Francesca Perretti

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.

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.

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 effect of thiorphan and epithelium removal on contractions and tachykinin release produced by activation of capsaicin-sensitive afferents in the guinea-pig isolated bronchus

Summary(1) We have studied the effect of epithelium removal (rubbing) and the endopeptidase 24.11 inhibitor, thiorphan, on the contractile response of the guinea-pig isolated bronchi (atropine and indomethacin in the bath) produced by electrical field stimulation, capsaicin or exogenously administered tachykinins (substance P and neurokinin A). (2) The response to field stimulation, thought to involve release of endogenous tachykinins, was potentiated by thiorphan in both epithelium-free and intact bronchi. However, at low frequencies (1–5 Hz), the effect of thiorphan was more evident in intact preparations. (3) The response to capsaicin was enhanced by both epithelium removal and thiorphan administration. (4) The response to exogenous substance P or neurokinin A was potentiated by thiorphan both in epithelium-free and intact bronchi. (5) Capsaicin (1 μM) evoked a consistent release of substance P-like immumoreactivity (determined by radioimmunoassay) and tachykinin-like immumoreactivity (determined by a novel immumoenzyme assay), which was enhanced by thiorphan in both epithelium-free and intact bronchi. (6) These findings suggest that a thiorphan-sensitive mechanism, presumably ‘enkephalinase’ (endopeptidase 24.11), plays a major role in inactivating endogenous tachykinins released from sensory nerves and that this enzymatic activity is still present after removal of the bronchial epithelium.

Vascular effects of capsaicin in isolated perfused rat mesentric bed

The effects of intra- and extraluminal capsaicin administration were evaluated in isolated perfused rat mesenteric bed. Capsaicin (10 nM-1 microM) produced a potent concentration-dependent relaxation of the tonic vasoconstriction induced by norepinephrine (1 microM) but not by high-K+ (60 mM). The capsaicin-induced relaxation was nearly abolished in preparations pretreated in vitro with a high concentration of capsaicin (1 microM, for 10 min, 1 h before). Capsaicin-induced relaxation was reduced but not abolished in preparations obtained from rats pretreated neonatally with capsaicin. The capsaicin effects were unaffected by atropine, guanethidine, propranolol, hexamethonium or tetrodotoxin. The observation that capsaicin (0.1 microM)-induced relaxation was virtually abolished in presence of the proteolytic enzyme alpha-chymotrypsin (1 U/ml) supports the involvement of neuropeptide(s) in this response. Bolus injections of calcitonin gene-related peptide (CGRP) elicited a potent and rapidly ensuing relaxation which underwent tachyphylaxis. However, no cross-desensitization with capsaicin was observed. It is concluded that activation of capsaicin-sensitive sensory fibers could release neuropeptides locally with a potent effect on intestinal blood flow.

Antibronchospastic activity of MEN10,627, a novel tachykinin NK2 receptor antagonist, in guinea-pig airways

The antibronchospastic activity against acetylcholine, antigen, histamine plus platelet-activating factor (PAF) or the selective tachykinin neurokinin (NK)1 and NK2 receptor agonists of the novel tachykinin NK2 receptor antagonist, MEN10,627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta)), was studied in anesthetized guinea-pigs. MEN10,627 (30-100 nmol/kg i.v.) reduced in a dose-dependent manner the bronchospasm induced by the tachykinin NK2 receptor agonist [beta Ala8]neurokinin A-(4-10) and the effect of the highest dose lasted up to 5 h from its administration. Conversely, airway constriction induced by the NK1 receptor agonist [Sar9]substance P sulfone or acetylcholine was unaffected by MEN10,627 up to a dose of 3 mumol/kg i.v. In animals sensitized with ovalbumin and pretreated with the endopeptidase inhibitor phosphoramidon, the aerosolized antigen produced a bronchospasm which was inhibited by MEN10,627 (30-100 nmol/kg i.v.) but not by the tachykinin NK1 receptor antagonist, (+/-)-CP96,345 ([2R,3R-cis- and [2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine]) (3 mumol/kg i.v.). Both MEN10,627 (30-100 nmol/kg i.v.) and (+/-)-CP96,345 (30-300 nmol/kg i.v.) reduced the PAF-induced hyperresponsiveness to histamine, without affecting the hypotension induced by PAF or the bronchospasm induced by histamine in guinea-pigs not exposed to PAF, showing the involvement of both tachykinin NK1 and NK2 receptors in this model. In summary, MEN10,627 behaves as a potent, selective and long-lasting tachykinin NK2 receptor antagonist in vivo. Further, tachykinin NK2 receptors could be activated during allergic responses and in the development of airway hyperresponsiveness.

Isbufylline, a new xanthine derivative, inhibits airway hyperresponsiveness and airway inflammation in guinea pigs

The pharmacological actions of the new xanthine, isbufylline, were evaluated in several models of airway hyperresponsiveness and airway inflammation in guinea pigs. At a dose (106 mumol kg-1 i.p.) providing complete protection against acetylcholine aerosol-induced dyspnea in the guinea pig, isbufylline inhibited platelet activating factor (PAF)- and antigen-induced eosinophil infiltration into bronchoalveolar lavage fluid 24 h after challenge of normal and actively immunized guinea pigs, respectively. In addition, this dose of isbufylline also inhibited capsaicin-induced extravasation of protein into bronchoalveolar lavage fluid. Isbufylline, 4.2 mumol kg-1 i.v., significantly inhibited PAF-induced bronchial hyper-responsiveness to i.v. histamine, without exerting evident bronchodilator activity. On the other hand the bronchodilator, salbutamol, at a dose (10.4 mumol kg-1 i.p.) shown to be equieffective to isbufylline (106 mumol kg-1 i.p.) for blocking acetylcholine aerosol-induced dyspnea, had no protective action against PAF- or antigen-induced eosinophil recruitment in bronchoalveolar lavage fluid, or against capsaicin-induced plasma protein extravasation. Furthermore, salbutamol (3.5 mumol kg-1) significantly potentiated allergen-induced cell infiltration and PAF-induced bronchial hyperresponsiveness. The results suggest that isbufylline can exert significant anti-inflammatory actions in guinea pig airways, in addition to its bronchodilator activity. These pharmacological activities are not shared by the beta 2-adrenoceptor agonist, salbutamol.

Arachidonic acid-induced bronchomotor responses are partially mediated by release of sensory neuropeptides from capsaicin-sensitive structures.

Administration of arachidonic acid (AA) both in vitro and in vivo elicited prominent contractile responses in guinea‐pig airways, which were markedly reduced after capsaicin desensitization. Furthermore, AA superfusion elicited a significant calcitonin gene‐related peptide‐like immunoreactivity release from isolated bronchi. It is suggested that at least part of the bronchomotor actions of AA rely upon stimulation of capsaicin‐sensitive primary afferents.

Neurochemical evidence of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) release from capsaicin-sensitive nerves in rat mesenteric arteries and veins.

1. The ability of capsaicin and antidromic stimulation of perivascular nerve fibers to release sensory neuropeptides (SP-LI and CGRP-LI) have been investigated in rat mesenteric arteries and veins. 2. Both in mesenteric arteries and veins substantial SP-LI and CGRP-LI tissue levels were measured. A significant reduction in sensory neuropeptides levels was observed in tissues obtained from capsaicin-pretreated animals. 3. Superfusion of isolated vessels with capsaicin (1 microM) produced a prompt and remarkable release of both SP-LI and CGRP-LI, which can be evoked only once in each preparation. 4. Electrical field stimulation (EFS, 20 Hz, 50 V, 0.5 msec, trains of 10 sec every 20 sec for 15 min) of isolated vessels resulted in a significant release of CGRP-LI. This release was significantly greater in veins as compared to arteries. EFS-induced CGRP-LI release was unaffected by atropine or guanethidine and absent in preparations obtained from capsaicin-pretreated rats. 5. These neurochemical findings further suggest that the local release of sensory neuropeptides from capsaicin-sensitive nerve endings might be important in the regulation of mesenteric circulation.

Calcitonin gene-related peptide in rat arterial and venous vessels: Sensitivity to capsaicin, bradykinin and FMLP

Sensitivity of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) to capsaicin was investigated in different arterial and venous tissues (mesenteric renal and femoral, artery and vein and carotid artery and jugular vein) of the rat.In vivo capsaicin pre-treatment depleted or reduced tissue CGRP-LI in all the vessels examined with the exception of the carotid artery. Likewise,in vitro exposure to capsaicin evoked release of CGRP-LI from all vessels, excluding the carotid artery. Remarkable amount of CGRP-LI sensitive to capsaicin was present in both mesenteric artery and vein as compared to the other vascular tissues. Endogenous (bradykinin) or exogenous (N-formyl-methionyl-leucyl-phenylalanine) proinflammatory agents, were found to release CGRP-LI from mesenteric veins.