercules, CA, USA). Potentials of the analytical electrodes of the detector were set at −200 mV (E₁) and +200 mV (E₂) for catecholamine determination. For 5-HT measurement, potentials of E₁ and E₂ were set at +70 and +300 mV, respectively. In both cases, the signal generated at the second electrode (E₂) was considered.

The mobile phase used for catecholamine assay consisted of a 50-mm potassium phosphate buffer, pH 3.4, containing 0.3 mm Na₂EDTA and 0.7 mm sodium dodecylsulphate. The mobile phase used for 5-HT analysis consisted of a 0.1-m sodium acetate buffer, pH 3.5, containing 0.3 mm Na₂EDTA and 0.6 mm octylsulphate.

Before the chromatographic analysis, brain and plasma catecholamines were extracted onto activated alumina and subsequently eluted in perchloric acid, as described previously (36). Fifty µl of the final eluate were then injected into the HPLC system. As for 5-HT, 50 µl of homogenate supernatant were directly injected into the HPLC system.

Protein assay

In order to express monoamine results as quantity of neurotransmitter per mg of brain proteins, protein content of brain samples was estimated using a commercially available kit (Pierce, Rockford, IL, USA) after dissolving the perchlorate precipitates in 0.5 N NaOH.

Statistics

Effects of treatments and time in the various groups of animals were analysed using the analysis of variance (anova). The Fisher's post hoc t-test was used to analyse differences between the two groups of animals (nitroglycerin vs. vehicle) at each time point. The level of statistical significance was set at P< 0.05.

Results

Brain catecholamines and 5-HT

Tissue levels of E bordered on the detection limit of our method in all the areas examined. Thus, only NE, DA and 5-HT levels are being presented (Table 1).

Table 1

Tissue levels of norepinephrine, dopamine and serotonin in distinct subcortical areas of rats killed 1, 2 or 4 h after systemic injection of nitroglycerin 10 mg/kg vs. controls

Subcortical area/	Norepinephrine (ng/mg protein)		Dopamine (ng/mg protein)		Serotonin (ng/mg protein)
Time (hours)	Nitroglycerin	Control	Nitroglycerin	Control	Nitroglycerin	Control
Hypothalamus
1	18.3 ± 2.3	22.3 ± 2.9	3.9 ± 1.0	3.7 ± 0.6	3.2 ± 1.1	5.0 ± 0.8
2	17.1 ± 3.3	19.4 ± 2.9	3.3 ± 0.8	3.0 ± 0.5	2.2 ± 0.7	1.8 ± 0.2
4	21.1 ± 3.0	17.4 ± 2.6	4.6 ± 0.6	3.2 ± 0.5	3.9 ± 0.9	5.6 ± 1.1
Mesencephalon
1	5.3 ± 0.6	5.0 ± 0.5	2.2 ± 0.4	1.9 ± 0.3	8.9 ± 2.4	7.5 ± 1.2
2	4.8 ± 0.5	6.2 ± 0.6	1.2 ± 0.2	2.1 ± 0.5	5.7 ± 0.5	6.0 ± 1.0
4	4.9 ± 0.8	5.0 ± 0.5	1.9 ± 0.4	1.4 ± 0.4	4.8 ± 1.0	4.5 ± 0.5
Pons
1	7.0 ± 0.9∗	4.8 ± 0.3	0.8 ± 0.2	0.4 ± 0.1	6.6 ± 1.9	6.9 ± 0.6
2	6.4 ± 0.5	5.8 ± 0.2	1.0 ± 0.1	0.7 ± 0.1	7.5 ± 1.5	6.2 ± 0.8
4	6.2 ± 1.3	4.4 ± 0.7	0.6 ± 0.1	0.5 ± 0.1	4.2 ± 0.3∗∗	6.8 ± 1.1
Medulla
1	3.9 ± 0.5	4.6 ± 0.8	0.6 ± 0.2	0.5 ± 0.1	3.7 ± 0.8	4.1 ± 0.3
2	6.1 ± 0.7	5.5 ± 1.1	0.6 ± 0.1	0.6 ± 0.1	4.8 ± 0.9	4.5 ± 0.6
4	5.5 ± 1.3	4.1 ± 0.7	0.6 ± 0.2	0.4 ± 0.1	3.2 ± 0.3∗∗∗	5.2 ± 0.8

Values are the mean±sem results obtained from groups of 7 animals for each time point. anova for norepinephrine: F=2.371, P< 0.05; dopamine: nonsignificant; serotonin: F=2.468, P< 0.04. Fisher's t test:

^∗ P = 0.039 vs. vehicle,

^∗∗ P = 0.042,

^∗∗∗ P = 0.037.

Nitroglycerin-injected rats showed a significant increase in NE levels in the pons at one hour postinjection (m ± sem: 31.4 ± 9.2%), compared to the rats that received vehicle. As for DA levels, no significant differences were found between the two groups of animals, at any of the time points considered.

Rats that received nitroglycerin showed significantly decreased levels of 5-HT, compared to control rats, in medullary (m ± sem: −38.5 ± 9.1%) and pontine regions (m ± sem: −38.2 ± 8.7%), four hours after the injection.

Plasma catecholamines

Changes in plasma concentrations of NE, E and DA are shown in Fig. 1. Rats that received nitroglycerin showed significantly higher levels of NE and DA, compared to control rats, at one hour postinjection. After two hours, nitroglycerin-treated rats showed significantly lower levels of both NE and E with respect to controls. They also showed a reduction in DA levels that, however, did not reach statistical significance. NE, E and DA concentrations in rats treated with nitroglycerin returned to values similar to controls at the fourth hour.

Figure 1

Plasma levels of (a) norepinephrine, (b) epinephrine and (c) dopamine 1, 2 and 4 h after systemic administration of ▪ nitroglycerin (10 mg/kg b.w.; n = 7) or □ vehicle (n = 7). Values are expressed as mean±sem. anova for norepinephrine, F=3.473, P< 0.05; epinephrine, F=4.578, P< 0.03; dopamine, F=3.398, P< 0.05. Fisher's t test: ∗P < 0.05; ∗∗P<0.02.

Behavioural response

Rat behaviour was similar to that observed in previous studies (13, 20). In particular, animals injected with nitroglycerin showed a marked decrease in motor behaviour up to four hours after the drug administration. They lay still, without attempting to drink or eat for the entire period of observation. No licking or scratching of the injection site was observed. Vehicle-injected animals behaved normally.

Discussion

We have previously shown that systemic administration of nitroglycerin to rats elicits peculiar neuronal activation in selected subcortical areas. The drug induces marked Fos expression in medullary, pontine, mesencephalic and hypothalamic nuclei, with a bimodal pattern. Nuclei involved in cardiovascular control show early activation. This phase is followed by a long-lasting activation of areas involved in the control of sensory-nociceptive, integrative and neuroendocrine functions or nociceptive and autonomic activities (10, 13–15, 20). The majority of the neurones of these nuclei use catecholamines or serotonin as neurotransmitters (15). We therefore investigated whether nitroglycerin-induced neuronal activation is paralleled by specific changes in brain monoamines.

Brain monoamine levels

Nitroglycerin induced discrete changes in the cerebral levels of monoamines. The changes followed a bimodal pattern, which was reminiscent of the Fos response to the drug. One hour postinjection, nitroglycerin induced a significant increase of NE levels in the pons. Such increase was followed, at the fourth hour, by a decrease in the tissue levels of 5-HT in the pons and medulla.

Different mechanisms are likely to underlie the opposite changes that affected brain levels of NE or 5-HT in response to nitroglycerin. The drug causes peripheral vasodilatation – and consequent hypotension – via the intravascular release of NO (1–3). It is known, from the literature, that the dose of nitroglycerin used in our study induces, in the rat, a moderate hypotension (− 37 ± 2%) and that such effect recedes within 75 min (37). The drug may also elicit a direct hypotensive response by acting locally, at the brainstem level (38). The NE changes observed at the first hour postinjection might therefore reflect an early activation of pontine, noradrenergic nuclei involved in the antihypotensive response to the drug, leading to increased NE synthesis. This is also in agreement with previous in vitro studies, showing that nitroglycerin induces the synthesis of NE in medulla-pons slices, as well as in striatal and atrial preparations (38, 39).

Conversely, the late reduction (4 h postnitroglycerin administration) in 5-HT levels observed in pontine and medullary regions of nitroglycerin-treated rats cannot be simply ascribed to changes in blood pressure. In fact, as mentioned above, nitroglycerin-induced hypotension lasts less than 2 h (37) and we did not detect significant changes in 5-HT levels until the fourth hour. An alternative explanation for the 5-HT reduction must be therefore considered. Such explanation may encompass the pivotal role that 5-HT plays in the central modulation of pain perception and, particularly, in the pathogenesis of migraine (32–34). Noxious stimuli decrease the content of brain 5-HT (40) or the release of the transmitter from the raphe magnus nucleus (41); conversely, analgesic substances – such as morphine or nonsteroid anti-inflammatory drugs – increase 5-HT levels in the rat brain (42–44). In migraine patients, systemic administration of nitroglycerin induces a spontaneous-like migraine attack, with a latency of several hours (4–6). Although the exact mechanism underlying this response remains elusive, Iversen and Olesen have suggested that the nitroglycerin-induced attack depends on the direct nociceptive action of the NO released from the drug (9). NO interacts with 5-HT in the modulation of antinociception elicited, for example, by electrical stimulation of the periaqueductal grey (45). In addition, recent studies show that NO modulates the release of 5-HT both in the hypothalamus and the locus coeruleus of rats (46, 47). Therefore, a possible explanation is that, in our experimental setting, the NO formed from nitroglycerin elicited a noxious stimulus, with consequent activation of brainstem areas electively involved in the processing of nociceptive information modulated by 5-HT, such as the periaqueductal grey and nucleus trigeminalis caudalis. Indeed, we have previously shown that neurones of the periaqueductal grey and nucleus trigeminalis caudalis are activated four hours after systemic injection of nitroglycerin (13). This hypothesis is further supported by preliminary findings that show how nitroglycerin induces a hyperalgesic state in rats exposed to the formalin test (Tassorelli et al., unpublished observations). It is difficult to compare the 5-HT changes of combined brain areas with the Fos expression detected in discrete nuclei. Nevertheless, the fact that the decrease in 5-HT found in pontine and medullary regions occurred at the same time point as the maximal Fos expression in the periaqueductal grey and nucleus trigeminalis caudalis is intriguing (13).

Plasma catecholamine levels

Nitroglycerin induced, at the first hour, consistent increases in plasma levels of NE and its direct precursor, DA. This finding supports the existence of an early phase of noradrenergic activation. The increase in NE and DA levels may reflect the sympathetic compensatory response to the drug-induced hypotension. Indeed, Adams et al. (48) have previously shown that nitroglycerin-induced hypotension increases plasma levels of NE in humans. This is further supported by the fact that E, which does not respond to hypotension (49, 50), did not increase in response to the drug.

In the nitroglycerin-treated group, the initial increase in plasma NE and DA was followed by a significant reduction of NE and E levels at the second hour. Dopamine levels decreased as well, although the difference between treated rats and controls did not reach statistical significance. At the fourth hour, similar levels of plasma catecholamines were found in the two groups of animals. This pattern of catecholamine release may reflect the ‘exhaustion’ of the secretory pool of peripheral catecholamines, secondary to the initial release stimulated by nitroglycerin.

Whether the changes in plasma catecholamines depended solely on the hypotension induced by nitroglycerin, or a direct effect of drug-released NO was also involved, is unclear.

In conclusion, systemic administration of nitroglycerin to rats caused a biphasic pattern of changes in brain monoamines. The early increase in pontine (and plasma) levels of catecholamines is likely to reflect the central autonomic response to the rapid cardiovascular effects of the drug. Conversely, the delayed decrease in medul-lary and pontine 5-HT may result from the interaction between nitroglycerin-released NO and 5-HT in central areas devoted to the modulation of nociception. These data might help to better understand the mechanisms underlying the delayed migraine attack that occurs in migraine sufferers after systemic administration of nitroglycerin.

Footnotes

Acknowledgements

The authors wish to thank Roberta Ricotti, Deborah Rivellini and Andrea Quadrelli for their expert technical support.

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Nitroglycerin-Induced Activation of Monoaminergic Transmission in the Rat

Abstract

Keywords

Introduction

Materials and methods

Assay of brain and plasma monoamine levels

HPLC assays

Protein assay

Statistics

Results

Brain catecholamines and 5-HT

Plasma catecholamines

Behavioural response

Discussion

Brain monoamine levels

Plasma catecholamine levels

Footnotes

Acknowledgements

References