Manuela Tramontana

Similarities and differences in the action of resiniferatoxin and capsaicin on central and peripheral endings of primary sensory neurons.

We have compared the ability of capsaicin and resiniferatoxin, a natural diterpene present in the latex of plants of the Euphorbia family to excite and desensitize capsaicin-sensitive primary afferents in a variety of models. Both capsaicin and resiniferatoxin inhibited the twitch contractions of the rat isolated vas deferens and prevented, in a concentration-related manner, the effect of a subsequent challenge with 1 microM capsaicin (desensitization). Resiniferatoxin was 1000-10,000 times more potent than capsaicin in both cases. The time course of action of resiniferatoxin was much slower than that of capsaicin, suggesting a slower penetration rate in the tissue. The action of resiniferatoxin was blocked by Ruthenium Red, a proposed antagonist at the cation channel coupled to the capsaicin receptor. Both capsaicin and resiniferatoxin produced a contraction of the rat isolated urinary bladder. Resiniferatoxin was about as potent as capsaicin in this assay although it was 500-1000 times more potent than capsaicin in desensitizing the primary afferents to a subsequent challenge with capsaicin itself. Resiniferatoxin did not affect motility in the isolated vasa deferentia or urinary bladder from capsaicin-pretreated rats. After topical application onto the rat urinary bladder both resiniferatoxin (10 nM) and capsaicin (10 microM) increased bladder capacity as assessed in a volume-evoked micturition reflex model in rats without affecting micturition contraction. Intrarterial injection of resiniferatoxin or capsaicin in the ear of anesthetized rabbits evoked a systemic depressor reflex due to activation of paravascular nociceptors, resiniferatoxin being about three times more potent than capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological profile of the novel mammalian tachykinin, hemokinin 1

The effects of the novel mammalian tachykinin, hemokinin 1 (HEK‐1), have been investigated by radioligand binding and functional in vitro and in vivo experiments. Similar to SP (Ki=0.13 nM), HEK‐1 inhibited in a concentration‐dependent manner and with high affinity [3H]‐substance P (SP) binding to human NK1 receptor (Ki=0.175 nM) while its affinity for [125I]‐neurokinin A (NKA) binding at human NK2 receptor was markedly lower (Ki=560 nM). In isolated bioassays HEK‐1 was a full agonist at tachykinin NK1, NK2 and NK3 receptors. In the rat urinary bladder (RUB) HEK‐1 was about 3 fold less potent than SP. In the rabbit pulmonary artery (RPA) HEK‐1 and in the guinea‐pig ileum (GPI), HEK‐1 was about 500 fold less potent than NKA and NKB, respectively. The responses to HEK‐1 were antagonized by GR 82334 in RUB (pKB=5.6±0.07), by nepadutant in RPA (pKB=8.6±0.04) and by SR 142801 in GPI (pKB=9.0±0.2) with apparent affinities comparable to that measured against tachykinin NK1, NK2 and NK3 receptor‐selective agonists, respectively. Intravenous HEK‐1 produced dose‐related decrease of blood pressure in anaesthetized guinea‐pigs (ED50=0.1 nmol kg−1) and salivary secretion in anaesthetized rats (ED50=6 nmol kg−1) with potencies similar to that of SP. All these effects were blocked by the selective tachykinin NK1 receptor antagonist, SR 140333. We conclude that HEK‐1 is a full agonist at tachykinin NK1, NK2 and NK3 receptors, possesses a remarkable selectivity for NK1 as compared to NK2 or NK3 receptors and acts in vivo experiments with potency similar to that of SP.

Effect of nociceptin on heart rate and blood pressure ananaesthetized rats

We now report on the effects of nociceptin, the endogenous ligand for the orphan opioid-like ORL1 receptor, on cardiovascular parameters (blood pressure and heart rate) in urethane-anaesthetized rats. Nociceptin dose dependently (10-100 nmol/kg i.v.) produced a transient (< 10 min) hypotension and bradycardia. The reduction in blood pressure was inhibited by guanethidine pretreatment and unaffected by bilateral cervical vagotomy. The bradycardia elicited by nociceptin was reduced after bilateral vagotomy and by guanethidine and was abolished by the combination of the two treatments. These findings indicate that nociceptin exerts a pronounced depressant effect on cardiovascular function which is produced indirectly through a concomitant inhibition and activation, respectively, of the sympathetic and parasympathetic outflows to the cardiovascular system.

Neurochemical evidence for the involvement of N-type calcium channels in transmitter secretion from peripheral endings of sensory nerves in guinea pigs

In the guinea pig ureter, substance P-(SP) and calcitonin gene-related peptide-(CGRP) like immunoreactivity (LI) were depleted by systemic capsaicin pretreatment, indicating that they are entirely stored in peripheral endings of primary afferent neurons. Electrical field stimulation (20 Hz, 60 V, 0.5 ms) evoked the simultaneous release of SP- and CGRP-LI from superfused guinea pig ureters which was abolished by tetrodotoxin (0.3 microM). omega-Conotoxin (0.1 microM), a potent blocker of N-type voltage-sensitive calcium channels, reduced by 50-70% the evoked release of both peptides. These findings provide direct neurochemical evidence indicating that conotoxin-sensitive calcium channels play a role in transmitter secretion evoked by antidromic invasion of peripheral terminals of capsaicin-sensitive primary afferents.

MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist

The pharmacological profile was studied of MEN 11420, or cyclo{[Asn(β‐D‐GlcNAc)‐Asp‐Trp‐Phe‐Dap‐Leu]cyclo(2β‐5β)}, a glycosylated derivative of the potent, selective, conformationally‐constrained tachykinin NK2 receptor antagonist MEN 10627 (cyclo(Met‐Asp‐Trp‐Phe‐Dap‐Leu)cyclo(2β‐5β)). MEN 11420 competitively bound with high affinity to the human NK2 receptor stably transfected in CHO cells, displacing radiolabelled [125I]‐neurokinin A and [3H]‐SR 48968 with Ki values of 2.5±0.7 nM (n=6) and 2.6±0.4 nM (n=3), respectively. MEN 11420 showed negligible binding affinity (pIC50<6) at 50 different receptors (including tachykinin NK1 and NK3 receptors) and ion channels. In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2 receptor‐mediated contractions (pKB=8.6±0.07, n=10, and 9.0±0.04, n=12; Schild plot slope=−1.06 (95% c.l.=−1.3; −0.8) and −1.17 (95% c.l.=−1.3; −1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2 receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pKB of 10.2±0.14, n= 9, and 9.8±0.15, n=9, was calculated in the hamster trachea and mouse urinary bladder, respectively. MEN 11420 showed low affinity (pKB<6) at guinea‐pig and rat tachykinin NK1 (guinea‐pig ileum and rat urinary bladder) and NK3 (guinea‐pig ileum and rat portal vein) receptors. On the whole, the affinities (potency and selectivity) showed by MEN 11420 for different tachykinin receptors, measured either in binding or in functional bioassays, were similar to those shown by the parent compound, MEN 10627. The in vivo antagonism of the contractions produced by [βAla8]neurokinin A(4–10) (1 nmol kg−1) was observed after intravenous (dose range: 1–10 nmol kg−1), intranasal (3–10 nmol kg−1), intrarectal (30–100 nmol kg−1) and intraduodenal (100–300 nmol kg−1) administration of MEN 11420. MEN 11420 was more potent (about 10 fold) and longer lasting than its parent compound MEN 10627, possibly due to a greater metabolic stability. A dose of MEN 11420 (100 nmol kg−1, i.v.), that produced potent and long lasting inhibition of the contraction of the rat urinary bladder induced by challenge with the NK2 selective receptor agonist [βAla8]neurokinin A(4–10) (10–300 nmol kg−1), was without effect on the responses produced by the NK1 receptor selective agonist [Sar9]substance P sulphone (1–10 nmol kg−1). These findings indicate that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist. The introduction of a sugar moiety did not produce major changes in the affinity profile of this antagonist as compared to MEN 10627, but markedly improved its in vivo potency and duration of action. With these characteristics, MEN 11420 is a suitable candidate for studying the pathophysiological significance of tachykinin NK2 receptors in humans.

Release of calcitonin gene-related peptide like-immunoreactivity induced by electrical field stimulation from rat spinal afferents is mediated by conotoxin-sensitive calcium channels

Electrical field stimulation (EFS, 10 V, 50 mA/cm2, 5-50 Hz, 1 ms pulse duration, 10 s train every 20 s for 5 min) produced a rapid and reproducible outflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) from superfused slices from the dorsal half of the rat spinal cord which is abolished by tetrodotoxin (TTX, 0.3 microM), in vitro capsaicin desensitization (10 microM for 30 min) and in Ca-free medium. The response was unaffected by ruthenium red (10 microM), indomethacin (10 microM) and nifedipine (1 microM) while it was abolished by omega-conotoxin (omega-CTX, 0.1 microM) and, in a naloxone-sensitive manner, by morphine (3 microM). Since CGRP release from capsaicin-sensitive afferents evoked by EFS in rat peripheral tissues is conotoxin-resistant, these findings provide direct evidence for a qualitative difference between central and peripheral endings of capsaicin-sensitive primary afferents in the mechanisms regulating transmitter release in the same species.

Topical capsaicin administration protects against trinitrobenzene sulfonic acid-induced colitis in the rat

We used the [3H]resiniferatoxin binding assay to demonstrate for the first time the existence of vanilloid receptors in the rat colon and to explore their expression during trinitrobenzene sulfonic acid-induced colitis. Membranes obtained from control colon bound [3H]resiniferatoxin with an affinity of 3 nM; the receptor density was 450 fmol/mg protein or 9 fmol/mg wet weight. Capsaicin and capsazepine, a competitive antagonist of capsaicin, inhibited specific resiniferatoxin binding with Ki values of 3 microM and 0.1 microM, respectively. Trinitrobenzene sulfonic acid induced a very rapid ulceration in the colon: 1 h after treatment 90% of the colon showed ulcerative damage. Coadministration of 640 microM capsaicin diminished the ulcerative effect of trinitrobenzene sulfonic acid to 64% when examined 1 h after trinitrobenzene sulfonic acid challenge; however, this protective action was lost 23 h later. Colon samples obtained 4 h, 24 h, and 1 week after trinitrobenzene sulfonic acid challenge bound resiniferatoxin, capsaicin, and capsazepine with affinities similar to those of control samples. The receptor density remained at an essentially constant level when expressed in fmol/mg protein but, in keeping with the increased wet weights, showed a reduction when expressed in fmol/mg wet weight. We conclude that acute capsaicin administration protects against the ulcerative action of trinitrobenzene sulfonic acid, most likely via the release of protective neuropeptides from capsaicin-sensitive nerve endings. The loss of this protective action is presumably due to a depletion of the protective neuropeptides rather than to a loss of vanilloid (capsaicin) receptors.

Sensory nerves, vascular endothelium and neurogenic relaxation of the guinea-pig isolated pulmonary artery

Summary1. In the presence of atropine and guanethidine (3 μmol/l each), electrical field stimulation (1–20 Hz) produced frequency-dependent relaxations of the histamine-(3 μmol/l) induced vascular tone in isolated rings from the guinea-pig pulmonary artery. The electrically-evoked relaxations were abolished by tetrodotoxin (1 μmol/l). The amplitude of these nerve-mediated, nonadrenergic non-cholinergic (NANC) relaxations was unaffected by removal of the vascular endothelium produced through rubbing of the internal surface. 2. Capsaicin (1 μmol/l) produced a prompt and sustained relaxation of the histamine-induced tone which was unaffected by removal of the endothelium. A second application of capsaicin 60–120 min later had no further relaxant effect, indicating desensitization. After in vitro capsaicin desensitization, the electrically-evoked NANC relaxations were abolished, both in the presence or absence of the vascular endothelium. 3. Substance P evoked a prompt and transient relaxation in precontracted arterial rings with intact endothelium and a transient small contraction in rings in which the endothelium had been mechanically removed. The selective NK-1 receptor agonist, [Pro9]-substance P sulfone closely mimicked the relaxation produced by substance P while the selective NK-2 or NK-3 receptor agonists had no relaxant effect. Tachyphylaxis to substance P did not modify the amplitude of the capsaicin-induced relaxation. 4. Human alpha calcitonin gene-related peptide (CGRP) produced a prompt and sustained relaxation both in the presence and absence of the vascular endothelium. 5. Ruthenium red (10 μmol/l) blocked the relaxation to capsaicin while leaving unaffected the relaxation to electrical field stimulation or CGRP (0.1 μmol/l). 6. Both substance P (SP)-and CGRP-like immunoreactivities (LI) were detected in extracts of the guinea-pig pulmonary artery. Capsaicin (1 μmol/l) evoked a prompt and simultaneous outflow of both SP- and CGRP-LI. A second application of capsaicin 60 min later failed to increase SP- or CGRP-LI outflow, indicating complete desensitization. A small but clearly detectable release of both SP-LI and CGRP-LI was also evoked by electrical field stimulation. 7. These findings provide evidence that the neurogenic NANC vasodilation in the guinea-pig pulmonary artery is due to antidromic activation of peripheral endings of capsaicin-sensitive primary afferents. Endogenous CGRP is a likely mediator for this vasodilation. No evidence was found that endogenous SP might contribute to vasodilation by activating NK-1 receptors on endothelial cells.

The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats.

1 We have investigated the effect of nociceptin on the micturition reflex evoked by distension or topical application of capsaicin on the urinary bladder of urethane‐anaesthetized rats. 2 Nociceptin produced a dose‐dependent (3–100 nmol kg−1 i.v.) transient suppression of the distension‐evoked micturition reflex: its effect was not modified by guanethidine (68 μmol kg−1 s.c.) nor by bilateral cervical vagotomy, alone or in combination, and by naloxone (1.2 μmol kg−1 i.v.). 3 Nociceptin (100 nmol/kg i.v.) slightly (about 30%) inhibited the contractions of the rat bladder produced by pre‐ or postganglionic electrical stimulation of the pelvic nerve. 4 Nociceptin almost totally abolished the reflex component of the response to topical capsaicin (1 μg in 50 μl). 5 In the rat isolated bladder, submaximal contractions produced by electrical field stimulation were slightly reduced (25±4% inhibition) by 1 μm nociceptin. Nociceptin did not affect the contraction of the rat bladder induced by acetylcholine (10 μm) or ATP (1 mm). 6 These findings indicate that nociceptin exerts a naloxone‐resistant suppression of the volume‐evoked micturition reflex which involves inhibition of transmitter release from postganglionic bladder nerves. An inhibitory effect on bladder afferent nerves is also suggested.

Adenosine inhibits action potential-dependent release of calcitonin gene-related peptide- and substance P-like immunoreactivities from primary afferents in rat spinal cord

Electrical field stimulation (5 Hz) evoked a prompt outflow of calcitonin gene-related peptide- and substance P-like immunoreactivities (CGRP-LI and SP-LI, respectively) from superfused slices of the dorsal but not ventral half of the rat spinal cord. The evoked outflow was abolished by tetrodotoxin, calcium-free medium or previous exposure to capsaicin, indicating that it is produced through action potentials invading the central terminals of capsaicin-sensitive primary afferents. Adenosine as well as gamma-aminobutyric acid (GABA) or the GABAB receptor agonist (-)-baclofen produced a concentration-dependent inhibition of the evoked CGRP-LI outflow. Adenosine also inhibited the evoked SP-LI outflow. These findings demonstrate that inhibition of transmitter release from primary afferent neurons should be considered as a possible mechanism of the antinociceptive action of adenosine and adenosine analogs.