Stefano Manzini

Cardioprotective effects of zofenopril, a new angiotensin-converting enzyme inhibitor, on doxorubicin-induced cardiotoxicity in the rat.

We have studied the effect of zofenopril, a new angiotensin-converting enzyme inhibitor in preventing cardiac injury induced by chronic doxorubicin treatment in rats. Cardiac function was assessed by measuring changes in electrocardiogram (ECG) tracings, haemodynamics and cardiac responses in vivo to isoprenaline, 4 weeks after suspension of doxorubicin treatment, in vehicle-treated rats and in animals receiving zofenopril (15 mg/kg/os/day) alone, doxorubicin (1.5 mg/kg i.v. once a week for 5 weeks) or zofenopril+doxorubicin treatment. Doxorubicin induced a significant lengthening of the QalphaT interval, which was completely prevented by zofenopril treatment. The cardiac positive inotropic effect induced by i.v. isoprenaline was selectively depressed by doxorubicin (no changes in chronotropic responses) and this adverse effect of doxorubicin was also prevented in zofenopril+doxorubicin pretreated rats. Doxorubicin induced a significant increase in relative heart weight, which was likewise prevented in zofenopril+doxorubicin treated rats. In separate experiments, zofenopril did not interfere with the antitumor activity of doxorubicin (inhibition of tumor growth in nude mice xenografted with A2780 human tumor line). In conclusion, the oral administration of zofenopril is able to significantly ameliorate, up to 4 weeks after the end of doxorubicin administration, doxorubicin-induced cardiotoxicity without affecting the antitumor activity of this anthracycline.

Functional characterization of substance P receptors on cultured human spinal cord astrocytes: synergism of substance P with cytokines in inducing interleukin-6 and prostaglandin E2 production.

Following brain injury, astrocytes express receptors for cytokines and neuropeptides and secrete several regulatory mediators that have a well established role in inflammation, immunity, and tissue development or repair. To elucidate the role of substance P (SP), a neurotransmitter peptide of the tachykinin family, in inducing astrocyte secretory activities, we have examined the expression of SP receptors and the functional consequences of their activation in cultured astrocytes from the human embryonic brain or spinal cord. Radioligand binding studies revealed that only one type of SP receptors, the high affinity NK‐1 receptor, was present on human astrocytes and that spinal cord astrocytes expressed about 6 times as many SP binding sites as brain astrocytes. Following SP treatment, a substantial inositol phosphate formation was observed in spinal cord astrocytes only. Stimulation of spinal cord astrocytes with SP alone did not induce secretion of cytokines [interleukin‐6 (IL‐6), granulocyte‐macrophage‐CSF, macrophage chemoattractant protein‐1 or leukemia inhibitory factor] or prostaglandin E2 (PGE2). Interestingly, however, SP selectively potentiated the inducing effect of IL‐1β on IL‐6 and PGE2 secretion by spinal cord astrocytes without affecting the IL‐1‐β‐evoked secretion of other cytokines. SP also enhanced the small inducing effect of tumor necrosis factor‐α (TNF‐α) on IL‐6 and PGE2 secretion and that of transforming growth factor‐β on PGE2 secretion. These results suggest that SP can enhance immunoregulatory and neurotrophic astroglial functions mediated by IL‐6 and PGE2 by acting in concert with a set of cytokines whose cerebral expression has been reported during development and in a variety of diseases. GLIA 21:183–193, 1997.

Simultaneous release by bradykinin of substance P- and calcitonin gene-related peptide immunoreactivities from capsaicin-sensitive structures in guinea-pig heart

Both bradykinin and capsaicin infusion evoked a marked increase in the outflow of substance P‐ (SP‐LI) and calcitonin gene‐related peptide‐like immunoreactivity (CGRP‐LI) from guinea‐pig isolated, perfused heart. After acute exposure to capsaicin in vitro, or in hearts taken from animals pretreated in vivo with capsaicin, bradykinin failed to induce any release. The positive chronotropic effect of bradykinin was reduced after acute capsaicin administration. The effect of bradykinin in the guinea‐pig heart could be mediated, at least partly, by release of neuropeptides from peripheral endings of capsaicin‐sensitive sensory neurones.

Extrinsic origin of the capsaicin-sensitive innervation of rat duodenum: possible involvement of calcitonin gene-related peptide (CGRP) in the capsaicin-induced activation of intramural non-adrenergic non-cholinergic neurons

Summary1.Capsaicin produces a concentration-related relaxation of the longitudinal muscle of the rat isolated duodenum in the presence of atropine (3 μM) plus guanethidine (3 μM). This effect of capsaicin is partly (about 40%) antagonized by tetrodotoxin (1.0 μM) suggesting the involvement of intramural non-adrenergic non-cholinergic (NANC) neurons.2.The capsaicin-induced relaxations are unaffected by previous bilateral vagotomy or removal of the inferior mesenteric ganglion but are completely prevented by removal of the coeliac ganglia plus the superior mesenteric ganglion (72 h before). Acute duodenal denervation did not modify the response to capsaicin.3.Unlike various neuropeptides (substance P, kassinin, neurokinin A, cholecystokinin octapeptide, somatostatin, vasoactive intestinal polypeptide) only the calcitonin gene-related peptide (CGRP) closely mimicked, both qualitatively and quantitatively, the capsaicin-induced relaxations. The CGRP-induced relaxations were unaffected by hexamethonium and partly reduced (about 40%) by tetrodotoxin.4.In preparations desensitized to adenosine-triphosphate (ATP) a putative NANC inhibitory neurotransmitter of the rat duodenum, the effects of CGRP were reduced (about 30%) as compared to controls. After ATP-desensitization tetrodotoxin did not produce any further reduction of the CGRP-induced relaxations suggesting the involvement of endogenous ATP in the neuronal (tetrodotoxin-sensitive) component of the CGRP-induced relaxations.5.Either ATP-or CGRP-desensitization reduced (about 50 and 65% respectively) the amplitude of the capsaicin-induced relaxations. In the presence of both ATP and CGRP capsaicin did not affect motor activity of the rat isolated duodenum indicating a complete desensitization of the effect of endogenously released substance(s).6.These findings indicate that the capsaicin-induced relaxations of the rat isolated duodenum are due to activation of a neural mechanism of extrinsic origin, presumably primary afferent fibers. CGRP is a likely candidate for the role of neuropeptide released by capsaicin in this preparation. Endogenous CGRP may produce NANC relaxation both directly and by releasing the endogenous NANC neurotransmitter(s).

Cutaneous lesions in capsaicin-pretreated rats. A trophic role of capsaicin-sensitive afferents?

Summary1. The time course and regional distribution of ‘spontaneous’ cutaneous lesions in rats desensitized to capsaicin as newborns was correlated to behavioural observations and regional distribution of substance P-like immunoreactivity (SP-LI) and tachykinin-like immunoreactivity (TK-LI) in various skin areas. 2. ‘Spontaneous’ skin lesions in the form of wounds, scabs and areas of alopecia were observed in 80–90% of rats desensitized to capsaicin. No major sex-related differences were observed with regard to incidence and distribution of the lesions with the possible exception of a lesser tendency to bilateral lesions in female rats. 3. ‘Spontaneous’ skin lesions were almost restricted to the head: the areas most frequently affected were snouts, periocular and retroauricular regions and ventral area of the neck. 4. No major differences were observed between capsaicin- or vehicle-treated animals in spontaneous or novelty-induced grooming as well as in open-field gross behaviour. Likewise, no differences were observed in the mouse-killing behaviour. 5. Both SP-LI and TK-LI in various skin areas were significantly reduced by systemic capsaicin pretreatment. The rank order of various skin areas for SP-LI or TK-LI levels was: snouts > thigh > neck > abdomen ≃ retroauricular region. 6. Intradermal injection of Arg-neurokinin B, a potent and water soluble derivative of neurokinin B, produced a similar plasma extravasation (Evans blue leakage technique) in the skin of vehicle- or capsaicin-pretreated rats. 7. In capsaicin-desensitized rats fur regrowth (measured at abdominal level, 28 days after shaving) was significantly less than in vehicle-treated animals. 8. The s. c. injection of 1 N HCl in the dorsal thoracic region (an area devoid of ‘spontaneous’ lesions in capsaicin-desensitized animals) produced cutaneous ulcers whose area and depth were greater in capsaicin- than vehicle-treated rats. 9. These findings are consistent with the hypothesis that capsaicin-sensitive nerves play a trophic role in the rat skin and contribute to its ability to react and repair injuries. The most consistent explanation for the restricted localization of ‘spontaneous’ skin lesions to the head seems to be that ‘normal’ injurious factors (such as grooming) operate on a distrophic skin to induce lesions by repeated microtrauma.

Bronchodilatation by tachykinins and capsaicin in the mouse main bronchus.

1 The effect of sensory neuropeptides and capsaicin on basal and stimulated tone of mouse bronchial smooth muscle has been evaluated. 2 In basal conditions neither sensory neuropeptides (subtance P, neurokinin A or calcitonin gene‐related peptide (CGRP)) nor capsaicin exerted any contractile effects. However, when a tonic contraction was induced with carbachol (1 μm) a prompt relaxation was induced by substance P (1– 100 nm) and by neurokinin A (1– 100 nm), with substance P being more potent. A second application of substance P was without effect. CGRP (10 nm) produced only a very small and erratic relaxation. Relaxation was also induced by capsaicin (1 μm), and this response could be evoked only once in each preparation. In 4 out of 6 preparations a cross‐desensitization between substance P and capsaicin was observed. 3 The selective NK1 tachykinin agonist, [Pro9]‐SP sulphone (1 μm), exerted potent bronchodilator actions on carbachol‐contracted mouse bronchial preparations. In contrast, neither [β Ala8]‐NKA (4–10) nor [MePhe7]‐NKB (both at a concentration of 1 μm), selective synthetic agonists for NK2 and NK3 receptors, exerted significant relaxant effects. Furthermore, the selective NK1 tachykinin antagonist, (±)‐CP 96,345 (1 μm), abolished substance P (1 nm)‐ but not isoprenaline (0.1 μm)‐induced relaxations. 4 Application of electrical field stimulation (EFS) (20 Hz, supramaximal voltage, 0.5 ms for 10 s) to carbachol‐contracted preparations evoked a transient contraction followed by a relaxation. The tetrodotoxin‐sensitive slow component of this relaxation was reduced following capsaicin desensitization. 5 In the presence of indomethacin (5 μm) the relaxation induced by substance P, capsaicin or EFS was suppressed. 6 In conclusion, the mouse main bronchus appears to be a monoreceptorial tissue containing only NK1 receptors which subserve bronchodilator functions. Such receptors could be activated by exogenous or endogenously (capsaicin or EFS) released tachykinins and the consequent relaxation is probably mediated by the generation of prostanoids.

A comparison of bradykinin- and capsaicin-induced myocardial and coronary effects in isolated perfused heart of guinea-pig: involvement of substance P and calcitonin gene-related peptide release

1 Bradykinin and capsaicin were compared for their ability to elicit functional effects and to release sensory neuropeptides from guinea‐pig isolated perfused hearts. 2 Both bradykinin (10 μm) and capsaicin (1 μm) produced a marked increase in coronary flow, a large positive chronotropic effect and a significant reduction in contractile strength. These actions were associated with a marked release of substance P‐like immunoreactivity (SP‐LI) and calcitonin gene‐related‐like immunoreactivity (CGRP‐LI). The percentage of the tissue content of SP‐LI and CGRP‐LI released by each agent was similar, although bradykinin was less effective than capsaicin. The ratio of SP‐LI/CGRP‐LI released by both agents was similar to that present in cardiac tissue. 3 Neuropeptide release could be evoked only once with capsaicin but at least four times with bradykinin. Also, functional responses to capsaicin underwent desensitization. After either in vitro or systemic capsaicin pretreatment, the release of SP‐LI and CGRP‐LI by bradykinin was reduced and the positive chronotropic effect of bradykinin was significantly reduced, while the increase in coronary flow and negative inotropic responses remained unchanged. 4 Pretreatment with indomethacin (10 μm) strongly antagonized the release of SP‐LI and CGRP‐LI by bradykinin and reduced the increase in heart rate. 5 These findings suggest that activation by bradykinin (probably through indirect mechanisms) of capsaicin‐sensitive sensory nerves in the heart, leads to a local release of sensory neuropeptides. These neuropeptides, in turn, could participate in determining the complex functional effects of this kinin on cardiac performance.

Effect of ricinoleic acid in acute and subchronic experimental models of inflammation

Observational studies indicate that topical application of ricinoleic acid (RA), the main component of castor oil, exerts remarkable analgesic and anti-inflammatory effects. Pharmacological characterization has shown similarities between the effects of RA and those of capsaicin, suggesting a potential interaction of this drug on sensory neuropeptide-mediated neurogenic inflammation. The aim of this study was to assess RA anti-inflammatory activities in comparison with capsaicin in several models of acute and subchronic inflammation. The acute inflammation was induced by intradermal injection of carrageenan in the mouse or by histamine in the guinea-pig eyelid. In either experiment, the extent of the oedema thickness was measured. Subchronic oedema was induced by complete Freunds adjuvant injection in the ventral right paw of mice. Tissue substance P (SP) was measured in the carrageenan experiments by radioimmunoassay (RIA). It was found that the acute topical application of RA (0.9 mg/mouse) or capsaicin (0.09 mg/mouse) significantly increased the mouse paw oedema induced by carrageenan, while an 8-day repeated topical treatment with the same doses of both compounds resulted in a marked inhibition of carrageenan-induced paw oedema matched by a reduction in SP tissue levels. Similar effects were found against histamine-induced eyelid oedema in guinea-pigs after acute or repeated application of RA or capsaicin. RA and capsaicin given for 1-3 weeks reduced the established oedema induced by Freunds adjuvant, a subchronic model of inflammation, particularly if given by the intradermal route. Either in mouse paw or in guinea-pig eyelid, capsaicin but not RA by itself produced a slight hyperemia and activation of a behavioural response (e.g. scratching of the eyelids). On the basis of the present results, RA may be seen as a new capsaicin-like, non-pungent anti-inflammatory agent suitable for peripheral application.

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.