Lithium Inhibits Substance P and Vasoactive Intestinal Peptide-Induced Relaxations on Isolated Porcine Ophthalmic Artery
Maurice B. Vincent, M.D.
Department of Neurology, Trondheim University Hospital, N-7006, Trondheim, Norway. Permanent address: Department of Neurology, University Hospital, Federal University of Rio de Janeiro, 21949 Rio de Janeiro, Brazil. Reprint requests to: M.B. Vincent, M.D., Department of Neurology, Trondheim University, Hospital, N-7006, Trondheim, Norway Accepted for Publication April 18, 1992 SYNOPSIS
Lithium is currently a major drug used as a treatment for effective disorders and cluster headache, among other conditions. Its mechanism of action remains unknown. The trigeminovascular system may be involved in the pathophysiology of cluster headache and related diseases by liberating neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) in the eye-forehead region. The objective of this study was to investigate whether or not a low concentration of lithium may interfere with peptidergic neurotransmission at this level. Vasoactive intestinal peptide (VIP), SP, CGRP, capsaicin, and SP+CGRP concentration-response relaxation curves were obtained in the presence and absence of 2 x 10-4M lithium in isolated porcine ophthalmic arteries. Lithium inhibited the SP and VIP, but not the CGRP responses. Capsaicin- (a neurotoxin causing release of stored sensory neuropeptides) induced relaxations were partially inhibited by lithium. it is concluded that lithium may interfere with SP and VIP-induced relaxation. If SP and VIP are of pathogenic significance in cluster headache, lithium may possibly work by counteracting unwanted effects of such peptides. Key Words: Cluster headache, neuropeptides, lithium, porcine ophthalmic artery. Abbreviations: SP, substance P, CGRP, calcitonin gene-related peptide. VIP, vasoactive intestinal peptide. (Headache 1992; 32:335-339) INTRODUCTION Trigeminal sensory C fibres are known to contain neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) as neurotransmitters1. There is also evidence suggesting that such fibres do not just carry pain information to the central nervous system, but may also liberate vasoactive neuropeptides at the periphery.2,3 This means that the sensory component of the trigeminal nerve together with the blood vessels and other structures located within the trigeminal sensory innervation areas may constitute an important anatomical substrate for headache.4 By releasing vasoactive neuropeptides at the periphery, trigeminal sensory fibres may induce a neurogenic inflammation (redness, miosis and increase of the intraocular pressure), which is reminiscent of signs and symptoms of cluster headache.2,3 Indeed, vasoactive trigeminal neuropeptides have been implicated in the pathophysiology of headaches.4-7 CGRP, a 37-aminoacid peptide encoded by the calcitonin gene, is a potent vasodilator in different species and vascular beds, including human cerebral arteries, and is known to occur in trigeminal sensory fibres.8 The tachykinin undecapeptide SP is also a transmitter that has vasodilatory properties; it is present in sensory trigeminal fibres, where it coexists with CGRP.9-11 Vasoactive intestinal polypeptide (VIP) is one of the most widely distributed vasodilatory peptides. Although VIP immunoreactivity has been demonstrated in trigeminal sensory fibres, it is mainly a parasympathetic peptide, where it coexists with acetylcholine. VIP is also a potent vasodilator of human cerebral arteries.12 Capsaicin, the pungent agent in pepper, is a well known neurotoxin. It induces tachykinin and CGRP liberation from sensory C fibres, and has therefore been used as an important neuropharmacological tool.13,14 Capsaicin has been shown to induce biphasic responses in isolated arteries. At lower concentrations, it probably acts via sensory fibres peptide release, but also seems to have an intrinsic vasoactivity at higher concentrations.15 Since lithium was introduced for the treatment of bipolar disorders16 it has been considered one of the drugs of choice for the such conditions.17 It has also been tried in other diseases, both in psychiatry and internal medicine, such as movement disorders, schizo-affective disorders and hyperthyroidism, among others.18,19 Both chronic and nonchronic forms of cluster headache have been treated with lithium with some success.20-23 How
ever, the mechanism of action of lithium in cluster headache and psychiatric disorders remains unknown. Lithium may interfere with neurotransmission at the level of transmitters, receptors and second messengers, the effects possibly changing at different synaptic sites and over time.19 For instance, it has been suggested that the antidepressant, anti-manic or prophylactic effects of lithium could involve serotoninergic transmission.19 Lithium may also change the concentration of peptides in different regions of the rat brain.24 It has been shown that chronic administration of lithium increases SP-like immunoreactivity in the striatum, nucleus accumbens and frontal cortex, but not in the hypothalamus, hippocampus or brain stem.24.25 The vascular bed of the eye-forehead region may be of special interest concerning the putative implications of neuropeptides (such as SP, CGRP and VIP) in the pathophysiology of cluster headache and related disorders. Due to the possibility that lithium acts at the level of peptide neurotransmission, either pre- or post-junctionally, the objective of this study was to investigate the effects of lithium on dilatory neuropeptides and capsaicin-induced vasoactivity in porcine ophthalmic arteries in vitro. MATERIAL AND METHODS The entire orbital contents were removed from pigs (15-26 kg) immediately after a lethal dose of pentobarbital i.v., placed in iced buffer solution (NaCI 119, KCI 4.6, CaCI2 1.5, MgCI2 1.2, NaHCO3 10, NaH2PO4 1.2, glucose 11. Values in mM) and kept at 4°C for 15-17 h, or used immediately in the case of all capsaicin experiments. Retro-ocular segments of the ophthalmic artery were gently dissected with the aid of a stereomicroscope. Circular artery segments (1 to 3 mm) were mounted on two L-shaped steel holders (0.1 mm diameter), one of the holders being connected to a Grass FT03C force-displacement transducer. The holders were immersed in 5 ml buffer solution through which a 5% CO2, 95% O2 mixture was continuously bubbled. For recording the arterial constriction forces, the transducers were linked to an Analog Digital Instruments MacLab analog-digital convertor through a Transbridge TBM4 preamplifier (World Precision Instruments, Inc, New Haven CT, USA). The digitized data were analyzed by MacLab's Chart/4 software on an Apple Macintosh SE 1/40 computer. After the equilibration period (1-1.5 hour, 4-5 mN passive tension), the arteries were first contracted by 10-5M PGF2a and when a stable reaction was reached, some of the arterial segments were treated with 2 x 10-4M lithium chloride, a concentration within the same order of magnitude of plasma therapeutic levels following oral intake.19 Ten minutes after lithium addition, the relaxation provoked by SP, CGRP, VIP, capsaicin, and SP together with CGRP was studied and compared with similar experiments performed in lithium-free arteries. Consecutive segments of the same artery with and without lithium pre-treatment were tested in parallel. A new animal was used for each concentration-response curve in every series of experiments. Peptide-induced responses were not tested twice in the same arterial segment. Evidence for the integrity of the endothelium in this preparation was obtained by reproducible 10-6 M acetylcholine-induced dilations (data not shown). SP, VIP, CGRP, and capsaicin experiments. Concentration-response relaxation curves were obtained by adding the peptides or capsaicin cumulatively in logarithmically increasing concentrations. (SP: 10-10 to 10-5 M. CGRP and VIP: 10-10 to 10-7M. Capsaicin: 10-14 to 10-4M). The maximum relaxation obtained after each addition was considered. SP + CGRP experiments. For obtaining concentration-response relaxation curves induced by a combination of peptides, SP was added together with CGRP in equal molar concentrations (10-10 to 10-7M). The maximum effect induced by each simultaneous addition was considered. Drugs were added in 50 µl aliquots. Substance P (acetate salt), VIP (porcine, synthetic) and a-CGRP (human, synthetic), as well as all other drugs were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were diluted in 0.9% NaCI containing 10-3M ascorbic acid. Capsaicin was dissolved in 100% ethanol and diluted in 0.9% NaCI containing 10-3M ascorbic acid. Appropriate controls showed that these vehicle substances did not significantly affect the contractions at the dilutions used. Fresh dilutions were prepared daily from stock solutions stored at -70°C. The data are presented as the mean ± S.E.M. Statistical analysis was carried out with Student's t-test and values of p
Maurice B. Vincent, M.D.
Department of Neurology, Trondheim University Hospital, N-7006, Trondheim, Norway. Permanent address: Department of Neurology, University Hospital, Federal University of Rio de Janeiro, 21949 Rio de Janeiro, Brazil. Reprint requests to: M.B. Vincent, M.D., Department of Neurology, Trondheim University, Hospital, N-7006, Trondheim, Norway Accepted for Publication April 18, 1992 SYNOPSIS
Lithium is currently a major drug used as a treatment for effective disorders and cluster headache, among other conditions. Its mechanism of action remains unknown. The trigeminovascular system may be involved in the pathophysiology of cluster headache and related diseases by liberating neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) in the eye-forehead region. The objective of this study was to investigate whether or not a low concentration of lithium may interfere with peptidergic neurotransmission at this level. Vasoactive intestinal peptide (VIP), SP, CGRP, capsaicin, and SP+CGRP concentration-response relaxation curves were obtained in the presence and absence of 2 x 10-4M lithium in isolated porcine ophthalmic arteries. Lithium inhibited the SP and VIP, but not the CGRP responses. Capsaicin- (a neurotoxin causing release of stored sensory neuropeptides) induced relaxations were partially inhibited by lithium. it is concluded that lithium may interfere with SP and VIP-induced relaxation. If SP and VIP are of pathogenic significance in cluster headache, lithium may possibly work by counteracting unwanted effects of such peptides. Key Words: Cluster headache, neuropeptides, lithium, porcine ophthalmic artery. Abbreviations: SP, substance P, CGRP, calcitonin gene-related peptide. VIP, vasoactive intestinal peptide. (Headache 1992; 32:335-339) INTRODUCTION Trigeminal sensory C fibres are known to contain neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) as neurotransmitters1. There is also evidence suggesting that such fibres do not just carry pain information to the central nervous system, but may also liberate vasoactive neuropeptides at the periphery.2,3 This means that the sensory component of the trigeminal nerve together with the blood vessels and other structures located within the trigeminal sensory innervation areas may constitute an important anatomical substrate for headache.4 By releasing vasoactive neuropeptides at the periphery, trigeminal sensory fibres may induce a neurogenic inflammation (redness, miosis and increase of the intraocular pressure), which is reminiscent of signs and symptoms of cluster headache.2,3 Indeed, vasoactive trigeminal neuropeptides have been implicated in the pathophysiology of headaches.4-7 CGRP, a 37-aminoacid peptide encoded by the calcitonin gene, is a potent vasodilator in different species and vascular beds, including human cerebral arteries, and is known to occur in trigeminal sensory fibres.8 The tachykinin undecapeptide SP is also a transmitter that has vasodilatory properties; it is present in sensory trigeminal fibres, where it coexists with CGRP.9-11 Vasoactive intestinal polypeptide (VIP) is one of the most widely distributed vasodilatory peptides. Although VIP immunoreactivity has been demonstrated in trigeminal sensory fibres, it is mainly a parasympathetic peptide, where it coexists with acetylcholine. VIP is also a potent vasodilator of human cerebral arteries.12 Capsaicin, the pungent agent in pepper, is a well known neurotoxin. It induces tachykinin and CGRP liberation from sensory C fibres, and has therefore been used as an important neuropharmacological tool.13,14 Capsaicin has been shown to induce biphasic responses in isolated arteries. At lower concentrations, it probably acts via sensory fibres peptide release, but also seems to have an intrinsic vasoactivity at higher concentrations.15 Since lithium was introduced for the treatment of bipolar disorders16 it has been considered one of the drugs of choice for the such conditions.17 It has also been tried in other diseases, both in psychiatry and internal medicine, such as movement disorders, schizo-affective disorders and hyperthyroidism, among others.18,19 Both chronic and nonchronic forms of cluster headache have been treated with lithium with some success.20-23 How
ever, the mechanism of action of lithium in cluster headache and psychiatric disorders remains unknown. Lithium may interfere with neurotransmission at the level of transmitters, receptors and second messengers, the effects possibly changing at different synaptic sites and over time.19 For instance, it has been suggested that the antidepressant, anti-manic or prophylactic effects of lithium could involve serotoninergic transmission.19 Lithium may also change the concentration of peptides in different regions of the rat brain.24 It has been shown that chronic administration of lithium increases SP-like immunoreactivity in the striatum, nucleus accumbens and frontal cortex, but not in the hypothalamus, hippocampus or brain stem.24.25 The vascular bed of the eye-forehead region may be of special interest concerning the putative implications of neuropeptides (such as SP, CGRP and VIP) in the pathophysiology of cluster headache and related disorders. Due to the possibility that lithium acts at the level of peptide neurotransmission, either pre- or post-junctionally, the objective of this study was to investigate the effects of lithium on dilatory neuropeptides and capsaicin-induced vasoactivity in porcine ophthalmic arteries in vitro. MATERIAL AND METHODS The entire orbital contents were removed from pigs (15-26 kg) immediately after a lethal dose of pentobarbital i.v., placed in iced buffer solution (NaCI 119, KCI 4.6, CaCI2 1.5, MgCI2 1.2, NaHCO3 10, NaH2PO4 1.2, glucose 11. Values in mM) and kept at 4°C for 15-17 h, or used immediately in the case of all capsaicin experiments. Retro-ocular segments of the ophthalmic artery were gently dissected with the aid of a stereomicroscope. Circular artery segments (1 to 3 mm) were mounted on two L-shaped steel holders (0.1 mm diameter), one of the holders being connected to a Grass FT03C force-displacement transducer. The holders were immersed in 5 ml buffer solution through which a 5% CO2, 95% O2 mixture was continuously bubbled. For recording the arterial constriction forces, the transducers were linked to an Analog Digital Instruments MacLab analog-digital convertor through a Transbridge TBM4 preamplifier (World Precision Instruments, Inc, New Haven CT, USA). The digitized data were analyzed by MacLab's Chart/4 software on an Apple Macintosh SE 1/40 computer. After the equilibration period (1-1.5 hour, 4-5 mN passive tension), the arteries were first contracted by 10-5M PGF2a and when a stable reaction was reached, some of the arterial segments were treated with 2 x 10-4M lithium chloride, a concentration within the same order of magnitude of plasma therapeutic levels following oral intake.19 Ten minutes after lithium addition, the relaxation provoked by SP, CGRP, VIP, capsaicin, and SP together with CGRP was studied and compared with similar experiments performed in lithium-free arteries. Consecutive segments of the same artery with and without lithium pre-treatment were tested in parallel. A new animal was used for each concentration-response curve in every series of experiments. Peptide-induced responses were not tested twice in the same arterial segment. Evidence for the integrity of the endothelium in this preparation was obtained by reproducible 10-6 M acetylcholine-induced dilations (data not shown). SP, VIP, CGRP, and capsaicin experiments. Concentration-response relaxation curves were obtained by adding the peptides or capsaicin cumulatively in logarithmically increasing concentrations. (SP: 10-10 to 10-5 M. CGRP and VIP: 10-10 to 10-7M. Capsaicin: 10-14 to 10-4M). The maximum relaxation obtained after each addition was considered. SP + CGRP experiments. For obtaining concentration-response relaxation curves induced by a combination of peptides, SP was added together with CGRP in equal molar concentrations (10-10 to 10-7M). The maximum effect induced by each simultaneous addition was considered. Drugs were added in 50 µl aliquots. Substance P (acetate salt), VIP (porcine, synthetic) and a-CGRP (human, synthetic), as well as all other drugs were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were diluted in 0.9% NaCI containing 10-3M ascorbic acid. Capsaicin was dissolved in 100% ethanol and diluted in 0.9% NaCI containing 10-3M ascorbic acid. Appropriate controls showed that these vehicle substances did not significantly affect the contractions at the dilutions used. Fresh dilutions were prepared daily from stock solutions stored at -70°C. The data are presented as the mean ± S.E.M. Statistical analysis was carried out with Student's t-test and values of p
